Hyaluronic acid derivative composition, pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition

Abstract

To provide a hyaluronic acid derivative composition that resists aggregation compared to the conventional art.SOLUTION: A hyaluronic acid derivative composition has (A) a hyaluronic acid derivative having a steryl group introduced thereto, and (B) at least one organic solvent selected from the group consisting of alcohol, sulfoxide, ketone, ether, and ester. A rate of introduction of the steryl group to the (A) hyaluronic acid derivative is 35% or more to less than 60%.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to hyaluronic acid derivative compositions, pharmaceutical compositions and hyaluronic acid derivative-drug conjugate compositions. [Background technology]

[0002] In recent years, biopharmaceuticals, which are pharmaceuticals that contain proteins, peptides, and nucleic acids as active ingredients, have been put to practical use, and their number is increasing year by year. Biopharmaceuticals can meet unmet medical needs that could not be met by conventional small molecule drugs. However, they have issues such as being difficult to absorb from the digestive tract or mucous membranes, being unstable in the body, and having a short half-life in the blood. For this reason, biopharmaceuticals must be administered frequently by injection, which is a heavy burden for both patients and medical professionals. Therefore, there is a demand for a drug matrix (sustained release drug delivery system matrix) that can encapsulate biopharmaceuticals without impairing their pharmacological activity and gradually release the active ingredient in the body.

[0003] In light of this background, Patent Document 1 proposes a sustained release drug delivery system substrate made of a hyaluronic acid derivative with excellent safety. This hyaluronic acid derivative spontaneously aggregates in an aqueous solution, and can efficiently encapsulate drugs, especially biopharmaceuticals, while maintaining their biological activity, and has good blood retention. This hyaluronic acid derivative can be used as a carrier that can efficiently encapsulate many drugs while maintaining pharmacological activity, and as a blood sustained release carrier and targeting carrier that have excellent blood retention, especially when using a biopharmaceutical as an active ingredient, and can also be a local (e.g., subcutaneous) sustained release carrier that can sustainably release drugs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2010 / 053140 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the hyaluronic acid derivative described in Patent Document 1, it is difficult to adjust the particle size, and the sterilization filterability of the drug composition containing hyaluronic acid derivative may deteriorate.The inventors have verified that the main cause of the particle size of hyaluronic acid derivative becoming large and the sterilization filterability becoming worse is the aggregation of hyaluronic acid derivative.

[0006] The present invention has been made in consideration of the above circumstances, and provides a hyaluronic acid derivative composition in which aggregation is suppressed more than conventional ones, as well as a pharmaceutical composition and a hyaluronic acid derivative-drug conjugate composition using the hyaluronic acid derivative composition. [Means for solving the problem]

[0007] That is, the present invention includes the following aspects. (1) A hyaluronic acid derivative composition comprising (A) a hyaluronic acid derivative having an introduced steryl group and (B) one or more organic solvents selected from the group consisting of alcohols, sulfoxides, ketones, ethers, and esters, wherein the introduction rate of the steryl group in the (A) hyaluronic acid derivative is 35% or more and less than 60%. (2) The hyaluronic acid derivative composition according to (1), wherein the content of the organic solvent (B) is 0.001 ppm by mass or more and less than 200,000 ppm by mass relative to the total mass of the hyaluronic acid derivative composition. (3) The hyaluronic acid derivative composition according to (1) or (2), wherein the (B) organic solvent is one or more organic solvents selected from the group consisting of dimethyl sulfoxide, acetone, tetrahydrofuran, ethanol, isopropanol, ethylene glycol, and ethyl acetate. (4) The hyaluronic acid derivative composition according to any one of (1) to (3), wherein the hyaluronic acid derivative (A) has a repeating unit represented by the following general formula (I):

[0008] [ka]

[0009] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X 1 is the following formula: -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, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f -, optionally with an inserted group selected from the group consisting of; R f is a hydrogen atom, C 1-12Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

[0010] (5) The hyaluronic acid derivative composition according to (4), wherein R is a cholesteryl group. (6) The hyaluronic acid derivative composition according to any one of (1) to (5), wherein the molecular weight of the hyaluronic acid derivative (A) is 1,000 or more and less than 1,000,000. (7) A pharmaceutical composition comprising the hyaluronic acid derivative composition according to any one of (1) to (6) as a carrier. (8) The pharmaceutical composition described in (7), wherein the drug forms a complex with the hyaluronic acid derivative (A). (9) The pharmaceutical composition according to (7) or (8), wherein the drug is a protein, peptide or nucleic acid having pharmacological activity. (10) A hyaluronic acid derivative-drug conjugate composition in which one or more drugs are bound to the hyaluronic acid derivative (A) contained in the hyaluronic acid derivative composition according to any one of (1) to (6). Effect of the Invention

[0011] According to the hyaluronic acid derivative composition of the above aspect, it is possible to provide a hyaluronic acid derivative composition in which aggregation is suppressed more than conventionally. [Brief description of the drawings]

[0012] [Figure 1] 1 is a 1H-NMR spectrum of cholesteryl 6-aminohexylcarbamate hydrochloride in Example 1. [Diagram 2] 1 is a 1H-NMR spectrum of the tetrabutylammonium (TBA) salt of hyaluronic acid (HA) in Example 1. [Diagram 3] 1H-NMR spectrum of the HA derivative (HA-C6-Chol) into which 6-aminohexylcarbamate was introduced in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] An embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail below, but the present invention is not limited to this embodiment, and various modifications are possible without departing from the gist of the present invention.

[0014] The terms used in this specification are explained below.

[0015] As used herein, "C 1-20 The term "alkyl" refers to a straight or branched chain alkyl group having from 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, etc. 1-4alkyl" and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, and the like. 1-20 Alkyl has 1 to 12 carbon atoms. 1-12 Alkyl, C with 1 to 6 carbon atoms 1-6 Alkyl groups are also included.

[0016] As used herein, "C 1-6 The term "alkylcarbonyl" refers to the alkyl group of the C 1-6 The term "C" refers to an alkylcarbonyl group, such as acetyl, propionyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, iso-butylcarbonyl, tert-butylcarbonyl, etc. 1-4 "Alkylcarbonyl" is included.

[0017] As used herein, "amino C 2-20 The term "alkyl" means a linear or branched alkyl having from 2 to 20 carbon atoms and an amino group as a substituent, for example, the amino group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl includes amino C with 2 to 12 carbon atoms. 2-12 Alkyl is also included.

[0018] As used herein, "hydroxy C 2-20 The term "alkyl" means a linear or branched alkyl group having from 2 to 20 carbon atoms and a hydroxyl group as a substituent, for example, the hydroxyl group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl has 2 to 12 carbon atoms and hydroxyl C 2-12 Alkyl is also included.

[0019] As used herein, "C 2-30The term "alkylene" means a linear or branched, divalent, saturated hydrocarbon group having from 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc., and has from 2 to 20 carbon atoms. 2-20 Alkylene, C with 2 to 8 carbon atoms 2-8 Alkylene, group -(CH2) n -" (wherein n is 2 or more and 30 or less, preferably 2 or more and 20 or less, and more preferably 2 or more and 15 or less).

[0020] As used herein, "C 1-5 The term "alkylene" means a linear or branched, divalent saturated hydrocarbon group having from 1 to 5 carbon atoms and includes, for example, methylene, ethylene, propylene, and the like.

[0021] The term "C 2-8 "Alkenylene" means a linear or branched, divalent saturated hydrocarbon group containing 2 to 8 carbon atoms and containing one or more double bonds, and includes, for example, -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomerism exists, each isomer and mixtures thereof are also included.

[0022] <Hyaluronic acid derivative composition> The hyaluronic acid derivative composition of the present embodiment includes (A) a hyaluronic acid derivative with a steryl group introduced therein (hereinafter, sometimes referred to as "(A) hyaluronic acid derivative") and (B) an organic solvent. (B) The organic solvent is one or more organic solvents selected from the group consisting of alcohols, sulfoxides, ketones, ethers, and esters. The introduction rate of steryl groups to (A) the hyaluronic acid derivative is 35% or more and less than 60%.

[0023] In the hyaluronic acid derivative composition of the present embodiment, (A) the hyaluronic acid derivative having an introduction rate of steryl groups within the above-mentioned range is treated with (B) the organic solvent, which prevents intermolecular interactions between the hyaluronic acid derivatives, making the intramolecular interactions relatively stronger and promoting the formation of nanoparticles, thereby suppressing aggregation.

[0024] In the hyaluronic acid derivative composition of the present embodiment, the particle size is not particularly limited, but is preferably 500nm or less, more preferably 400nm or less, even more preferably 300nm or less, and particularly preferably 200nm or less.If the particle size is below the upper limit, it can be sterilized by filtration using a filter.Moreover, the particle size is most preferably less than 100nm.By having a particle size of less than 100nm, it is easy to be taken up by the reticuloendothelial system in vivo.In addition, in the hyaluronic acid derivative composition of the present embodiment, the particle size can be measured by the method described in the examples below.

[0025] <(A) Hyaluronic acid derivatives with steryl groups> In the (A) hyaluronic acid derivative, the steryl group may be directly bound to the hyaluronic acid or may be bound via a linker. The "linker" referred to here can be any peptide linker or synthetic compound linker that can be introduced by genetic engineering, but in the (A) hyaluronic acid derivative of this embodiment, a peptide linker is preferred. The length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose, but the preferred length is 2 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. The peptide linkers contained in the (A) hyaluronic acid derivative may all be peptide linkers of the same length, or peptide linkers of different lengths may be used.

[0026] [Steryl group] The term "steryl group" used in this specification is not particularly limited as long as it is a group having a steroid skeleton.Specific examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simiarenolol, bile acid, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycortisterone, etc.Steryl groups include cholesteryl groups, stigmasteryl groups, lanosteryl groups, ergosteryl groups, etc., and among them, cholesteryl groups (particularly, cholest-5-en-3β-yl groups) are preferred.

[0027] [Steryl group introduction rate] The introduction rate of steryl group to the (A) hyaluronic acid derivative (hereinafter, sometimes simply referred to as "steryl group introduction rate") is 35% or more and less than 60%, preferably 36% or more and less than 55%, more preferably 38% or more and less than 50%, and even more preferably 38% or more and less than 45%. When the introduction rate of steryl group is within the above range, the hyaluronic acid derivative has the property of forming nanoparticles well in water and dispersing. When the introduction rate of steryl group is within the above range, the hyaluronic acid derivative-drug conjugate composition, which is the (A) hyaluronic acid derivative in the hyaluronic acid derivative composition complexed with a drug, can become a sustained release preparation that can be dissolved in water and sterilized and filtered.

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

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

[0030] [(A) Molecular weight of hyaluronic acid derivative] The molecular weight of (A) hyaluronic acid derivative is not particularly limited, but from the viewpoint of increasing the number of steryl groups introduced per molecule of hyaluronic acid derivative and forming a drug conjugate, the (A) hyaluronic acid derivative with a relatively large molecular weight is preferred, and from the viewpoint of expecting sterilization filtration performance and from the viewpoint of syringeability when the final dosage form is a solution preparation, the (A) hyaluronic acid derivative with a relatively small molecular weight is preferred.The molecular weight of such (A) hyaluronic acid derivative is preferably 800 (0.8k) or more and 1,000,000 (1,000k) or less, more preferably 1k or more and 1,000k or less, even more preferably 1k or more and 300k or less, particularly preferably 2k or more and 120k or less, and most preferably 3k or more and 100k or less.The molecular weight of (A) hyaluronic acid derivative can generally be adjusted by using a raw material with a corresponding molecular weight.

[0031] Here, "the molecular weight of (A) hyaluronic acid derivative" refers to the weight-average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS).Specifically, it can be measured according to the method described in the following examples.

[0032] Specific examples of preferred (A) hyaluronic acid derivatives include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter, sometimes referred to as "repeating units (I)").

[0033] [ka]

[0034] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X 1 is the following formula: -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, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f -, optionally with an inserted group selected from the group consisting of; R f is a hydrogen atom, C 1-12 Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

[0035] The (A) hyaluronic acid derivative preferably contains a hyaluronic acid derivative having one or more repeating units represented by the following general formula (Ia) (hereinafter, sometimes referred to as "repeating unit (Ia)").

[0036] [ka]

[0037] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X is -NR a -Y-NR b- a hydrophobic group represented by COO-R; R a and R b each independently represents a hydrogen atom and C 1-6 selected from the group consisting of alkyl; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, m is an integer between 1 and 100.

[0038] Here, when the hyaluronic acid derivative (A) contains two or more repeating units (I) or two or more repeating units (Ia), the repeating units may be the same or different.

[0039] The (A) hyaluronic acid derivative may be modified at a position other than the repeating unit (I) or the repeating unit (Ia), for example, the hydroxy group may be modified by -O(C 1-6 Alkyl), -O(Formyl), -O(C 1-6 The carboxy group may be converted to an amide or ester, or may form a salt.

[0040] Repeating unit (I) The group "-ZN(R a )YX 1 " has the following formula: -NH-(CH2) mz -NH-R; -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; -NH-(CH2) mz -COO-R; -NH-(CH2CH2O) m -CH2CH2-COO-R, -NH-(CH2) mz -O-COO-R; -NH-(CH2CH2O) m -CH2CH2-O-COO-R, -NH-(CH2) mz -SR; -NH-(CH2CH2O) m -CH2CH2-SR; -NH-(CH2) mz -O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -SSR; and -Z-NR a -Y-NR b -COO-R (where mz is an integer between 2 and 30, and R 8 is a hydrogen atom or a methyl group, and R and m are as previously defined herein. The group includes a group selected from the group consisting of groups represented by: The group is, -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -SSR (wherein mz, ​​R, and m are as previously defined herein.) A group selected from the group consisting of:

[0041] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 Ha-NR b In yet another embodiment, Z is -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a )-CO-, where n is an integer of 2 to 30, a are each independently H2N-CH(-Z a The peptide linker is attached to the carboxy group of the glucuronic acid moiety at the N-terminus and to the group -N(-R)-COOH at the C-terminus. a )-YX 1 Examples of amino acids that can be used as the amino acid residue of the peptide linker include α-amino acids, such as natural (L-type) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, and D-forms thereof, and all α-amino acids, including synthetic amino acids, can be used. That is, Z a Examples of the linker include -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, and the like. Furthermore, 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 the peptide linker include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, and the like.

[0042] (Y) In the general formula (I), Y is -(CH2) n1 -and-(CHCHO) m1-CH2CH2- (wherein 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; and m1 is an integer of 1 or more and 4 or less) is preferred. Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12 From the viewpoint of realizing high solubility in pure water or at a low salt concentration and exhibiting high precipitate-forming ability at a physiological salt concentration, Y is preferably -(CH2)2-, -(CH2)6-, -(CH2)8-, or -(CH2) 12 A group selected from the group consisting of - is preferred, with -(CH2)6- being more preferred.

[0043] Y may be, for example, -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, and the like.

[0044] (Y a ) Y a As the alkyl group, -CH2- or -CH2-CH2- is preferable.

[0045] (Y b ) Y b As the alkyl group, -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 is preferable, and -CH2-CH2- or -CH(CH3)CH2- is more preferable.

[0046] Group "-ZN(R a )YX 1Specific examples of " are -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C H2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-NH-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-cholesteryl, and the like are preferred. a )YX 1 " As for R a , R b and R c is a hydrogen atom, and Y is a linear C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, and Y a But linear C 1-5 is alkylene, or Y b But linear C 2-8 Alkylene or linear C 2-8 It is alkenylene.

[0047] [Repeating unit (Ia)] In the general formula (Ia), X is -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl, -NH-(CH2) 12-NH-COO-cholesteryl or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl is preferred, and -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl is more preferred.

[0048] The hyaluronic acid derivative (A) may further contain, in addition to the repeating unit (I), a repeating unit represented by general formula (II) (hereinafter sometimes referred to as "repeating unit (II)").

[0049] [ka]

[0050] (In the formula, R 1a , R 2a , R 3a , and R 4a are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X a Hydroxy and -OQ + wherein Q is selected from the group consisting of + is the counter cation.)

[0051] Here, when the hyaluronic acid derivative (A) contains two or more repeating units (II), the repeating units may be the same or different. In another embodiment, the hyaluronic acid derivative (A) may be a hyaluronic acid derivative essentially consisting of the repeating unit (I), the repeating unit (Ia) and the repeating unit (II).

[0052] [Repeating unit (II)] In the general formula (II), Q +is not particularly limited as long as it is a counter cation that forms a salt with a carboxy group in water, and when it is divalent or higher, it forms salts with multiple carboxy groups depending on the valence. Examples of the counter cation include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; + R j R k R l R m (In the formula, R j , R k , R l and R m each independently represents a hydrogen atom and C 1-6 Among them, the ammonium ion represented by Q + is preferably a sodium ion, a potassium ion, or a tetraalkylammonium ion (e.g., a tetra-n-butylammonium ion, etc.). j , R k , R l and R m is C 1-6 Preferably, they are identical groups selected from the group consisting of alkyl, and preferably n-butyl.

[0053] R 1 , R 2 , R 3 , and R 4 , and R 1a , R 2a , R 3a , and R 4a are preferably all hydrogen atoms. a and R b are preferably all hydrogen atoms.

[0054] Among them, the (A) hyaluronic acid derivative is preferably a hyaluronic acid derivative substantially composed of repeating unit (I) and repeating unit (II).The (A) hyaluronic acid derivative is such that, for example, 80% or more, preferably 90% or more, more preferably 95% or more of the repeating units of the disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative are repeating units (I) and repeating units (II).The (A) hyaluronic acid derivative may be composed only of repeating units (I) and repeating units (II).

[0055] [(B) Organic solvent] (B) The organic solvent functions as a nanoparticle formation promoter for the hyaluronic acid derivative in water, suppressing the aggregation of the hyaluronic acid derivative in water, and as a result improving the sterilization filterability.

[0056] The content of (B) the organic solvent is preferably 0.001 ppm by mass or more and less than 200,000 ppm by mass, more preferably 0.001 ppm by mass or more and 150,000 ppm by mass or less, even more preferably 0.005 ppm by mass or more and 120,000 ppm by mass or less, and particularly preferably 0.006 ppm by mass or more and 100,000 ppm by mass or less, relative to the total mass of the hyaluronic acid derivative composition. (B) The content of organic solvent is above the lower limit, so that the nanoparticle forming ability of hyaluronic acid derivative composition in water can be improved, while the content is below the upper limit, so that the aggregation of hyaluronic acid derivative in water can be more effectively suppressed.As a result, the content of organic solvent is within the above-mentioned range, so that the particle size can be relatively small, and the sterilization filterability is necessarily improved. The content of the (B) organic solvent can be measured, for example, according to the method described in the Examples below.

[0057] (B) The organic solvent is one or more organic solvents selected from the group consisting of alcohols, sulfoxides, ketones, ethers, and esters.

[0058] The alcohol may be a monoalcohol or a polyhydric alcohol. Examples of the monoalcohol include methanol, ethanol, 1-propanol, 2-propanol (isopropanol), 1-butanol, 2-butanol, isobutanol, 1-pentanol, 2-pentanol, isoamyl alcohol, 1-hexanol, 2-hexanol, 1-heptanol, 1-octanol, 2-ethyl-1-hexanol, 3,3,5-trimethyl-1-hexanol, tridecanol, pentadecanol, palmityl alcohol, stearyl alcohol, cyclopentanol, cyclohexanol, methylcyclohexanol, and trimethylcyclohexanol. Examples of the polyhydric alcohol include dihydric alcohol and trihydric alcohol. Examples of the dihydric alcohol include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of trihydric alcohols include glycerin and trimethylolpropane. Among them, the alcohol is preferably one or more alcohols selected from the group consisting of ethanol, isopropanol, and polyhydric alcohols, more preferably polyhydric alcohols, and further preferably ethylene glycol.

[0059] Examples of sulfoxides include diethyl sulfoxide, butyl sulfoxide, tetramethylene sulfoxide, sulfolane, dimethyl sulfone, and dimethyl sulfoxide (DMSO). Among these, as the sulfoxide, dialkyl sulfoxides are preferable, and dimethyl sulfoxide is more preferable.

[0060] Examples of ketones include acetone, methyl ethyl ketone, methyl butyl ketone, methyl isobutyl ketone, diethyl ketone, diisobutyl ketone, cyclopentanone, cyclohexanone, and methylcyclohexanone. Among these, the ketone is preferably acetone, methyl ethyl ketone, or methyl isobutyl ketone, and more preferably acetone.

[0061] Examples of the ether include diethyl ether, diisopropyl ether, dimethoxymethane, 1,2-dimethoxyethane, 1,4-dioxane, 1,3-dioxolane, tetrahydrofuran, anisole, penethol, and tert-butyl methyl ether. Among these, the ether is preferably diethyl ether, 1,4-dioxane, tetrahydrofuran, or tert-butyl methyl ether, and more preferably tetrahydrofuran.

[0062] Examples of esters include methyl acetate, ethyl acetate, n-butyl acetate, isobutyl acetate, isopropyl acetate, propyl acetate, ethyl formate, methyl formate, ethyl formate, propyl formate, pentyl formate, methyl acetate, ethyl acetate, and pentyl acetate. Among these, the ester is preferably ethyl formate, methyl acetate, ethyl acetate, n-butyl acetate, isobutyl acetate, isopropyl acetate, or propyl acetate, and more preferably ethyl acetate.

[0063] <Method of producing hyaluronic acid derivative composition> The hyaluronic acid derivative composition of the present embodiment can be produced by producing (A) a hyaluronic acid derivative, and then adding (B) an organic solvent to the (A) hyaluronic acid derivative.

[0064] First, as the method for producing (A) hyaluronic acid derivative, for example, the carboxyl group of glucuronic acid is converted to amide, and steryl group is introduced to obtain (A) hyaluronic acid derivative.In addition, by adjusting the amount of the compound having steryl group reacted with raw material hyaluronic acid or its derivative, the introduction rate of steryl group can be 35% or more and less than 60%.

[0065] Specifically, the method of converting the carboxy group of glucuronic acid into an amide and introducing a steryl group is, for example, to ion-exchange the raw material hyaluronic acid or a derivative thereof, preferably hyaluronic acid or a derivative thereof composed only of the repeating unit (II), with a tetraalkylammonium salt (e.g., a tetrabutylammonium (TBA) salt), and then reacting the hyaluronic acid salt with a compound of the formula: "HNR a -Y-NR b -R, NHR a -Y-NR b -COO-R, HNR a -Y-NR b -COO-R, HNR a -Y-NR b -CO-R, HNR a -Y-NR b -CO-NR c -R, HNR a -Y-COO-R, HNR a -YO-COO-R, HNR a -YSR, HNR a -Y-CO-Y a -SR, HNR a -YO-CO-Y b -SR, HNR a -Y-NR b -CO-Y b -SR, HNR a -YSSR or -Z-NR a -Y-NR b -COO-R(wherein, R a , R b , R c , Y, Y a , Y b , Z and R are as defined above) with an amine having a steryl group (particularly, a cholesteryl group) introduced therein.

[0066] Condensing agents that can be used in the above reaction are not particularly limited, and examples thereof include 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), and benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium. Examples include hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0067] In particular, DMT-MM is preferred because it can react efficiently even in a mixed solvent of water and an organic solvent, but is not limited thereto.In addition, by using DMT-MM as a condensing agent, in a system in which a large number of hydroxyl groups coexist, it is possible to suppress the formation of ester bonds and to highly selectively form amide bonds between amino groups and carboxyl groups.By using this condensing agent, it is possible to prevent, for example, the reaction of the alcohol solvent with the carboxyl group of the hyaluronic acid portion, and the intramolecular or intermolecular bonding of the carboxyl group and hydroxyl groups that simultaneously exist in the hyaluronic acid portion to form undesired crosslinks.

[0068] Examples of the solvent used in the steryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyhydric alcohols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixed solvents thereof. Examples of the polyhydric alcohol include the same as those exemplified in "(B) Alcohol" above.

[0069] Alternatively, raw hyaluronic acid or its derivative may be ion-exchanged with tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and in the presence of a suitable condensing agent, the hyaluronic acid salt may be reacted with a spacer moiety in a solvent (at this time, protection and deprotection reactions may be performed as necessary), converting the carboxy group (-COOH) of raw hyaluronic acid or its derivative, and then reacting with a suitable reagent. Examples of combinations of groups derived from carboxy groups and reaction reagents are shown below. -CONR a -Y-NR b H + Hal-R; -CONR a -Y-NR b H + Hal-COOR; -CONR a -Y-NR b H+HOCO-R; -CONR a -Y-NR b H + Hal-CO-R; -CONR a -Y-NR b -COOH + HNR c -R; -CONR a -Y-NR b -CO-NR c H + Hal-R; -CONR a -Y-NR b H+HOCO-NR c -R; -CONR a -Y-NR b H + Hal-CO-NR c -R; -CONR a -Y-COOH + HO-R; -CONR a -Y-OH + Hal-COO-R; -CONR a -Y-OCOOH+HO-R; -CONR a -Y-OCOOH + Hal-R; -CONRa -Y-OCO-Hal + HO-R; -CONR a -Y-SH + Hal-R; -CONR a -Y-Hal + HS-R; -CONR a -Y-CO-Y a -Hal + HS-R; -CONR a -Y-CO-Y a -SH + Hal-R; -CONR a -YO-CO-CH=CH2+ HS-R; -CONR a -Y-NR b -CO-CH(CH3)=CH2+ HS-R; -CONR a -Y-SH + HS-R; -COZ-OH + HNR a -Y-NR b -COO-R; -COZ-NR a -Y-NR b H + Hal-COO-R (In the formula, R a , R b , R c , Y, Y a , Y b and Z are as previously defined herein, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0070] Examples of reaction modes include dehydrohalogenation reaction, condensation reaction, dehydration reaction, nucleophilic addition reaction such as Michael addition, oxidative disulfide formation reaction, etc. These are well-known reactions, and can be appropriately selected by those skilled in the art and carried out under preferred reaction conditions. When the conversion product or reaction product has a carboxy group, it may be reacted as an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester.

[0071] Another method is to react the carboxy group of the raw material hyaluronic acid or its derivative with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative having a spacer having a mercapto group modified with a leaving group at the end, and then subject this to a nucleophilic substitution reaction with thiocholesterol to form a disulfide bond.

[0072] Furthermore, the method of preparing the one in which a part of spacer is introduced into the carboxyl group of hyaluronic acid or its derivative and the one in which a part of spacer is introduced into the steryl group and reacting them can also be mentioned.Some of the specific examples have been mentioned above, but furthermore, when -SS- is inserted into Y, the method of preparing the hyaluronic acid derivative in which a spacer having a mercapto group at the end is introduced into the carboxyl group of hyaluronic acid and the steryl group in which a spacer having a mercapto group at the end is introduced can also be mentioned, and the method of forming disulfide bond by oxidative reaction of these can also be mentioned.At this time, one mercapto group can be reacted with 2-mercaptopyridine to form disulfide, and then replaced with the other mercapto group.

[0073] In addition, after preparing the hyaluronic acid derivative of the present invention, other substituents may be further introduced. For example, 0.1% to 99.5%, preferably 40% to 65%, of the carboxyl groups in the hyaluronic acid derivative essentially consisting of the repeating unit (I) and the repeating unit (II) may be replaced with -CO-X z , where X z is the following group: -NH-(CH2) p1 -O-CO-C(R 17 )=CH2; -NH-(CH2) p1 -O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -SH; -NH-(CH2) p1 -NH-CO-C(R 17 )=CH2; -NH-(CH2) p1-NH-C(=NH)-(CH2)3-SH; -NH-(CH2) p1 -NH-CO-(CH2) r -SH; -NH-(CH2) p1 -NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-(CH2)2-SH; -NH-NH-CO-(CH2)4-CO-NH-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-(CH2) r -SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-(CH2)2-SH; -NH-CH(CO2H)-(CH2)-SH; -NH-CH(COH)-(CH)-SH; and -NH-CH(CO2H)-(CH2)2-CONH-CH(CONH-CH2-CO2H)-CH2-SH (where R 17 is a hydrogen atom or C 1-6 p1 is an integer of 2 or more and 10 or less, q is an integer of 1 or more and 200 or less, and r is an integer of 1 or more and 3 or less. By converting the compound into the above, cross-linking can be effected within a molecule or between molecules including other molecules to form a gel.

[0074] (A) The condition of the step of gelling the hyaluronic acid derivative by chemical crosslinking may be appropriately selected. The crosslinking conditions include the crosslinking method, polymer concentration, crosslinking agent concentration, solvent, solvent pH, salt concentration, temperature, time, etc.

[0075] (A) In the process of gelling the hyaluronic acid derivative, the crosslinking density of the resulting gel can be increased by increasing the reaction conditions for crosslinking, for example, the polymer concentration during chemical crosslinking and the introduction rate of groups capable of crosslinking.

[0076] (A) In the step of gelling the hyaluronic acid derivative, when using a crosslinking agent having groups capable of forming crosslinks at both ends, it is preferable to add the crosslinking agent at a concentration such that the groups can participate in the crosslinking reaction quickly and without excess or deficiency. For example, when a polymer having a methacryloyl group (MA group) introduced therein is crosslinked by Michael addition reaction using DTT, the ratio of MA group:SH group is preferably 3:1 to 1:3, and particularly preferably 2:1 to 1:2.

[0077] The solvent in the step of gelling the (A) hyaluronic acid derivative is preferably one that can dissolve the polymer and the crosslinking agent sufficiently, and is not particularly limited, but is preferably water, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dimethylformamide (DMF), N-methylpyrrolidone (NMP) and a mixed solvent selected from these. It is also possible to use an organic solvent that is miscible with these solvents by mixing them. Although not particularly limited, examples of the miscible organic solvent include methanol, ethanol, propanol, isopropanol, butanol, polyhydric alcohol, acetone, acetonitrile, etc. As the polyhydric alcohol, the same as those exemplified in the above "(B) alcohol" are included, and among them, ethylene glycol is preferred.

[0078] (A) Hyaluronic acid derivative forms nanoparticles in aqueous solution, so that it can be crosslinked under dilute conditions to form nano-sized microparticle gel, and can be used as a blood sustained release carrier or targeting carrier. Dilute conditions are 10mg / mL or less, preferably 5mg / mL or less, and more preferably 1mg / mL or less. On the other hand, crosslinking under high concentration conditions can form bulk gel in which microparticles are crosslinked with each other. This is useful as a subcutaneous sustained release carrier. High concentration conditions are 5mg / mL or more, preferably 20mg / mL or more, and more preferably 40mg / mL.

[0079] (A) The step of gelling the hyaluronic acid derivative may be carried out in bulk, or in a discontinuous phase such as in an emulsion or in sprayed droplets. For example, when carrying out in a W / O emulsion, the aqueous phase in which the polymer, crosslinking agent, etc. are dissolved may be emulsified in a solvent that is not miscible with water, and the gelling reaction may be carried out. The solvent that is not miscible with water is not particularly limited, but examples thereof include hexane, chloroform, dichloromethane, ethyl acetate, medium chain triglyceride (MCT), liquid paraffin, soybean oil, etc. A surfactant may be added to stabilize the emulsion. In addition, the step may be carried out in a solvent that can be desolvated, such as in supercritical carbon dioxide or PEG. In this case, the aqueous phase or organic solvent phase in which the polymer, crosslinking agent, etc. are dissolved is emulsified and dispersed in the solvent of the previous example, and the polymer is concentrated due to desolvation (solvent diffusion), so that a gel with a higher crosslink density can be obtained.

[0080] (A) The step of gelling the hyaluronic acid derivative and thereafter, an operation of stopping the crosslinking reaction and an operation of deactivating or washing the remaining crosslinking functional group may be performed. It is preferable to remove the crosslinking functional group not involved in the reaction, the group bound to only one end of the crosslinking agent, the remaining crosslinking agent, etc. from the viewpoints of safety, stability during storage, and side reactions with the drug to be encapsulated. Although not particularly limited, for example, if the unreacted crosslinking agent remains, it may be removed by washing with excess water or the like. In addition, for example, if the methacryloyl group substituted on the polymer remains, it may be removed by adding excess mercaptoethanol or the like to deactivate the methacryloyl group, and then washing the excess mercaptoethanol with excess water or the like. Furthermore, for example, if the mercapto group remains, it may be removed by adding excess 3-maleimidopropionic acid, iodoacetic acid, etc. to deactivate the mercapto group, and then washing the excess 3-maleimidopropionic acid and iodoacetic acid with excess water or the like.

[0081] (A) After the step of gelling the hyaluronic acid derivative, a pulverization step may be carried out. As the pulverization method, pulverization using a pestle and a mortar or mill can be mentioned, but mill pulverization is preferred. As the mill pulverization device, there are rotary disk type pulverization devices such as centrifugal pulverizer (Nihon Seiki Seisakusho) and impact mill (Dalton Co., Ltd.), screen mill pulverization devices such as atomizer (Tokyo Atomizer Manufacturing Co., Ltd.), sample mill (Tokyo Atomizer Manufacturing Co., Ltd.), bantam mill (Tokyo Atomizer Manufacturing Co., Ltd.), and SK mill (Tokken), jet pulverization devices such as ultra-low-volume lab jet mill (AO jet mill, Seishin Enterprise Co., Ltd.), and Rinrex mill (Liquid Gas Co., Ltd.) that can be pulverized at ultra-low temperature, but SK mill and Rinrex mill are preferred.

[0082] After the process of gelling (A) hyaluronic acid derivative, drying process can be carried out.Drying method can be, for example, ventilation drying, drying in thermostatic chamber, drying under reduced pressure, hot air circulation drying, etc.Wind speed, drying time, temperature, pressure, etc. are appropriately selected within the range that does not cause decomposition or deterioration of the gel of (A) hyaluronic acid derivative.

[0083] In the above-mentioned (A) hyaluronic acid derivative production, when the (B) organic solvent derived from the solvent used in production remains, it is possible to obtain a hyaluronic acid derivative composition without adding (B) organic solvent.Alternatively, it is possible to obtain a hyaluronic acid derivative composition by adding (B) organic solvent after the above-mentioned (A) hyaluronic acid derivative production so that the content of (B) organic solvent is desired.When (B) organic solvent is added, it is preferable to add it before the above-mentioned drying process.

[0084] Pharmaceutical Composition The pharmaceutical composition of this embodiment comprises the above-mentioned hyaluronic acid derivative composition as a carrier.In the pharmaceutical composition of this embodiment, the carrier and the drug are directly or indirectly bound to each other to form a complex, and it is desirable that they are not in a state of being separated from each other.When this pharmaceutical composition is administered to a living body, the drug is gradually released from the carrier, and good sustained release can be expected.The bond between the carrier and the drug can be either covalent or non-covalent, but non-covalent bond is preferred from the viewpoint of maintaining the activity of the drug.

[0085] In the pharmaceutical composition of the present embodiment, it is preferred that the drug forms a complex with the (A) hyaluronic acid derivative in the hyaluronic acid derivative composition as a carrier.It is considered that in a solvent, the steryl group of the (A) hyaluronic acid derivative and the drug present in the system spontaneously associate with each other through hydrophobic interaction, forming a complex between the drug and the (A) hyaluronic acid derivative.By forming this complex, it is expected to improve the storage stability of the drug, maintain biological activity, improve sustained release, and when the drug is poorly water-soluble, it is expected to solubilize the drug in water, improve resistance to stimuli such as heat and light, and suppress aggregation and precipitation.

[0086] <Drugs> The drug contained in the pharmaceutical composition of the present embodiment is not particularly limited as long as it can be used as a pharmaceutical for humans and animals.For example, it can be protein, peptide, polysaccharide, nucleic acid, low molecular weight compound, etc.The pharmaceutical composition of the present embodiment preferably contains the biopharmaceutical drug or low molecular weight compound such as protein, peptide, nucleic acid, etc. that has pharmacological activity, and the (A) hyaluronic acid derivative in the hyaluronic acid derivative composition to form a complex.

[0087] [Low molecular compounds] Examples of low molecular weight compounds include anticancer agents (e.g., alkylating agents, metabolic antagonists, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroids, non-steroidal anti-inflammatory agents, etc.), antirheumatic agents, antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfa drugs, etc.).

[0088] [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), serial neutrophic 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 (IL-1), and the like. These include IL-1ra, brain-derived neurotrophic factor (BDNF), keratinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth and differentiation factor (MGDF), osteoprotegerin (OPG), leptin, parathyroid hormone (PTH), basic fibroblast growth factor (b-FGF), bone morphogenetic protein (BMP), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), glucagon-like peptide-1 (GLP-1), antigens for vaccines, antibodies, diabodies, minibodies, and fragmented antibodies.

[0089] [Nucleic acid] Examples of nucleic acids include DNA, RNA, antisense nucleic acids, decoy nucleic acids, ribozymes, small interfering RNA, and nucleic acid aptamers.

[0090] <Form> The pharmaceutical composition of the present embodiment may be a dispersible microparticle solution, a precipitated suspension, or a freeze-dried product. In addition, in the case of a freeze-dried product, it may be a sustained release formulation in which a doctor adds an isotonic solution such as physiological saline before administration to prepare the administration solution when needed. In this case, it is considered to be suitable for pharmaceutical compositions containing active ingredients that are unstable in a solution state.

[0091] When the pharmaceutical composition of the present embodiment is a dispersible microparticle solution or a precipitated suspension, the concentration of the (A) hyaluronic acid derivative in the pharmaceutical composition is preferably 0.01 mg / mL or more and 500 mg / mL or less, more preferably 0.1 mg / mL or more and 200 mg / mL or less, even more preferably 0.2 mg / mL or more and 100 mg / mL or less, and particularly preferably 0.5 mg / mL or more and 50 mg / mL or less. The concentration of the (A) hyaluronic acid derivative in the pharmaceutical composition is above the upper and lower limits, which can improve the storage stability of the drug and the sustained release of biological activity, and when the drug is poorly water-soluble, can make the drug more excellent in solubilization in water, and can also tend to increase the amount of drug administered. On the other hand, the concentration of the (A) hyaluronic acid derivative in the pharmaceutical composition is below the upper limit, which can improve the injectability when administered into the living body using a syringe needle, and can also tend to improve the sterilization filterability.

[0092] <Hyaluronic acid derivative-drug conjugate composition> In the hyaluronic acid derivative-drug conjugate composition of the present embodiment, one or more drugs are conjugated to the hyaluronic acid derivative (A) contained in the hyaluronic acid derivative composition.

[0093] Drugs suitable for forming the hyaluronic acid derivative-drug conjugate composition of this embodiment are biopharmaceuticals such as proteins, peptides, and nucleic acids, or low molecular weight compounds.

[0094] The pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment are not limited to the above-mentioned forms, and may be in the form of nanoparticles, microparticles, solution, emulsion, suspension, gel, micelle, implant, powder, or film. Powder may be produced by grinding solid obtained by freeze-drying or spray-drying, or may be produced from dried precipitate.

[0095] The pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment may be administered orally, parenterally, intranasally, intravaginally, intraocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally, or intraoral. The pharmaceutical composition and hyaluronic acid derivative-drug conjugate composition of this embodiment are not limited to injections, and may be patch preparations, microneedle preparations, ointments, eye drops, sprays, inhalants, etc. EXAMPLES

[0096] The present invention will be described in detail below with reference to examples, but it is not intended that the scope of the present invention be limited to these examples.

[0097] The methods for measuring and evaluating the physical properties of the hyaluronic acid derivative compositions prepared in the Examples and Comparative Examples are as follows.

[0098] [Physical Properties 1] (Molecular weight of hyaluronic acid derivative) The molecular weight of the hyaluronic acid derivative is the weight average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS). The hyaluronic acid derivative composition (20 mg) was dissolved in ultrapure water (10 mL) and stirred at room temperature for 12 hours or more to obtain an aqueous solution of hyaluronic acid derivative composition (2 mg / mL). A 300 mM aqueous solution of hydroxypropyl-β-cyclodextrin (HP-β-CD) (750 μL) was added to this aqueous solution of hyaluronic acid derivative composition (750 μL), mixed for 10 seconds using a shaker, and incubated at 37 ° C. for 1 hour. The obtained sample was then subjected to SEC-MALS measurement to determine the weight average molecular weight. The conditions of the SEC-MALS measurement are shown below.

[0099] (Measurement conditions) Column: TSKgel GMPWXL (Tosoh Corporation) x 2 Column temperature: 30℃ Eluent: 10mM HP-β-CD in phosphate buffered saline (pH 7.4) Flow rate: 1mL / min Injection volume: 200μL

[0100] [Physical Properties 2] (Steryl group introduction rate) The steryl group introduction rate of the hyaluronic acid derivative is 1 The H-NMR measurement was used to determine the concentration. First, dimethyl sulfoxide-d6 (99.9 v / v%, containing 0.05 v / v% trimethylsilyl (TMS), Fuji Film Wako Co., Ltd.) and 20% deuterium chloride (99.5 v / v%, Fuji Film Wako Co., Ltd.) were mixed in a mass ratio of 99:1 to prepare a measurement solvent. Next, the hyaluronic acid derivative composition (2 mg) was added to this measurement solvent (0.6 mL), and the mixture was treated in an ultrasonic bath for 30 minutes to completely dissolve the composition. 1 H-NMR measurement was performed. 1 H-NMR measurements were performed using a Fourier transform nuclear magnetic resonance (FT-NMR) (ECS400, manufactured by JEOL Ltd.) at a sample temperature of 85° C. The introduction rate of steryl groups into hyaluronic acid units was calculated using the following formula from the integral 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 integral value of the peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H). In addition, since the peak derived from the cholesteryl group (5H) overlaps with the peak in the vicinity of 1.6 ppm to 2.0 ppm, which includes the peak derived from the acetyl group of N-acetyl-D-glucosamine, the value calculated by subtracting 5 / 3 times the integral value of the peak derived from the cholesteryl group methyl (0.7 ppm) from the integral value of the peak in the vicinity of 1.6 ppm to 2.0 ppm (i.e., integral value (1.6 ppm to 2.0 ppm) - integral value (0.7 ppm) × 5 / 3) was used as the integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine to calculate the steryl group introduction rate.

[0101] [Steryl group introduction rate (%)] = [(peak integral value derived from methyl group in cholesteryl group) / (peak integral value derived from acetyl group of N-acetyl-D-glucosamine)] × 100 = [integral value (0.7 ppm) / {integral value (1.6 ppm or more and 2.0 ppm or less) - integral value (0.7 ppm) × 5 / 3}] × 100

[0102] [Physical Properties 3] (Organic solvent content) The content of organic solvent in the hyaluronic acid derivative composition was measured by GC / MS. The hyaluronic acid derivative composition (80mg) was dissolved in ultrapure water (10mL) and stirred at room temperature for 12 hours or more to obtain an aqueous solution of hyaluronic acid derivative composition (8mg / mL). This aqueous solution of hyaluronic acid derivative composition was subjected to GC / MS measurement to determine the content of organic solvent. The conditions of GC / MS measurement are shown below.

[0103] (Measurement conditions) GC device: Agilent Technologies, 7890B Column: DB-WAX (30m x 0.25mmφ, film thickness 0.25μm, manufactured by Agilent Technologies) Column temperature: 40℃ (5min), 10℃ / min heating, 250℃ (9min) Flow rate: 1mL / min Inlet temperature: 250℃ Split ratio: 1 / 50 Injection volume: 1μL MS equipment: Agilent Technologies, 5977B Ionization: EI, 70 eV Ion source temperature: 230℃

[0104] [Rating 1] (particle size) The particle size of the hyaluronic acid derivative composition was determined by the following procedure. First, one packet of 1 / 15 mol / L PB (phosphate buffer powder, Fujifilm Wako) was dissolved in 3.33 L of ultrapure water, and filtered through a 0.22 μm filter to prepare a 20 mM PB aqueous solution. Next, ultrapure water (5 mL) was added to the hyaluronic acid derivative composition (10 mg), and the mixture was stirred for 12 hours or more to dissolve, thereby obtaining an aqueous solution of the hyaluronic acid derivative composition. This aqueous solution (1 mL) was diluted with a 20 mM PB aqueous solution (1 mL) to obtain an aqueous solution of the hyaluronic acid derivative composition PB. The obtained aqueous solution of the hyaluronic acid derivative composition PB was then subjected to dynamic light scattering (DLS) measurement to determine the particle size of the hyaluronic acid derivative composition. The measurement conditions for DLS are shown below. The particle size is the harmonic mean particle size (diameter) based on the scattered light intensity standard, the unit is nanometers (nm), and is the average value of two measurements of the same sample.

[0105] (Measurement conditions) DLS device: Otsuka Electronics, ELSZ2000 Cell: Microparticle size cell Temperature: 37℃ Concentration of hyaluronic acid derivative composition: 2mg / mL Solution: 10 mmol / L phosphate buffer (pH 7.4)

[0106] <Production of hyaluronic acid derivative composition> [Example 1] (Production of hyaluronic acid derivative composition HA-a1) A hyaluronic acid derivative composition was prepared according to the following steps 1 to 3.

[0107] 1.Process 1 (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1 and then 1-2.

[0108] (1) Process 1-1 Triethylamine (TEA, 1.05 mL) was added to a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) under an argon atmosphere and stirred. 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise under ice cooling, and the mixture was stirred for 30 minutes under ice cooling, then warmed to room temperature, and stirred overnight. The reaction mixture was washed with ultrapure water and saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:4), and the fractions of the target product were combined and the solvent was distilled off under reduced pressure.

[0109] (2) Process 1-2 The resulting residue was dissolved in ethyl acetate (40 mL), and a 4N hydrochloric acid / ethyl acetate solution (40 mL) was added and stirred at room temperature overnight. The resulting precipitate was collected 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). 1 The H-NMR spectrum (ECS400 manufactured by JEOL, EtOH-d6) is shown in Figure 1.

[0110] 2.Process 2 (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) Hyaluronic acid TBA salt (HA-TBA) was prepared according to the following steps 2-1 and 2-2.

[0111] (1) Process 2-1 DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water about three times by decantation. A 40 wt% aqueous solution of tetrabutylammonium hydroxide (TBA-OH) (manufactured by Aldrich) was added in an amount of about 1.5 times the molar equivalent of the cation exchange capacity of the resin, and the mixture was stirred for 30 minutes. The excess TBA-OH solution was removed by decantation, and the resin was further washed with excess ultrapure water to obtain a cation exchange resin converted into a TBA salt.

[0112] (2) Process 2-2 The raw material hyaluronic acid sodium salt (HA-Na) with a molecular weight of 10,000 (10k) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of the cation exchange resin converted to a TBA salt in "(1) Step 2-1" was added in an amount of 5 times the molar equivalent of the ion exchange capacity of the resin relative to the number of moles of HA units (unit molecular weight 401.3). After stirring for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid. The product 1 The H-NMR spectrum (ECS400 manufactured by JEOL, EtOH-d6) is shown in Figure 2.

[0113] 3.Process 3 An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in "2. (2) Step 2-2" was prepared. Then, Chol hydrochloride was added so that the molar ratio of the disaccharide repeating unit (HA unit) present in the HA-TBA synthesized in "1. Step 1" was 41 / 100. Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added so that the molar ratio of the HA unit was 49.2 / 100, and the mixture was stirred at room temperature overnight. The reaction solution was dialyzed (Spectrapore 7, molecular weight cutoff (MWCO): 2,000) against a 0.3 M ammonium acetate / DMSO solution, a 0.15 M NaCl aqueous solution, and ultrapure water in that order. Ethylene glycol (EG) was added to the obtained dialysate so that the content in the composition was 0.006 ppm by mass, and then the mixture was freeze-dried to obtain the target product (HA-C6-Chol) as a white solid. 1 The H-NMR spectrum is shown in Figure 3. A peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed.

[0114] [Examples 2 to 14 and Comparative Examples 1 to 2] (Production of hyaluronic acid derivative compositions HA-a2 to HA-a14 and HA-b1) 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, and the type and amount of organic solvent added were adjusted to those shown in Tables 1 and 2. Except for this, the same method as in Example 1 was used to obtain each hyaluronic acid derivative composition. 1 H-NMR measurements confirmed that in all hyaluronic acid derivatives contained in each hyaluronic acid derivative composition, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were observed.

[0115] In Tables 1 and 2, the types and abbreviations of the organic solvents (B) are as follows:

[0116] (Type and abbreviation of organic solvent) Isopropanol: IPA Ethanol: EtOH Dimethyl sulfoxide: DMSO Tetrahydrofuran: THF

[0117] The hyaluronic acid derivative compositions obtained in the Examples and Comparative Examples were measured for various properties and evaluated using the methods described above. The results are shown in Tables 1 and 2.

[0118] [Table 1]

[0119] [Table 2]

[0120] As shown in Tables 1 and 2, the hyaluronic acid derivative compositions HA-a1 to HA-a14 (Examples 1 to 14) containing an organic solvent had a good particle size of 50.1 nm or more and 484.4 nm or less. On the other hand, the hyaluronic acid derivative composition HA-b1 (Comparative Example 1) not containing an organic solvent had a poor particle size of 764.9 nm.

[0121] Furthermore, in the hyaluronic acid derivative compositions HA-a1 to HA-a4 (Examples 1 to 4) and HA-a8 to HA-a11 (Examples 8 to 11) containing different amounts of organic solvent, there was a tendency for the particle size to become smaller as the amount of organic solvent decreased. [Industrial Applicability]

[0122] According to the hyaluronic acid derivative composition of the present embodiment, it is possible to provide a hyaluronic acid derivative composition in which aggregation is suppressed more than conventional compositions.

Claims

1. (A) a hyaluronic acid derivative having a steryl group introduced therein; (B) one or more organic solvents selected from the group consisting of alcohols, sulfoxides, ketones, ethers, and esters; A pharmaceutical composition comprising a hyaluronic acid derivative composition as a carrier, comprising: (A) A pharmaceutical composition, in which the introduction rate of the steryl group into a hyaluronic acid derivative is 35% or more and less than 60%.

2. The pharmaceutical composition according to claim 1, wherein the content of the organic solvent (B) is 0.001 ppm by mass or more and less than 200,000 ppm by mass relative to the total mass of the hyaluronic acid derivative composition.

3. 3. The pharmaceutical composition according to claim 1, wherein the organic solvent (B) is one or more organic solvents selected from the group consisting of dimethyl sulfoxide, acetone, tetrahydrofuran, ethanol, isopropanol, ethylene glycol, and ethyl acetate.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the hyaluronic acid derivative (A) has a repeating unit represented by the following general formula (I): 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ , and R ４ Each independently represents a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X １ is the following formula: -NR ｂ -R、 -NR ｂ -COO-R、 -NR ｂ -CO-R、 -NR ｂ -C-NR ｃ -R、 -COO-R, -O-COO-R, -S-R, -CO-Y ａ -S-R、 -O-C-Y ｂ -S-R、 -NR ｂ -CO-Y ｂ -S-R, and -S-S-R, is a group selected from the group consisting of groups represented by R ａ , R ｂ and R ｃ Each independently represents a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR ｆ - may be inserted with a group selected from the group consisting of; R ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and Hydroxy C ２－１２ alkyl, the alkyl portion of which may have an inserted group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, where the alkylene is -O-, -NR ｇ - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, the alkyl portion of which may have an inserted group selected from the group consisting of -O- and -NH-; Y ａ is C １－５ is alkylene; Y ｂ is C ２－８ Alkylene or C ２－８ alkenylene; m is an integer of 1 or more and 100 or less.

5. The pharmaceutical composition according to claim 4, wherein R is a cholesteryl group.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the molecular weight of the hyaluronic acid derivative (A) is 1,000 or more and less than 1,000,000.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the drug forms a complex with the hyaluronic acid derivative (A).

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

9. A hyaluronic acid derivative-drug conjugate composition, in which one or more drugs are bound to the hyaluronic acid derivative (A) contained in the pharmaceutical composition according to any one of claims 1 to 8.

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