Hyaluronic acid derivatives, pharmaceutical compositions and hyaluronic acid derivative-drug conjugates

Abstract

The present invention provides a hyaluronic acid derivative having a steroidal group incorporated therein, wherein the weight average molecular weight Mw of the hyaluronic acid derivative is 1.11 or more and 10.0 or less than the number average molecular weight Mn.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid derivative, a pharmaceutical composition, and a hyaluronic acid derivative-drug combination. This application claims priority based on Special Application No. 2020-019963 filed in Japan on February 7, 2020, and Special Application No. 2020-111674 filed in Japan on June 29, 2020, the contents of which are incorporated herein by reference. Prior Art

[0002] In recent years, pharmaceuticals using proteins, peptides, or nucleic acids as active ingredients—biopharmaceuticals—have become commercially available, with their number increasing year by year. Biopharmaceuticals can address unmet health needs previously unmet by low-molecular-weight drugs. However, they face challenges such as poor absorption through the digestive tract and mucous membranes, instability in the body, and a short half-life in the bloodstream. Consequently, biopharmaceuticals require frequent injections, placing a significant burden on both patients and healthcare professionals. Consequently, there is a growing demand for drug-based materials (sustained-release drug delivery system materials) that can encapsulate biopharmaceuticals without compromising their pharmacological activity, allowing them to slowly release their active ingredients within the body.

[0003] Against this backdrop, Patent Documents 1 and 2 propose sustained-release drug delivery system substrates containing hyaluronic acid derivatives, which exhibit excellent safety. These hyaluronic acid derivatives spontaneously associate in aqueous solutions, efficiently encapsulating drugs, particularly biopharmaceuticals, while maintaining their biological activity. They aggregate (or disperse) at physiological saline concentrations and exhibit excellent blood retention. It is believed that these hyaluronic acid derivatives, particularly when using biopharmaceuticals as active ingredients, can be used as carriers capable of efficiently encapsulating large amounts of drugs while maintaining pharmacological activity, as well as for sustained-release and targeted delivery systems with excellent blood retention. Furthermore, they can be used as localized (e.g., subcutaneous) sustained-release carriers capable of sustained drug release. [Prior Art Literature] [Patent Document]

[0004] [Patent Document 1] International Publication No. 2010 / 053140 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-297542 Summary of the Invention

[0005] [Problems to be solved by the invention]

[0006] However, the hyaluronic acid derivatives described in Patent Documents 1 and 2 have the problem of high viscosity and insufficient permeability during injection.

[0007] The present invention is made in view of the above situation and provides a hyaluronic acid derivative with excellent needle-permeability, a pharmaceutical composition using the hyaluronic acid derivative, and a hyaluronic acid derivative-drug combination. [Technical means to solve the problem]

[0008] That is, the present invention includes the following aspects. (1) A hyaluronic acid derivative into which a steryl group is introduced, and The ratio Mw / Mn of the weight average molecular weight Mw to the number average molecular weight Mn of the hyaluronic acid derivative is 1.11 or more and 10.0 or less. (2) The hyaluronic acid derivative as described in (1) above, wherein the weight average molecular weight of the hyaluronic acid derivative is greater than or equal to 3,000 and less than or equal to 160,000. (3) The hyaluronic acid derivative as described in (1) or (2) above, wherein the Mw / Mn of the hyaluronic acid derivative is greater than 1.2 and less than 3.0. (4) The hyaluronic acid derivative as described in any one of (1) to (3) above, wherein the introduction rate of the steryl group relative to the hyaluronic acid derivative is greater than 6% and less than 60%. (5) The hyaluronic acid derivative according to any one of (1) to (4) above, wherein the hyaluronic acid derivative has a repeating unit represented by the following general formula (I): [Chemistry 1] (wherein, R 1, R 2, R 3 and R 4 are independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a methyl group and a C 1-6 alkylcarbonyl group; Z represents a direct bond, or a peptide linker containing 2 or more and 30 or less amino acid residues; X1 is a group selected from the group consisting of groups represented by the following formula: -NR bR、 -NR b-COO-R, -NR b-CO-R, -NR b-CO-NR cR, -COO-R, -O-COO-R, -SR, -CO-Y aSR, -O-CO-Y bSR, -NR b-CO-Y bSR, and -SSR; R a, R b and R c are independently selected from the group consisting of a hydrogen atom, a C 1-20 alkyl group, an amino C 2-20 alkyl group and a hydroxy C 2-20 alkyl group, wherein the alkyl portion of the group may be inserted with a group selected from the group consisting of -O- and -NR f-; Rf is selected from the group consisting of a hydrogen atom, a C 1-12 alkyl group, an amino C 2-12 alkyl group and a hydroxy C 2-12 alkyl group, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is a C 2-30 alkylene group, or -(CH 2CH 2O) m-CH 2CH 2-, wherein the alkylene group may be inserted with a group selected from the group consisting of -O-, -NR g- and -SS-; Rg is selected from the group consisting of a hydrogen atom, a C 1-20 alkyl group, an amino C 2-20 alkyl group and a hydroxy C 2-20 alkyl group, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; Y a is a C 1-5 alkyl group; Y b is C 2-8 alkyl or C 2-8 alkenyl; m is an integer of 1 to 100. (6) The hyaluronic acid derivative as described in (5) above, wherein the above R is a cholesterol group. (7) A hyaluronic acid derivative as described in any one of (1) to (6) above, wherein the ratio Mw / Mn of the weight average molecular weight Mw of the hyaluronic acid derivative to the number average molecular weight Mn is greater than 1.2 and less than 2.9. (8) The hyaluronic acid derivative as described in any one of (1) to (7) above, wherein the weight average molecular weight of the hyaluronic acid derivative is greater than or equal to 7,000 and less than or equal to 120,000. (9) A pharmaceutical composition comprising a hyaluronic acid derivative as described in any one of (1) to (8) above as a carrier. (10) The pharmaceutical composition as described in (9) above, wherein the drug forms a complex with the above-mentioned hyaluronic acid derivative. (11) The pharmaceutical composition as described in (9) or (10) above, wherein the drug is a protein, peptide or nucleic acid with pharmacological activity. (12) A hyaluronic acid derivative-drug conjugate, wherein one or more drugs are conjugated to a hyaluronic acid derivative as described in any one of (1) to (8) above. [Effects of the Invention]

[0009] According to the above-mentioned hyaluronic acid derivative, a hyaluronic acid derivative with excellent needle permeability can be provided. Simple diagram description

[0010] FIG1 is the 1H-NMR spectrum of 6-aminohexylcarbamate cholesteryl ester hydrochloride in Example 1. FIG2 is the 1H-NMR spectrum of the tetrabutylammonium (TBA) salt of hyaluronic acid (HA) in Example 1. FIG3 is the 1H-NMR spectrum of the HA derivative (HA-C 6-Chol) introduced with 6-aminohexylcarbamate in Example 1. Implementation Method

[0011] Hereinafter, an embodiment 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 the scope of the present invention.

[0012] The following is an explanation of the terms used in this manual.

[0013] The term "C1-20 alkyl" as used in this specification refers to a linear or branched alkyl group having 1 to 20 carbon atoms. Examples include "C1-4 alkyl" groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. These groups also include n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, and 2-ethylbutyl. C1-20 alkyl groups also include C1-12 alkyl groups having 1 to 12 carbon atoms and C1-6 alkyl groups having 1 to 6 carbon atoms.

[0014] The term "C 1-6 alkylcarbonyl" used in this specification refers to an alkylcarbonyl group whose alkyl portion is the aforementioned C 1-6 alkyl group, for example, including "C 1-4 alkylcarbonyl" such as acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, and tert-butylcarbonyl.

[0015] As used herein, the term "amino C2-20 alkyl" refers to a linear or branched alkyl group having 2 to 20 carbon atoms and having an amino group as a substituent. For example, the amino group may be located on the terminal carbon atom of the alkyl group. Amino C2-20 alkyl also includes amino C2-12 alkyl groups having 2 to 12 carbon atoms.

[0016] The term "hydroxy C 2-20 alkyl" as used herein refers to a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxyl group as a substituent. For example, the hydroxyl group may be located on the terminal carbon atom of the alkyl group. Hydroxy C 2-20 alkyl groups also include hydroxy C 2-12 alkyl groups having 2 to 12 carbon atoms.

[0017] The term "C2-30 alkylene" as used in this specification means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, including, for example, ethylene and propylene, as well as C2-20 alkylene groups having 2 to 20 carbon atoms, C2-8 alkylene groups having 2 to 8 carbon atoms, and the group "-(CH2)n-" (wherein n is 2 to 30, preferably 2 to 20, and more preferably 2 to 15).

[0018] The term "C 1-5 alkylene" used in this specification refers to a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, including, for example, methylene, ethylene, and propylene.

[0019] The term "C2-8 alkenyl" as used herein refers to a linear or branched divalent saturated hydrocarbon group having 2 or more and no more than 8 carbon atoms and containing one or more double bonds, including, for example, -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl. When geometric isomers exist, each isomer and mixtures thereof are included.

[0020] Hyaluronic acid derivatives The hyaluronic acid derivative of this embodiment is a steryl group-introduced hyaluronic acid derivative. The ratio Mw / Mn (hereinafter sometimes referred to as "polydispersity") of the weight average molecular weight Mw to the number average molecular weight Mn of the hyaluronic acid derivative of this embodiment is 1.11 or more and 10.0 or less.

[0021] By keeping the polydispersity of the hyaluronic acid derivative of this embodiment within the above range, it exhibits excellent low-viscosity response to shear during injection, achieving good syringeal permeability. In particular, by keeping the polydispersity of the hyaluronic acid derivative above the above lower limit, intermolecular cross-linking is relaxed during shear application, thereby improving syringeal permeability.

[0022] [Mw / Mn (polydispersity)] Mw / Mn (polydispersity) is 1.11 or more and 10.0 or less, preferably 1.2 or more and less than 3.0, preferably 1.2 or more and 2.9 or less, preferably 1.3 or more and 2.9 or less, more preferably 1.3 or more and 2.4 or less, more preferably 1.3 or more and 2.1 or less, and further preferably 1.5 or more and 2.1 or less. By keeping the Mw / Mn ratio above the lower limit, the hyaluronic acid derivative can be shear-thinning, reducing its viscosity in response to shear during injection, thereby improving injectability. When the Mw / Mn ratio is above the lower limit, the proportion of low-molecular-weight hyaluronic acid increases. However, low-molecular-weight hyaluronic acid has a reduced number of modifying groups per molecule, weakening the hydrophobic interactions between molecules. Therefore, it is speculated that the viscosity decreases more significantly when shear is applied, with the difference between weak and strong shear increasing. By keeping the Mw / Mn ratio below the upper limit (or below), the reduction in the local retention of the hyaluronic acid derivative in vivo can be suppressed. The term "shear-thinning" here refers to the property of decreasing viscosity with increasing shear rate.

[0023] Mw / Mn (polydispersity) can be calculated from the weight average molecular weight (Mw) and number average molecular weight (Mn) determined, for example, by size exclusion chromatography-multi-angle light scattering (SEC-MALS). Mw / Mn (polydispersity) can be specifically measured according to the method described in the Examples below.

[0024] [Weight average molecular weight Mw] The weight-average molecular weight (Mw) of the hyaluronic acid derivative is not particularly limited. To achieve sustained-release properties through delayed diffusion during topical administration, a relatively large molecular weight is preferred. To ensure good permeability when the final dosage form is a solution, a relatively small molecular weight is preferred. Therefore, the weight-average molecular weight (Mw) of the hyaluronic acid derivative is preferably 3,000 (3 kDa) to 160,000 (160 kDa), more preferably 7,000 (7 kDa) to 160,000 (160 kDa), even more preferably 7,000 (7 kDa) to 120,000 (120 kDa), still more preferably 7 kDa to 60 kDa, and most preferably 10 kDa to 50 kDa. The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives can generally be adjusted by using raw materials with corresponding molecular weights. Furthermore, molecular weight can also be controlled by adding a crosslinking agent or by intermolecular crosslinking. The weight average molecular weight (Mw) and number average molecular weight (Mn) of the hyaluronic acid derivative can be measured, for example, using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). Specifically, the weight average molecular weight (Mw) and number average molecular weight (Mn) of the hyaluronic acid derivative can be measured according to the methods described in the Examples below.

[0025] [Steryl] In hyaluronic acid derivatives, the steryl group can be directly bonded to hyaluronic acid or bonded to hyaluronic acid through a linker. The "linker" herein can be any peptide linker or synthetic compound linker that can be introduced through genetic engineering. In hyaluronic acid derivatives, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be selected by the industry based on the intended purpose. A preferred length is 2 amino acids or longer (the upper limit is not particularly limited, but is generally 30 amino acids or shorter, preferably 20 amino acids or shorter), and 15 amino acids is particularly preferred. The peptide linkers contained in the hyaluronic acid derivative can all be of the same length or of varying lengths.

[0026] The term "steryl" as used herein is not particularly limited as long as it refers to a group having a steroid skeleton. Specific examples of steroids include cholesterol, cholestanol, campesterol, ergostanol, stigmasterol, adrenosterol, stigmasterol, sitosterol, lanosterol, ergosterol, sagodole, bile acid, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycorticosterone. Examples of steryl groups include cholesterol, stigmasterol, lanosterol, and ergosterol. Among these, cholesterol (particularly cholestane-5-en-3β-yl) is preferred.

[0027] [Steryl incorporation rate] The incorporation rate of steryl groups relative to the hyaluronic acid derivative (hereinafter sometimes referred to as the "steryl incorporation rate") is preferably 6% or more and less than 60%, more preferably 6% or more and less than 50%, further preferably 6% or more and less than 40%, particularly preferably 6% or more and less than 35%, and most preferably 8% or more and less than 22%. By keeping the steryl incorporation rate within the above range, the hyaluronic acid derivative dissolves well in pure water or at low salt concentrations and exhibits shear-thinning properties, imparting acupuncture properties. Furthermore, by combining a hyaluronic acid derivative with a steryl incorporation rate within the above range with a drug and administering it in vivo (e.g., subcutaneously), a long-term sustained-release preparation can be obtained that remains at the administration site. Furthermore, from the perspective of improving local retention at the aforementioned administration site, the steroid group incorporation rate is preferably 6% or higher and less than 35%, more preferably 8% or higher and less than 33%, and even more preferably 12% or higher and less than 22%. Furthermore, from the perspective of inhibiting drug aggregation and achieving stable dispersion, the rate is preferably 35% or higher and less than 60%, and even more preferably 35% or higher and less than 50%.

[0028] The steryl group incorporation rate can be measured by 1H-NMR measurement. Specifically, the rate can be calculated using the integrated value of the peak derived from the steryl group of the hyaluronic acid derivative and the integrated value of the peak derived from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH3, 1.6 ppm to 2.0 ppm, 3H) in the 1H-NMR spectrum according to the following formula. In the formula, nH represents the number of hydrogen atoms corresponding to the peak. Specifically, the rate can be measured according to the method described in the Examples below.

[0029] [Steryl introduction rate] (%) =[(integral value of the peak derived from the steryl group × 3 / n H) / (integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0030] Preferred hyaluronic acid derivatives include, specifically, hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter sometimes referred to as "repeating unit (I)").

[0031] [Chemistry 2]

[0032] (wherein, R 1, R 2, R 3 and R 4 are independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a methyl group and a C 1-6 alkylcarbonyl group; Z represents a direct bond, or a peptide linker comprising 2 or more and 30 or less of any amino acid residues; X1 is a group selected from the group consisting of groups represented by the following formula: -NR bR、 -NR b-COO-R, -NR b-CO-R, -NR b-CO-NR cR, -COO-R, -O-COO-R, -SR, -CO-Y aSR, -O-CO-Y bSR, -NR b-CO-Y bSR, and -SSR; R a, R b and R c are independently selected from the group consisting of a hydrogen atom, a C 1-20 alkyl group, an amino C 2-20 alkyl group and a hydroxy C 2-20 alkyl group, wherein the alkyl portion of the group may be inserted with a group selected from the group consisting of -O- and -NR f-; Rf is selected from the group consisting of a hydrogen atom, a C 1-12 alkyl group, an amino C 2-12 alkyl group and a hydroxy C 2-12 alkyl group, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is a C 2-30 alkylene group, or -(CH 2CH 2O) m-CH 2CH 2-, wherein the alkylene group may be inserted with a group selected from the group consisting of -O-, -NR g- and -SS-; Rg is selected from the group consisting of a hydrogen atom, a C 1-20 alkyl group, an amino C 2-20 alkyl group and a hydroxy C 2-20 alkyl group, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; Y a is a C 1-5 alkyl group; Y b is C 2-8 alkyl or C 2-8 alkenyl; m is an integer greater than or equal to 1 and less than or equal to 100)

[0033] The hyaluronic acid derivative is preferably a hyaluronic acid derivative containing one or more repeating units represented by the following general formula (Ia) (hereinafter sometimes referred to as "repeating unit (Ia)").

[0034] [Chemistry 3]

[0035] (wherein, R 1, R 2, R 3 and R 4 are independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a methyl group and a C 1-6 alkylcarbonyl group; X is a hydrophobic group represented by -NR aY-NR b-COO-R; R a and R b are independently selected from the group consisting of a hydrogen atom and a C 1-6 alkyl group; R is a steryl group; Y is C 2-30 alkylene, or -(CH 2CH 2O) m-CH 2CH 2-, m is an integer greater than or equal to 1 and less than or equal to 100)

[0036] Here, when the hyaluronic acid derivative contains two or more repeating units (I) or repeating units (Ia), the repeating units may be the same or different.

[0037] The hyaluronic acid derivative may also be modified at positions other than the repeating unit (I) or the repeating unit (Ia). For example, the hydroxyl group may be converted into -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 alkylcarbonyl), etc., the carboxyl group may be converted into an amide or ester, and a salt may be formed.

[0038] [Repeating Unit (I)] The group "-ZN(Ra)YX1" in the general formula (I) includes a group selected from the group consisting of groups represented by the following formulae: -NH-(CH 2) mz-NH-R; -NH-(CH 2) mz-NH-COO-R; -NH-(CH 2CH 2O) m-CH 2CH 2-NH-COO-R; -NH-(CH 2) mz-COO-R; -NH-(CH 2CH 2O) m-CH 2CH 2-COO-R、 -NH-(CH 2) mz-O-COO-R; -NH-(CH 2CH 2O) m-CH 2CH 2-O-COO-R、 -NH-(CH 2) mz-SR; -NH-(CH 2CH 2O) m-CH 2CH 2-SR; -NH-(CH 2) mz-O-CO-CH(R 8)-CH 2-SR; -NH-(CH 2) mz-NHCO-CH(R 8)-CH 2-SR; -NH-(CH 2CH 2O) m-CH 2CH 2-NHCO-CH(R 8)-CH 2-SR; -NH-(CH 2CH 2O) m-CH 2CH 2-O-CO-CH(R 8)-CH 2-SR; -NH-(CH 2) mz-SSR; and -Z-NR aY-NR b-COO-R (Herein, mz is an integer from 2 to 30, R8 is a hydrogen atom or a methyl group, and R and m are as defined in this specification). As the base, it is better to select -NH-(CH 2) mz-NH-COO-R; -NH-(CH 2CH 2O) m-CH 2CH 2-NH-COO-R; and -NH-(CH 2) mz-SSR (Herein, mz, R and m are as defined in this manual) The basis of the group.

[0039] (Z) In general formula (I), Z is preferably a direct bond. Furthermore, in another embodiment, when Z is a peptide linker, X 1 is preferably -NR b -COO-R. Furthermore, in another embodiment, Z may be a peptide linker represented by -NH-[CH(-Z a)-CONH] n - 1-CH(-Z a)-CO-, where n is an integer from 2 to 30, and each Za independently represents a substituent in an α-amino acid represented by H 2 N-CH(-Z a)-COOH. This peptide linker is bonded to the carboxyl group of the glucuronic acid moiety at its N-terminus and to the group -N(-Ra)-YX 1 at its C-terminus. Examples of amino acids that can be used as amino acid residues in the peptide linker include natural (L-type) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamine, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as their D-isomeric α-amino acids. All α-amino acids, including synthetic amino acids, can be used. Specifically, examples of Z include -CH3, H2NC(NH)NH(CH2)3-, and H2NCOCH2-. Furthermore, the n Zs may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, and more preferably 2 to 4. Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, and Phe-Gly-.

[0040] (Y) In general formula (I), Y is preferably a group selected from the group consisting of -(CH 2) n1- and -(CH 2CH 2O) m1-CH 2CH 2- (where n1 is an integer from 2 to 20, preferably an integer from 2 to 15, more preferably an integer from 2 to 12, and even more preferably an integer from 2 to 6. m1 is an integer from 1 to 4). Specifically, -(CH 2) 2-, -(CH 2) 6-, -(CH 2) 8-, -(CH 2) 12-, or -(CH 2CH 2O) 2-CH 2CH 2-. Furthermore, from the viewpoint of achieving high solubility in pure water or at low salt concentrations and exhibiting a high precipitate-forming ability at physiological salt concentrations, Y is preferably a group selected from the group consisting of -(CH 2) 2-, -(CH 2) 6-, -(CH 2) 8-, and -(CH 2) 12-, more preferably -(CH 2) 6-.

[0041] Y can also be, for example, -CH 2CH 2O-CH 2CH 2-SS-CH 2CH 2O-CH 2CH 2-, -(CH 2CH 2O) 2-CH 2CH 2-SS-CH 2CH 2O-CH 2CH 2-, -CH 2CH 2O-CH 2CH 2-SS-(CH 2CH 2O) 2-CH 2CH. 2-, -(CH 2CH 2O) 2-CH 2CH 2-SS-(CH 2CH 2O) 2-CH 2CH 2-etc.

[0042] (Y a) As Ya, -CH2- or -CH2-CH2- is preferred.

[0043] (Y b) Yb is preferably -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl or octa-2,4,6-triene-1,8-diyl, more preferably -CH2-CH2- or -CH(CH3)CH2-.

[0044] Specific examples of the group "-ZN(Ra)YX1" include: -NH-(CH2)2-NH-CO-cholesterol group, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesterol group, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesterol group, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesterol group, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesterol group, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesterol group, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesterol group, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesterol group, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesterol group, 4-N(-(CH 2) 3-NH-(CH 2) 3-NH 2)-COO-cholesterol group, -NH-(CH 2) 3-NH-(CH 2) 4-N(-(CH 2) 3-NH 2)-CO-NH-cholesterol group, -NH-(CH 2) 3-NH-(CH 2) 4-N(-(CH 2) 3-NH 2)-CO-cholesterol group, -NH-(CH 2) 3-NH-(CH 2) 4-N(-(CH 2) 3-NH 2)-CO-cholesterol group, -NH-(CH 2) 3-NH-(CH 2) 4-N(-(CH 2) 3-NH 2)-cholesterol group, and the like. Preferred groups include "-ZN(Ra)YX1," wherein Ra, Rb, and Rc are hydrogen atoms, Y is a linear C2-30 alkylene group or -(CH2CH2O)m-CH2CH2-, Ya is a linear C1-5 alkylene group, or Yb is a linear C2-8 alkylene group or a linear C2-8 alkenylene group.

[0045] [Repeating Unit (Ia)] In general formula (Ia), X is preferably -NH-(CH2)2-NH-COO-cholesterol group, -NH-(CH2)6-NH-COO-cholesterol group, -NH-(CH2)12-NH-COO-cholesterol group or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesterol group, and more preferably -NH-(CH2)2-NH-COO-cholesterol group, -NH-(CH2)6-NH-COO-cholesterol group or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesterol group.

[0046] In addition to the repeating unit (I), the hyaluronic acid derivative may further contain a repeating unit represented by the general formula (II) (hereinafter sometimes referred to as "repeating unit (II)").

[0047] [Chemistry 4]

[0048] (wherein, R 1a, R 2a, R 3a and R 4a are independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a methyl group and a C 1-6 alkylcarbonyl group; Xa is selected from the group consisting of hydroxyl and -OQ+; where Q+ is a counter cation)

[0049] Here, when the hyaluronic acid derivative contains two or more repeating units (II), the repeating units may be the same or different. In another embodiment, the hyaluronic acid derivative may be a hyaluronic acid derivative substantially consisting of repeating unit (I), repeating unit (Ia) and repeating unit (II).

[0050] [Repeating unit (II)] In general formula (II), Q+ is not particularly limited as long as it is a counter cation that forms a salt with a carboxyl group in water. When the valence is greater than 2, it forms a salt with multiple carboxyl groups depending on the valence. Examples of counter cations include metal ions such as lithium ions, sodium ions, rubidium ions, cesium ions, magnesium ions, and calcium ions; and ammonium ions represented by the formula: N+RjRkRlRm (wherein Rj, Rk, Rl, and Rm are independently selected from the group consisting of a hydrogen atom and a C1-6 alkyl group). Q+ is preferably a sodium ion, a potassium ion, or a tetraalkylammonium ion (e.g., tetra-n-butylammonium ion). Rj, Rk, Rl, and Rm are preferably the same group selected from the group consisting of C1-6 alkyl groups, preferably an n-butyl group.

[0051] R1, R2, R3, and R4, as well as R1a, R2a, R3a, and R4a, are preferably all hydrogen atoms. Furthermore, Ra and Rb are preferably all hydrogen atoms.

[0052] The hyaluronic acid derivative is preferably one consisting essentially of repeating units (I) and (II). In the hyaluronic acid derivative, for example, at least 80%, preferably at least 90%, and even more preferably at least 95% of the repeating units comprising the disaccharide of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative are repeating units (I) and (II). The hyaluronic acid derivative may consist solely of repeating units (I) and (II).

[0053] <Method for producing hyaluronic acid derivatives> The hyaluronic acid derivative of this embodiment is obtained, for example, by converting the carboxyl groups of glucuronic acid into amides and then introducing steryl groups. Furthermore, by adjusting the amount of the compound containing steryl groups to be reacted with the starting hyaluronic acid or its derivative, the steryl group incorporation rate can be adjusted to 6% or higher and less than 60%.

[0054] As a method for converting the carboxyl group of glucuronic acid into an amide and introducing a steryl group, specifically, for example, the following method can be mentioned: hyaluronic acid or a derivative thereof as a starting material, preferably hyaluronic acid or a derivative thereof consisting solely of repeating unit (II), is subjected to ion exchange with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt); and the hyaluronic acid salt is reacted with a hydroxyl group of the formula: [HNRaY-NRbR, NHRaY-NRb-COO-R, HNRaY-NRb-COO-R, HNRaY-NRb-CO-R, HNRaY-NRb-CO-NRcR, HNRaY-COO-R, HNRaY-NRb-CO-NRcR, HNRaY-COO-R, HNRaYO-COO-R, HNRaYSR, HNRaY-CO-YaSR, HNRaYO-CO-YbSR, HNRaY-NRb-CO-YbSR, HNRaYSSR or -Z-NRaY-NRb The reaction is carried out with an amine introduced with a steryl group (especially a cholesterol group) represented by "b-COO-R (wherein, Ra, Rb, Rc, Y, Ya, Yb, Z and R are as defined in the present specification)".

[0055] The condensing agent that can be used in the above reaction is not particularly limited, and examples thereof include: 4-(4,6-dimethoxy-1,3,5-triazolium)-4-methylmorpholinium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroethylene, borate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazolium (HODhbt), benzotriazol-1-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), etc.

[0056] DMT-MM is preferred because the reaction proceeds efficiently even in mixed solvents of water and organic solvents, but is not particularly limited. Furthermore, using DMT-MM as a condensing agent can suppress ester bond formation in systems where multiple hydroxyl groups coexist, allowing for highly selective formation of amide bonds between amino and carboxyl groups. This condensing agent can prevent, for example, solvent alcohols from reacting with carboxyl groups in the hyaluronic acid moiety, or carboxyl groups and hydroxyl groups present in the hyaluronic acid moiety from forming unintended crosslinks within or between the molecules.

[0057] Examples of solvents used in the steroidal group introduction reaction include water, DMSO (dimethyl sulfoxide), methanol, ethanol, propanol, butanol, isopropanol, polyols, acetonitrile, DMF (dimethyl sulfoxide), THF (tetrahydrofuran), dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixed solvents thereof. Examples of polyols include the same as those exemplified in "(B) Alcohols" above, with ethylene glycol being preferred.

[0058] Alternatively, the starting hyaluronic acid or its derivative can be subjected to ion exchange with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt). In the presence of a suitable condensing agent, the hyaluronic acid salt is reacted with the spacer moiety in a solvent (protection and deprotection reactions may be performed as needed) to convert the carboxyl groups (-COOH) of the starting hyaluronic acid or its derivative, followed by reaction with a suitable reagent. The following illustrates examples of combinations of groups derived from carboxyl groups and reaction reagents. -CONR aY-NR bH+Hal-R; -CONR aY-NR bH+Hal-COOR; -CONR aY-NR bH+HOCO-R; -CONR aY-NR bH+Hal-CO-R; -CONR aY-NR b-COOH+HNR cR; -CONR aY-NR b-CO-NR cH+Hal-R; -CONR aY-NR bH+HOCO-NR cR; -CONR aY-NR bH+Hal-CO-NR cR; -CONR aY-COOH+HO-R; -CONR aY-OH+Hal-COO-R; -CONR aY-OCOOH+HO-R; -CONR aY-OCOOH+Hal-R; -CONR aY-OCO-Hal+HO-R; -CONR aY-SH+Hal-R; -CONR aY-Hal+HS-R; -CONR aY-CO-Y a-Hal+HS-R; -CONR aY-CO-Y a-SH+Hal-R; -CONR aYO-CO-CH=CH 2+HS-R; -CONR aY-NR b-CO-CH(CH 3)=CH 2+HS-R; -CONR aY-SH+HS-R; -COZ-OH+HNR aY-NR b-COO-R; -COZ-NR aY-NR bH+Hal-COO-R (wherein, Ra, Rb, Rc, Y, Ya, Yb and Z are as defined in this specification, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0059] Examples of reaction methods include dehydrohalogenation, condensation, dehydration, nucleophilic addition reactions such as Michael addition, and oxidation reactions to form disulfides. These reactions are well-known, and practitioners can select and perform them under optimal reaction conditions. If the transformant or reactant has a carboxyl group, it can also be converted to an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester before proceeding with the reaction.

[0060] Another example method is as follows: the carboxyl group of hyaluronic acid or its derivative as a raw material is reacted with 2-aminoethyl 2-pyridine disulfide to prepare a hyaluronic acid derivative having an introduced spacer having a terminal thiol group modified with a leaving group, and the spacer undergoes a nucleophilic substitution reaction with thiocholesterol to form a disulfide bond.

[0061] Furthermore, a method can be exemplified by introducing a spacer moiety into the carboxyl group of hyaluronic acid or its derivative, and introducing a spacer moiety into the steryl group, and then reacting these moieties. As described above, in the case of inserting -SS- into Y, a method can be exemplified by introducing a hyaluronic acid derivative having a terminal thiol spacer into the carboxyl group of hyaluronic acid, and introducing a terminal thiol spacer into the steryl group, and then subjecting these moieties to oxidation reactions to form a disulfide bond. In this case, the thiol group of one moiety can be reacted with 2-mercaptopyridine to form a disulfide, and then substituted with the thiol group of the other moiety.

[0062] Furthermore, after preparing the hyaluronic acid derivative of the present invention, other substituents may be introduced. For example, gelation can be achieved by converting 0.1% to 99.5%, preferably 10% to 40%, of the carboxyl groups in the hyaluronic acid derivative substantially composed of repeating units (I) and (II) into -CO-Xz, thereby crosslinking the molecule or other molecules. [Here, X z is selected from the group consisting of the following groups: -NH-(CH 2) p1-O-CO-C(R 17)=CH 2; -NH-(CH 2) p1-O-CO-CH(R 17)-CH 2-S-CH 2-CH(OH)-CH(OH)-CH 2-SH; -NH-(CH 2) p1-SH; -NH-(CH 2) p1-NH-CO-C(R 17)=CH 2; -NH-(CH 2) p1-NH-C(=NH)-(CH 2) 3-SH; -NH-(CH 2) p1-NH-CO-(CH 2) r-SH; -NH-(CH 2) p1-NH-CO-CH(R 17)-CH 2-S-CH 2-CH(OH)-CH(OH)-CH 2-SH; -NH-(CH 2) p1-NH-CO-CH(NH 2)-CH 2-SH; -NH-(CH 2) p1-NH-CO-CH(NH 2)-(CH 2) 2-SH; -NH-NH-CO-(CH 2) 4-CO-NH-NH-C(=NH)-(CH 2) 3-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-O-CO-C(R 17)=CH 2; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-O-CO-CH(R 17)-CH 2-S-CH 2-CH(OH)-CH(OH)-CH 2-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-CO-C(R 17)=CH 2; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-C(=NH)-(CH 2) 3-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-CO-(CH 2) r-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-CO-CH(R 17)-CH 2-S-CH 2-CH(OH)-CH(OH)-CH 2-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-CO-CH(NH 2)-CH 2-SH; -NH-(CH 2-CH 2-O) q-CH 2-CH 2-NH-CO-CH(NH 2)-(CH 2) 2-SH; -NH-CH(CO 2H)-(CH 2)-SH; -NH-CH(CO 2H)-(CH 2) 2-SH; -NH-CH(CO 2H)-(CH 2) 2-CONH-CH(CONH-CH 2-CO 2H)-CH 2-SH (Herein, R17 represents a hydrogen atom or a C1-6 alkyl group, p1 represents an integer from 2 to 10, q represents an integer from 1 to 200, and r represents an integer from 1 to 3)]

[0063] The steps for gelling the hyaluronic acid derivative by chemical crosslinking can be appropriately selected. Crosslinking conditions include the crosslinking method, polymer concentration, crosslinking agent concentration, solvent, solvent pH, salt concentration, temperature, and time.

[0064] In the step of gelling the hyaluronic acid derivative, the crosslinking density of the generated gel can be increased by increasing the reaction conditions for forming crosslinks, such as the polymer concentration during chemical crosslinking, and improving the introduction rate of crosslinkable groups.

[0065] Regarding the crosslinking agent concentration during the gelation step of the hyaluronic acid derivative, when using a hyaluronic acid derivative with crosslinkable groups at both ends, it is preferably added at a concentration that allows for rapid crosslinking without excess or deficiency of these groups. For example, when using DTT (dithiothreitol) to crosslink a polymer containing methacryloyl (MA) groups via the Michael addition reaction, the preferred ratio of MA groups to SH groups is 3:1 to 1:3, and more preferably 2:1 to 1:2.

[0066] The solvent used in the gelation step of the hyaluronic acid derivative is preferably one that fully dissolves the polymer and crosslinker, and is not particularly limited. Preferred solvents include water, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dimethylformamide (DMF), N-methylpyrrolidone (NMP), and mixed solvents selected from these. Furthermore, miscible organic solvents may be mixed with these solvents. Examples of miscible organic solvents include, but are not particularly limited to, methanol, ethanol, propanol, isopropanol, butanol, polyols, acetone, and acetonitrile. Examples of polyols include those listed above for "(B) Alcohols," with ethylene glycol being preferred.

[0067] Because hyaluronic acid derivatives form nanoparticles in aqueous solutions, cross-linking under dilute conditions can form nanosized particle gels, which can be used as sustained-release carriers in the blood or targeted delivery systems. Dilute concentrations refer to concentrations of 10 mg / mL or less, preferably 5 mg / mL or less, and even more preferably 1 mg / mL or less. On the other hand, cross-linking under high concentrations can form a monolithic gel composed of cross-linked particles. This can be used as a subcutaneous sustained-release carrier. High concentrations refer to concentrations of 5 mg / mL or more, preferably 20 mg / mL or more, and even more preferably 40 mg / mL.

[0068] The gelation step of the hyaluronic acid derivative can be performed in a bulk state or in a discontinuous phase such as an emulsion or spray droplets. For example, when performed in a W / O emulsion, an aqueous phase containing a dissolved polymer or crosslinker is emulsified in a water-immiscible solvent to allow the gelation reaction to proceed. The water-immiscible solvent is not particularly limited and includes, for example, hexane, chloroform, dichloromethane, ethyl acetate, medium-chain triglycerides (MCT), liquid paraffin, and soybean oil. A surfactant may also be added to stabilize the emulsion. Alternatively, the gelation step can be performed in supercritical carbon dioxide or a desolvable solvent such as PEG. In this case, by emulsifying and dispersing the aqueous or organic solvent phase containing a dissolved polymer or crosslinker in the solvent described above, polymer concentration is achieved with desolvation (solvent diffusion), resulting in a gel with a higher crosslinking density.

[0069] Alternatively, the following steps and operations may be performed: a gelation step of the hyaluronic acid derivative, followed by a step of terminating the cross-linking reaction and deactivating or washing away any remaining cross-linking functional groups. 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 bonded only to one end of the cross-linking agent, and any remaining cross-linking agent. For example, if there is residual unreacted cross-linking agent, this can be removed by washing with excess water, etc., but this is not particularly limited. Alternatively, for example, if there are residual methacryloyl groups substituted on the polymer, these can be deactivated by adding excess mercaptoethanol, etc., and then the remaining mercaptoethanol can be removed by washing with excess water, etc. Furthermore, for example, when thiol groups remain, they can be inactivated by adding an excess of 3-maleimide propionic acid, iodoacetic acid, or the like, and then the remaining 3-maleimide propionic acid or iodoacetic acid can be washed away with an excess of water or the like.

[0070] After the gelation step of the hyaluronic acid derivative, a pulverization step may be performed. Examples of pulverization methods include pulverization using a pestle and mortar or pulverization using a grinder, with pulverization using a grinder being preferred. Examples of the grinding and pulverizing apparatus include rotating disk-type pulverizing apparatuses such as a centrifugal pulverizer (Nippon Seiki Seisaku-sho) and an impact mill (DALTON Co., Ltd.), pulverizing apparatuses such as an atomizer (TOKYO ATOMIZER MFG CO., LTD.), a sample mill (TOKYO ATOMIZER MFG CO., LTD.), a small mill (TOKYO ATOMIZER MFG CO., LTD.), and a screen mill (SK Mill, TOKKEN Co., Ltd.), a jet pulverizing apparatus such as an ultra-fine laboratory jet mill (AO Jet Mill, Seishin Enterprise Co., Ltd.), and a Linlex Mill (Liquid Gas Co., Ltd.) capable of ultra-low temperature pulverization. SK Mill and Linlex Mill are preferred.

[0071] After the gelation step of the hyaluronic acid derivative, a drying step may be performed. Examples of drying methods include ventilation drying, drying in a constant temperature chamber, reduced pressure drying, and hot air circulation drying. The air velocity, drying time, temperature, and pressure should be appropriately selected within a range that does not decompose or deteriorate the gel of the hyaluronic acid derivative.

[0072] Pharmaceutical Compositions The pharmaceutical composition of this embodiment includes the aforementioned hyaluronic acid derivative as a carrier. In this embodiment, the carrier and drug are ideally bound directly or indirectly to form a complex, and are not free from each other. When the pharmaceutical composition is administered into a living organism, the drug is slowly released from the carrier, and good sustained-release properties are expected. The bond between the carrier and drug can be covalent or non-covalent, but non-covalent bonding is preferred to maintain drug activity.

[0073] In the pharmaceutical composition of this embodiment, the drug preferably forms a complex with a hyaluronic acid derivative serving as a carrier. It is believed that in a solvent, the steryl groups of the hyaluronic acid derivative spontaneously associate with the hydrophobic interaction of the drug present in the system, thereby forming a complex between the drug and the hyaluronic acid derivative. This complex is expected to enhance the drug's storage stability, maintain biological activity, improve sustained release, increase solubility in water, enhance resistance to stimuli such as heat and light, and inhibit aggregation and precipitation.

[0074] <Medication> The drug included in the pharmaceutical composition of this embodiment is not particularly limited, as long as it is a drug that can be used as a pharmaceutical for humans or animals. Examples include proteins, peptides, polysaccharides, nucleic acids, and low-molecular-weight compounds. The pharmaceutical composition of this embodiment preferably includes pharmacologically active biopharmaceuticals such as proteins, peptides, and nucleic acids, or low-molecular-weight compounds, in the form of a complex with a hyaluronic acid derivative. Formation of this complex is expected to improve the chemical stability of the drug, inhibit aggregation, and inhibit enzyme degradation.

[0075] [Low molecular weight compounds] Examples of low molecular weight compounds include anticancer agents (e.g., alkylating agents, antimetabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (e.g., steroids, non-steroidal anti-inflammatory agents), anti-rheumatic agents, antibacterial agents (e.g., β-lactamide antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfonamides, etc.), and the like.

[0076] [Proteins and peptides] Examples of proteins and peptides include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferon-α, β, γ (INF-α, β, γ), thrombopoietin (TPO), ciliary neurotrophic factor (CNTF), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNFbp), interleukin-10 (IL-10), FMS-like tyrosine kinase (Flt-3), growth hormone (GH), insulin, insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), and keratinocyte-derived growth factor (CGF). Keratinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth factor (MGDF), osteoprotegerin (OPG), leptin, parathyroid hormone (PTH), basic fibroblast growth factor (b-FGF), bone modeling protein (BMP), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), glucagon-like peptide-1 (GLP-1), vaccine antigens, antibodies, diabodies, miniantibodies, fragmented antibodies, PEGylated proteins (such as PEGylated interferon-α-2a, PEGylated interferon-α-2b, PEGylated GCSF, PEGylated hG11 antagonist B2036, etc.), etc.

[0077] [Nucleic Acids] Examples of nucleic acids include DNA, RNA, antisense nucleic acid, decoy nucleic acid, ribozyme, low-molecular-weight interfering RNA, and nucleic acid aptamers.

[0078] <Form> The pharmaceutical composition of this embodiment can be a dispersed microparticle solution, a precipitated suspension, or a freeze-dried form. Furthermore, in the case of a freeze-dried form, it can be a precipitated sustained-release preparation, where the physician can prepare the dosage solution immediately by adding a physiological saline isotonic solution prior to administration. This approach is believed to be suitable for pharmaceutical compositions containing active ingredients that are unstable in solution.

[0079] When the pharmaceutical composition of this embodiment is a dispersed microparticle solution or a precipitated suspension, the concentration of the hyaluronic acid derivative in the pharmaceutical composition is preferably 1 mg / mL to 200 mg / mL, more preferably 4 mg / mL to 100 mg / mL, even more preferably 4 mg / mL to 50 mg / mL, and even more preferably 4 mg / mL to 12 mg / mL. By maintaining the concentration of the hyaluronic acid derivative in the pharmaceutical composition within this range, the viscosity of the injection can be improved. Furthermore, the injectable properties of the injection using the hyaluronic acid derivative of this embodiment can be further optimized.

[0080] Hyaluronic acid derivative-drug combination The hyaluronic acid derivative-drug conjugate system of this embodiment has one or more drugs conjugated to the hyaluronic acid derivative.

[0081] The drugs suitable for forming the hyaluronic acid derivative-drug conjugate composition of this embodiment are biopharmaceuticals such as proteins, peptides, nucleic acids, or low molecular weight compounds.

[0082] The pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment are not limited to the forms described above and may also be in the form of nanoparticles, microparticles, solutions, emulsions, suspensions, gels, micelles, implants, powders, or films. Powders can be produced by pulverizing solids obtained by freeze-drying or spray-drying, or by drying precipitates.

[0083] The pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment can be administered orally, parenterally, intranasally, intravaginally, intraocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally, or intraorally. Furthermore, the pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment are not limited to injections and can also be in the form of patch or microneedle preparations, topicals, eye drops, sprays, inhalers, dermal fillers, and the like.

[0084] Furthermore, the hyaluronic acid derivative of this embodiment can be used not only as a pharmaceutical composition, but also as a cosmetic composition, a test drug composition, or a preservative or surface coating agent for medical devices.

[0085] Cosmetic compositions may contain physiologically active ingredients. The types of physiologically active ingredients are not particularly limited, and examples thereof include anti-aging agents, astringents, anti-inflammatory agents, whitening agents, moisturizers, blood circulation enhancers, vitamins, amino acids, peptides, cerebrosides, wound healing enhancers, UV protection agents, irritation alleviating agents, cell activators, and enzyme ingredients.

[0086] Drug compositions for testing may contain components for testing or other components. Antibody-loaded polymer particles, inorganic particles, flat membranes, and the like may be used as testing components. Other components may include a carrier, physiological saline, preservatives (such as sodium azide, benzalkonium chloride, and chlorobutanol), stabilizers (such as propylene glycol and ascorbic acid), excipients (such as lactose, starch, sorbitol, and D-mannitol), buffers (such as sodium lauryl sulfate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, glyceryl monostearate, and aluminum monostearate), and preservatives (such as benzalkonium chloride, benzyl alcohol, and methyl paraben).

[0087] Preservatives or surface coatings for medical devices may include thickeners, vitamins, amino acids, peptides, stabilizers, and bactericides. Examples of medical devices include endoscopes, artificial joints, dentures, intravascular stents, contact lenses, catheters, guidewires, finger structures, artificial hearts, and artificial muscle fibers. [Example]

[0088] Hereinafter, the present invention will be described in detail by way of examples, but these examples are not intended to limit the scope of the present invention to these examples.

[0089] The methods for measuring and evaluating the physical properties of the hyaluronic acid derivatives produced in the Examples and Comparative Examples are as follows.

[0090] [Physical properties 1] (Weight average molecular weight (Mw) of hyaluronic acid derivatives, and the ratio of weight average molecular weight to number average molecular weight (Mw / Mn)) The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives were measured using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). A hyaluronic acid derivative (20 mg) was dissolved in ultrapure water (10 mL) and stirred at room temperature for at least 12 hours to obtain a 2 mg / mL aqueous solution of the hyaluronic acid derivative. A 300 mM aqueous solution of hydroxypropyl-β-cyclodextrin (HP-β-CD) (750 μL) was added to the aqueous solution of the hyaluronic acid derivative (750 μL), mixed for 10 seconds using a shaker, and incubated at 37°C for 1 hour. The resulting sample was then subjected to SEC-MALS analysis to determine the weight-average molecular weight (Mw) and number-average molecular weight (Mn). The SEC-MALS measurement conditions are described below. The Mw / Mn ratio (weight-average molecular weight to number-average molecular weight) was calculated by dividing the weight-average molecular weight (Mw) by the number-average molecular weight (Mn) based on the SEC-MALS measurement results.

[0091] (Measurement conditions) Column: TSKgel GMPWXL (manufactured by Tosoh Corporation) 2 pieces Column temperature: 30℃ Eluent: Phosphate-buffered saline (pH 7.4) supplemented with 10 mM HP-β-CD Flow rate: 1 mL / min Injection volume: 200 μL

[0092] [Physical Properties 2] (Steryl introduction rate) The steryl incorporation rate of hyaluronic acid derivatives was determined by 1H-NMR measurement. First, a measurement solvent was prepared by mixing dimethylsulfoxide-d6 (99.9 v / v%, containing 0.05 v / v% trimethylsilane (TMS), manufactured by Fujifilm Wako Co., Ltd.) and 20% deuterated hydrochloric acid (99.5 v / v%, manufactured by Fujifilm Wako Co., Ltd.) at a mass ratio of 99:1. Next, 2 mg of the hyaluronic acid derivative was added to this measurement solvent (0.6 mL) and completely dissolved in an ultrasonic bath for 30 minutes before 1H-NMR measurement. 1H-NMR measurements were performed using a Fourier transform nuclear magnetic resonance (FT-NMR) instrument (ECS400, manufactured by JEOL Ltd.) at a sample temperature of 85°C. The steryl group incorporation rate was calculated using the formula below from the integrated value of the peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and the integrated value of the peak derived from the methyl group in the cholesterol group (CH3, 0.7 ppm, 3H). Furthermore, the peak (5H) derived from the cholesteryl group overlaps with the peak in the vicinity of 1.6 ppm to 2.0 ppm, including the peak derived from the acetyl group of N-acetyl-D-glucosamine. Therefore, the value calculated by subtracting the integrated value of the peak derived from the cholesteryl methyl group (0.7 ppm) multiplied by 5 / 3 from the integrated value of the peak in the vicinity of 1.6 ppm to 2.0 ppm (i.e., integrated value (1.6 ppm to 2.0 ppm) - integrated value (0.7 ppm) × 5 / 3) was used as the integrated value of the peak derived from the acetyl group of N-acetyl-D-glucosamine for calculation of the steryl group introduction rate.

[0093] [Steryl introduction rate (%)] =[(integral value of the peak originating from the methyl group in the cholesterol group) / (integral value of the peak originating from the acetyl group in N-acetyl-D-glucosamine)]×100 =[Integrated value (0.7 ppm) / {Integrated value (1.6 ppm or more and 2.0 ppm or less) - Integrated value (0.7 ppm) × 5 / 3}] × 100

[0094] [Evaluation 1] <Strain rate dependence test> The low viscosity of the hyaluronic acid derivative in response to shear, i.e., the shear thinning property, is determined by the following sequence. First, ultrapure water was added to the hyaluronic acid derivative and stirred for at least 12 hours to dissolve it, resulting in a 4 mg / mL aqueous solution of the hyaluronic acid derivative. This aqueous solution was then used in a strain rate dependence test. The following describes the strain rate dependence test conditions.

[0095] (Measurement conditions) Measurement device: ARES manufactured by TA Instruments Measurement temperature: 25℃ Geometry: 50 mm Φ cone & flat plate Shear rate: 0.25 s -1 or more and 1000 s -1 or less Measurement time: 60 s per point

[0096] The shear thinning property of hyaluronic acid derivatives was calculated based on the following formula.

[0097] 〔Shear thinning property〕 =(viscosity at shear rate 1 s -1 [mPa·s]) / (viscosity at shear rate 1000 s -1 [mPa·s])

[0098] <Manufacturing of Hyaluronic Acid Derivatives> [Example 1] (Manufacturing of HA-a1, a hyaluronic acid derivative) Prepare hyaluronic acid derivatives according to the following steps 1 to 3.

[0099] 1. Step 1 (Synthesis of 6-aminohexylcarbamate cholesterol ester hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1 and 1-2.

[0100] (Step 1-1) To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) was added triethylamine (TEA, 1.05 mL) under an argon atmosphere and stirred. 6-(tert-Butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise while cooling in an ice bath. The mixture was stirred for 30 minutes while cooling in an ice bath, then warmed to room temperature and stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:4). The fractions containing the target product were combined and the solvent was removed by distillation under reduced pressure.

[0101] (Steps 1-2) The resulting residue was dissolved in ethyl acetate (40 mL), and a 4N hydrochloric acid / ethyl acetate solution (40 mL) was added. The mixture was stirred at room temperature overnight. The resulting precipitate was recovered by centrifugation. The resulting solid was washed four times with ethyl acetate and then dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride). The 1H-NMR spectrum of the product (ECS400, manufactured by JEOL Ltd., EtOH-d 6) is shown in Figure 1.

[0102] 2. Step 2 (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) Prepare hyaluronic acid TBA salt (HA-TBA) by following steps 2-1 and 2-2.

[0103] (1) Step 2-1 DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water and washed approximately three times with ultrapure water by decantation. A 40 wt% aqueous solution of tetrabutylammonium hydroxide (TBA-OH) (manufactured by Aldrich) was added at a molar equivalent of approximately 1.5 times the cation exchange capacity of the resin and stirred for 30 minutes. The remaining TBA-OH solution was removed by decantation and then washed with excess ultrapure water to obtain a TBA-salified cation exchange resin.

[0104] (2) Step 2-2 Sodium hyaluronate (HA-Na) with a weight-average molecular weight of 50,000 (50 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of the cation exchange resin treated with TBA in "(1) Step 2-1" was added in an amount equivalent to 5 times the molar number of HA units (unit molecular weight 401.3) based on the resin's ion exchange energy. After stirring for 15 minutes, the mixture was filtered through a 0.45 μm filter and the filtrate was freeze-dried to obtain TBA salt of hyaluronic acid (HA-TBA) as a white solid. The 1H-NMR spectrum of the product (ECS400, manufactured by JEOL Ltd., EtOH-d 6) is shown in Figure 2.

[0105] 3. Step 3 Prepare an anhydrous DMSO solution (10 mg / mL) of the HA-TBA prepared in "2. (2) Step 2-2." Then, add Chol hydrochloride at a molar ratio of 15 / 100 relative to the disaccharide repeating units (HA units) present in the HA-TBA synthesized in "1. Step 1." Next, add 4-(4,6-dimethoxy-1,3,5-trioxan-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) at a molar ratio of 21.6 / 100 relative to the HA units, and stir overnight at room temperature. The reaction solution is dialyzed (Spectra / Por4, molecular weight cutoff (MWCO): 12,000 to 14,000) against a 0.3 M ammonium acetate / DMSO solution, a 0.15 M NaCl aqueous solution, and ultrapure water, in that order. The dialyzate is freeze-dried to obtain the target product (HA-C6-Chol) as a white solid. The 1H-NMR spectrum of the product is shown in Figure 3. A peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH 3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesterol group (CH 3, 0.7 ppm, 3H) were confirmed.

[0106] [Examples 2 to 31] (Manufacturing of hyaluronic acid derivatives HA-a2~HA-a31) Each hyaluronic acid derivative was obtained by the same method as in Example 1, except that in "2. (2) Step 2-2," the raw material sodium hyaluronic acid (HA-Na) had a molecular weight shown in Tables 1 to 7. In "3. Step 3," the amount of Chol hydrochloride added relative to the HA unit and the amount of DMT-MM added relative to the HA unit were adjusted to the molar ratios shown in Tables 1 and 2. 1H-NMR analysis of the obtained hyaluronic acid derivatives revealed peaks derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks derived from the methyl group of the cholesterol group (CH3, 0.7 ppm, 3H) in each hyaluronic acid derivative.

[0107] [Example 32] (Hyaluronic acid derivative HA-a32) Each hyaluronic acid derivative was obtained by the same method as in Example 1, except that in "2. (2) Step 2-2," a raw material of hyaluronic acid sodium salt (HA-Na) having a molecular weight shown in Table 8 was used, and in "3. Step 3," the amounts of Chol hydrochloride and DMT-MM added relative to the HA unit were adjusted to the molar ratios shown in Tables 1 and 2, and 1,6-diaminohexane was further added. 1H-NMR analysis of the obtained hyaluronic acid derivatives revealed peaks derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks derived from the methyl group of the cholesterol group (CH3, 0.7 ppm, 3H) in each hyaluronic acid derivative.

[0108] [Comparative Example 1] (Manufacturing of HA-b1, a hyaluronic acid derivative) Follow steps 1 to 3 below to prepare a hyaluronic acid derivative. 1. Step 1 (Synthesis of 6-aminohexylcarbamate cholesterol ester hydrochloride) Chol hydrochloride was synthesized by the same method as in Example 1.

[0109] 2. Step 2 (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) Prepare hyaluronic acid TBA salt (HA-TBA) by following steps 2-1, 2-2, and 2-3.

[0110] (1) Step 2-1 Sodium hyaluronate (HA-Na, 50 kDa, Mw / Mn 1.2) was dissolved in ultrapure water to a concentration of 10 mg / mL. The solution was dialyzed against ultrapure water (Spectra / Por 6, molecular weight cutoff (MWCO): 50,000). The dialyzate was freeze-dried to obtain the target product (HA-Na) as a white solid. The weight average molecular weight and number average molecular weight of the product were measured using size exclusion chromatography-multi-angle light scattering detection (SEC-MALS). The results showed that the weight average molecular weight Mw was 50,000 and the Mw / Mn (polydispersity) was 1.1.

[0111] (2) Step 2-2 A cation exchange resin treated with TBA was prepared by the same method as in "2. (1) Step 2-1" of Example 1.

[0112] (3) Steps 2-3 Using the hyaluronic acid sodium salt (HA-Na) with a weight average molecular weight Mw of 50,000 (50k) obtained in the above "(1) Step 2-1", the TBA salt of hyaluronic acid (HA-TBA) as a white solid was obtained by the same method as "2. (2) Step 2-2" of Example 1.

[0113] 3. Step 3 The target product (HA-C6-Chol) was obtained as a white solid by the same method as in "3. Step 3" of Example 1, except that the HA-TBA obtained in "2. (3) Step 2-3" was used. 1H-NMR analysis of the obtained HA-C6-Chol revealed peaks derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and from the methyl group of the cholesteryl group (CH3, 0.7 ppm, 3H).

[0114] [Comparative Examples 2 to 7] (Hyaluronic acid derivatives HA-b2~HA-b7) In "2. (1) Step 2-1" of Comparative Example 1, sodium hyaluronate having the weight average molecular weight and Mw / Mn (polydispersity) values shown in Table 2 was used. In "3. Step 3" of Comparative Example 1, the amounts of Chol hydrochloride added relative to the HA unit and DMT-MM added relative to the HA unit were adjusted to the molar ratios shown in Tables 2 to 7. Hyaluronic acid derivatives were obtained by the same method as in Comparative Example 1, except that the molar ratios were set as shown in Tables 2 to 7. 1H-NMR analysis of the obtained hyaluronic acid derivatives revealed peaks derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks derived from the methyl group of the cholesterol group (CH3, 0.7 ppm, 3H).

[0115] The above methods were used to measure the physical properties of the hyaluronic acid derivatives obtained in the Examples and Comparative Examples, and various evaluations were performed. The results are shown in Tables 1 to 8.

[0116] [Table 1] Example 1 Practical example 2 Example 3 Example 4 Example 5 Comparative Example 1 Hyaluronic acid derivatives HA-a1 HA-a2 HA-a3 HA-a4 HA-a5 HA-b1 composition Molecular weight of HA-Na 50 kDa 50 kDa 50 kDa 50 kDa 50 kDa 50 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 Steroid introduction rate (%) 15 15 15 15 15 15 Weight average molecular weight Mw of hyaluronic acid derivatives 53 kDa 52 kDa 52 kDa 51 kDa 51 kDa 53 kDa Mw / Mn 1.2 1.3 1.8 2.1 2.9 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 10.8 10.8 11 18.9 5.9 11.9 Viscosity at shear rate 1000 s -1 (mPa·s) 6 5.2 3.1 3.2 2.9 7.9 Shear thinning 1.8 2.1 3.5 6 2 1.5

[0117] [Table 2] Example 6 Example 7 Example 8 Example 9 Example 10 Comparative Example 2 Hyaluronic acid derivatives HA-a6 HA-a7 HA-a8 HA-a9 HA-a10 HA-b2 composition Molecular weight of HA-Na 120 kDa 120 kDa 120 kDa 120 kDa 120 kDa 120 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 100 / 15 / 21.6 Steroid introduction rate (%) 15 15 15 15 15 15 Weight average molecular weight Mw of hyaluronic acid derivatives 132 kDa 131 kDa 131 kDa 130 kDa 129 kDa 134 kDa Mw / Mn 1.2 1.3 1.5 1.7 2.4 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 872 756.3 732.1 925.5 557.4 881 Viscosity at shear rate 1000 s -1 (mPa·s) 34.7 24.5 20.5 15.1 14.7 59 Shear thinning 25.1 30.9 35.7 61.3 37.9 14.9

[0118] [Table 3] Example 11 Comparative Example 3 Hyaluronic acid derivatives HA-a11 HA-b3 composition Molecular weight of HA-Na 35 kDa 35 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 15 / 21.6 100 / 15 / 21.6 Steroid introduction rate (%) 15 15 Weight average molecular weight Mw of hyaluronic acid derivatives 35 kDa 35 kDa Mw / Mn 1.7 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 5.4 6.6 Viscosity at shear rate 1000 s -1 (mPa·s) 2.7 5 Shear thinning 2 1.3

[0119] [Table 4] Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 Comparative Example 4 Hyaluronic acid derivatives HA-a12 HA-a13 HA-a14 HA-a15 HA-a16 HA-a17 HA-b4 composition Molecular weight of HA-Na 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 19 / 26.6 100 / 19 / 26.6 100 / 19 / 26.6 100 / 19 / 26.6 100 / 19 / 26.6 100 / 19 / 26.6 100 / 19 / 26.6 Steroid introduction rate (%) 19 19 19 19 19 19 19 Weight average molecular weight Mw of hyaluronic acid derivatives 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa 35 kDa Mw / Mn 1.2 1.3 1.5 1.7 2.1 2.9 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 5.2 5.2 5.1 5 6.3 4.7 6 Viscosity at shear rate 1000 s -1 (mPa·s) 2.8 2.6 2.4 2.3 2.3 2.2 4.5 Shear thinning 1.9 2 2.1 2.2 2.7 2.1 1.3

[0120] [Table 5] Example 18 Example 19 Example 20 Example 21 Example 22 Comparative Example 5 Hyaluronic acid derivatives HA-a18 HA-a19 HA-a20 HA-a21 HA-a22 HA-b5 composition Molecular weight of HA-Na 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 44 / 58 100 / 44 / 58 100 / 44 / 58 100 / 44 / 58 100 / 44 / 58 100 / 44 / 58 Steroid introduction rate (%) 43 43 43 43 43 43 Weight average molecular weight Mw of hyaluronic acid derivatives 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa Mw / Mn 1.3 1.5 1.7 2.1 2.9 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 3 3.3 1.8 1.9 1.9 1.9 Viscosity at shear rate 1000 s -1 (mPa·s) 1.5 1.5 1.4 1.3 1.3 1.9 Shear thinning 2 2.1 1.3 1.5 1.5 1

[0121] [Table 6] Example 23 Example 24 Example 25 Example 26 Example 27 Comparative Example 6 Hyaluronic acid derivatives HA-a23 HA-a24 HA-a25 HA-a26 HA-a27 HA-b6 composition Molecular weight of HA-Na 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa Molar ratio of HA unit / Chol hydrochloride / DMT-MM 100 / 34 / 45 100 / 34 / 45 100 / 34 / 45 100 / 34 / 45 100 / 34 / 45 100 / 34 / 45 Steroid introduction rate (%) 33 33 33 33 33 33 Weight average molecular weight Mw of hyaluronic acid derivatives 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa Mw / Mn 1.3 1.5 1.7 2.1 2.9 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 3.6 3.7 4.3 4.2 3.8 2.7 Viscosity at shear rate 1000 s -1 (mPa·s) 2.4 2.4 2.3 2.3 2.1 2.6 Shear thinning 1.5 1.5 1.9 1.8 1.8 1

[0122] [Table 7] Example 28 Example 29 Example 30 Example 31 Comparative Example 7 Hyaluronic acid derivatives HA-a28 HA-a29 HA-a30 HA-a31 HA-b7 composition Molecular weight of HA-Na 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa HA unit / Molar ratio of Chol hydrochloride / DMT-MM 100 / 8 / 11.2 100 / 8 / 11.2 100 / 8 / 11.2 100 / 8 / 11.2 100 / 8 / 11.2 Steroid introduction rate (%) 8 8 8 8 8 Weight average molecular weight Mw of hyaluronic acid derivatives 10 kDa 10 kDa 10 kDa 10 kDa 10 kDa Mw / Mn 1.3 1.5 2.1 2.9 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 3.3 3.4 3.7 3.5 2.5 Viscosity at shear rate 1000 s -1 (mPa·s) 2.1 2 2 1.9 2.4 Shear thinning 1.6 1.7 1.9 1.8 1

[0123] [Table 8] Example 32 Comparative Example 4 Hyaluronic acid derivatives HA-a32 HA-b4 composition Molecular weight of HA-Na 10 kDa 35 kDa Molar ratio of HA unit / Chol hydrochloride / 1,6-diaminohexane / DMT-MM 100 / 19 / 0.3 / 26.6 100 / 19 / 0 / 26.6 Steroid introduction rate (%) 19 19 Weight average molecular weight Mw of hyaluronic acid derivatives 35 kDa 35 kDa Mw / Mn 2.1 1.1 evaluate Viscosity at shear rate 1 s -1 (mPa·s) 5.5 6 Viscosity at shear rate 1000 s -1 (mPa·s) 2.6 4.5 Shear thinning 2.1 1.3

[0124] Table 1 shows that the hyaluronic acid derivatives HA-a1 to HA-a5 (Examples 1-5) with a weight-average molecular weight (Mw) of 50 kDa and an Mw / Mn (polydispersity) of 1.11 or greater exhibit excellent shear-thinning properties, ranging from 1.8 to 6.0. Furthermore, the viscosity at high shear (rate of 1000 s⁻¹) is relatively low, ranging from 2.9 mPa·s to 6.0 mPa·s, contributing to improved needle permeability. On the other hand, the hyaluronic acid derivative HA-b1 (Comparative Example 1), with a weight-average molecular weight (Mw) of 50 kDa and a polydispersity of 1.1, exhibits a shear-thinning property of 1.5, with a poor viscosity at high shear (rate of 1000 s⁻¹) of 7.9 mPa·s. Furthermore, the shear-thinning property is better with a polydispersity of 1.3 (Example 2) than with a polydispersity of 1.2 (Example 1).

[0125] Furthermore, as shown in Table 2, the hyaluronic acid derivatives HA-a6 to HA-a10 (Examples 5 and 6) with a weight-average molecular weight (Mw) of 120 kDa and a polydispersity of 1.11 or greater exhibited superior shear-thinning properties compared to Comparative Example 2, which had a polydispersity of 1.1. Furthermore, for hyaluronic acid derivatives HA-a3, HA-a9, and HA-a10 (Examples 3, 9, and 10) with the same degree of polydispersity but different weight-average molecular weights, the following trend was observed: the larger the weight-average molecular weight, the better the shear-thinning properties.

[0126] As shown in Table 4, even when the steryl group introduction rate is changed to 19%, compared with Comparative Example 4 with a polydispersity of 1.1, when the polydispersity is greater than 1.11, the viscosity at a shear rate of 1000 s -1 is lower and the shear thinning property is also good.

[0127] Table 5 shows that the hyaluronic acid derivatives HA-a19 and HA-a20 (Examples 19 and 20), with a weight-average molecular weight (Mw) of 10 kDa and a polydispersity range of 1.5 to 1.7, exhibit shear-thinning properties of 1.3 to 2.1, and exhibit good viscosities at high shear (1000 s⁻¹) of 1.4 to 1.5 mPa⁻¹. Meanwhile, the hyaluronic acid derivative HA-b5 (Comparative Example 5), with a polydispersity of 1.1, exhibits a shear-thinning property of 1.0 and poor viscosity at high shear (1000 s⁻¹) of 1.9 mPa⁻¹. Furthermore, Table 6 shows that the hyaluronic acid derivative HA-a25 (Example 25), with a weight-average molecular weight (Mw) of 10 kDa, a steryl incorporation rate of 33%, and a polydispersity of 1.7, has a shear-thinning property of 1.9 and a good viscosity of 2.3 mPa·s at high shear (1000 s -1 ). Meanwhile, the hyaluronic acid derivative HA-b6 (Comparative Example 6), with a polydispersity of 1.1, has a shear-thinning property of 1.0 and a poor viscosity of 2.6 mPa·s at high shear (1000 s -1 ). Furthermore, as shown in Table 7, when the weight average molecular weight Mw is 10 kDa and the steryl group introduction rate is set to 8%, the polydispersity of 1.11 or higher exhibits better shear thinning properties than the polydispersity of 1.1. Furthermore, for hyaluronic acid derivatives HA-a20, HA-a25, and HA-a30 (Examples 20, 25, and 30), which have the same degree of polydispersity but different steryl incorporation rates, the following trend was observed: the lower the steryl incorporation rate, the better the shear thinning properties.

[0128] Table 8 shows that even when HA-a32, a hyaluronic acid derivative with a weight-average molecular weight of 35 kDa, is obtained by crosslinking HA-Na, a starting material with a weight-average molecular weight of 10 kDa, it exhibits superior shear-thinning properties compared to HA-b4 (Comparative Example 4), which has the same weight-average molecular weight but a polydispersity as low as 1.1. This demonstrates that the final molecular weight and molecular weight distribution, rather than the starting material, are crucial.

[0129] [Example 33] (Complexation of hyaluronic acid derivatives and proteins) The hyaluronic acid derivative HA-a25 obtained in Example 25 was dissolved in pure water at a concentration of 45.0 mg / mL. 1.0 mL of the resulting aqueous solution of the hyaluronic acid derivative HA-a25 was mixed with 0.5 mL of a separately obtained aqueous solution of hGH (human growth hormone) (9 mg / mL). The mixture was incubated at 37°C for 24 hours to obtain a complex solution containing 30 mg / mL of the hyaluronic acid derivative HA-a25 and 3 mg / mL of hGH. Subsequently, 1.5 mL of 2× PBS was added to precipitate a complex of the hyaluronic acid derivative HA-a10 and hGH. The supernatant was centrifuged at 9000 g for 10 minutes, and gel permeation chromatography (GPC) analysis of the supernatant (analysis conditions are shown below) revealed no peak attributable to hGH, confirming complete complexation.

[0130] (GPC conditions) Apparatus: HLC8320-GPC (manufactured by Tosoh Corporation) Column:G2000SWXL Eluent: 10 mM PBS (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI or UV 280 nm Temperature: 30℃

[0131] [Example 34] (Complexation of hyaluronic acid derivatives and PEGylated proteins) A complex of the hyaluronic acid derivative HA-a25 and PEGylated G-CSF was obtained by the same method as in Example 33, except that PEGylated G-CSF was used instead of hGH. Furthermore, GPC analysis under the same conditions as in Example 33 revealed no peak attributable to PEGylated G-CSF in the supernatant, confirming complete complexation.

[0132] [Example 35] (Combination of hyaluronic acid derivatives and poorly soluble drugs) The hyaluronic acid derivative HA-a25 obtained in Example 25 was dissolved in pure water at a concentration of 31.6 mg / mL. 0.05 mL of a methanol solution (30 mg / mL) of cyclosporine A, also obtained, was mixed with 0.95 mL of the hyaluronic acid derivative HA-a25 aqueous solution. The mixture was incubated at 4°C for 16 hours to obtain a complex solution containing 30 mg / mL of the hyaluronic acid derivative HA-a25 and 3 mg / mL of cyclosporine A. Subsequently, 1.5 mL of 2× PBS was added to precipitate the complex of the hyaluronic acid derivative HA-a25 and cyclosporine A. The supernatant was centrifuged at 9000 g for 10 minutes, and high-performance liquid chromatography (HPLC) analysis revealed no peak attributable to cyclosporine A, confirming complete complexation.

[0133] (HPLC conditions) Apparatus: HPLC system Extrema (manufactured by JASCO Corporation) Column: Inertsil ODS 5 μm Mobile phase: 0.1 v / v% TFA: 90 v / v% acetonitrile: 10 v / v% water Flow rate: 1 mL / min Injection volume: 30 μL Detector: UV (210 nm) Temperature: 40℃ [Industrial Applicability]

[0134] According to the hyaluronic acid derivative of this embodiment, a hyaluronic acid derivative with excellent needle permeability can be provided.

Claims

1. A hyaluronic acid derivative having a steroidal group incorporated therein, wherein the weight-average molecular weight Mw of the hyaluronic acid derivative is in the ratio Mw / Mn of its number-average molecular weight Mw to 1.2 or more and 2.9 or less, and the hyaluronic acid derivative has a repeating unit represented by the following general formula (I), [Chemical 1] (wherein, R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen atoms, C1-6 alkyl groups, methyl groups and C1-6 alkyl carbonyl groups, respectively; Z represents a direct bond or a peptide linker containing 2 or more and 30 or fewer amino acid residues; X1 is a group selected from the group consisting of the following groups: -NR bR, -NR b-COO-R, -NR b-CO-R, -NR b-CO-NR cR, -COO-R, -O-COO-R, -SR, -CO-Y aSR, -O-CO-Y bSR, -NR b-CO-Y bSR and -SSR; Ra, Rb and Rc are each independently selected from the group consisting of hydrogen atoms, C1-20 alkyl, amino C2-20 alkyl and hydroxy C2-20 alkyl, where the alkyl part of the group may be inserted into the group consisting of -O- and -NRf-; Rf is selected from the group consisting of hydrogen atoms, C1-12 alkyl, amino C2-12 alkyl and hydroxy C2-12 alkyl, where the alkyl part of the group may be inserted into the group consisting of -O- and -NH-; R is steroidal; Y is C2-30 alkylene or -(CH2CH2O)m-CH2CH2-, where the alkylene may be inserted into the group consisting of -O-, -NRg- and -SS-; Rg is selected from hydrogen atoms, C1-20 alkyl, amino C2-20 alkyl and hydroxy C2-20 alkyl. The group consisting of 2-20 alkyl groups, wherein the alkyl portion of the group may be inserted into a group selected from the group consisting of -O- and -NH-; Ya is a C1-5 alkyl group; Yb is a C2-8 alkyl group or a C2-8 alkenyl group; m is an integer from 1 to 100.

2. The hyaluronic acid derivative of claim 1, wherein the weight average molecular weight of the hyaluronic acid derivative is more than 3,000 and less than 160,000.

3. The hyaluronic acid derivative of claim 2, wherein the weight average molecular weight of the hyaluronic acid derivative is more than 10,000 and less than 50,000.

4. The hyaluronic acid derivative of any one of claims 1 to 3, wherein the steroidal group has an infusion rate of 6% or more but less than 60% relative to the hyaluronic acid derivative.

5. The hyaluronic acid derivative of claim 1, wherein the R mentioned above is a cholesterol group.

6. The hyaluronic acid derivative of any one of claims 1 to 3, wherein the weight average molecular weight of the hyaluronic acid derivative is more than 7,000 and less than 120,000.

7. A pharmaceutical composition comprising a hyaluronic acid derivative as claimed in any one of claims 1 to 6 as a carrier.

8. The pharmaceutical composition of claim 7, wherein the drug forms a complex with the aforementioned hyaluronic acid derivative.

9. The pharmaceutical composition of claim 7 or 8, wherein the drug is a pharmacologically active protein, peptide or nucleic acid.

10. A hyaluronic acid derivative-drug conjugate having one or more drugs conjugated to the hyaluronic acid derivative of any one of claims 1 to 6.

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