Hyaluronic acid derivative pharmaceutical composition and method for producing the pharmaceutical composition

Abstract

This hyaluronic acid derivative pharmaceutical composition comprises (A) a hyaluronic acid derivative to which a hydrophobic group is introduced, (B) an association accelerator, and (C) an active ingredient. It is preferable that the (B) association accelerator include at least four ether structures (R-O-R) and have four or more carbon atoms.

Description

[Technical Field]

[0001] This invention relates to a hyaluronic acid derivative pharmaceutical composition and a method for producing the pharmaceutical composition. This application claims priority based on Japanese Patent Application No. 2022-115839, filed in Japan on July 20, 2022, Japanese Patent Application No. 2022-151823, filed in Japan on September 22, 2022, and Japanese Patent Application No. 2022-115841, filed in Japan on July 20, 2022, and the contents thereof are incorporated herein by reference. [Background technology]

[0002] In recent years, biopharmaceuticals, which are medicines that use proteins, peptides, and nucleic acids as active ingredients, have been put into practical use, and their number continues to increase year by year. Biopharmaceuticals can meet unmet medical needs that could not be met by conventional small-molecule drugs.

[0003] However, biopharmaceuticals have challenges such as poor absorption from the gastrointestinal tract and mucous membranes, instability in the body, and a short half-life in the blood. Therefore, frequent administration by injection is necessary, which is burdensome for both patients and healthcare professionals. Thus, there is a need for drug substrates (sustained-release drug delivery system substrates) that can encapsulate biopharmaceuticals without compromising their pharmacological activity, allowing for gradual release of the active ingredient within the body.

[0004] Against this backdrop, Patent Document 1 proposes a sustained-release drug delivery system substrate made of a hyaluronic acid derivative with excellent safety. The hyaluronic acid derivative disclosed in Patent Document 1 spontaneously associates in aqueous solution and can efficiently encapsulate drugs, particularly biopharmaceuticals, while maintaining their biological activity. As a result, it aggregates (or disperses even at physiological salt concentrations) and has good blood retention properties. This hyaluronic acid derivative can be used as a carrier that can efficiently encapsulate many drugs while maintaining their pharmacological activity, especially when biopharmaceuticals are used as the active ingredient, as well as a blood-sustaining carrier and targeting carrier with excellent blood retention properties, and can also serve as a local (e.g., subcutaneous) sustained-release carrier that can continuously release drugs.

[0005] On the other hand, in the pharmaceutical industry, even if a drug active ingredient possesses strong biological activity, its low water solubility prevents it from exerting its effects. As a result, development is abandoned, or the product is marketed as a formulation that exhibits less activity than its original potential.

[0006] Methods for solubilizing pharmaceutical active ingredients that are insoluble or poorly soluble in water include the following methods (a) to (c).

[0007] (a) Method of creating soluble derivatives by altering part of the drug structure: Soluble derivatives such as hydrochloride, hydrobromide, sulfate, methanesulfonate, sodium salt, potassium salt, and sodium sulfonate.

[0008] (b) Methods using an organic solvent alone or a mixed solvent of an aqueous solvent and an organic solvent: Methods using methanol, ethanol, etc.

[0009] (c) Methods involving the addition of solubilizers: Methods involving the addition of surfactants to solubilize by micellization and emulsification. Methods using serum albumin or plasma proteins.

[0010] However, method (a) above involves altering a part of the structure of the active pharmaceutical ingredient (API) itself, and therefore cannot increase the solubility of the API itself. Furthermore, creating a derivative can lead to various problems, such as a decrease in pharmaceutical activity or drug precipitation due to changes in pH, making it an undesirable method.

[0011] The method using organic solvents such as methanol described in (b) above is not widely used in the pharmaceutical field because there are very few safe organic solvents that are biologically inert and do not cause hemolysis. For example, Patent Document 2 describes a manufacturing method for a poorly water-soluble cyclosporine composition, which involves dissolving it in an organic solvent, a solubilizing agent, or a mixed solvent of water and an organic solvent, or water and a solubilizing agent, or water, an organic solvent, and a solubilizing agent.

[0012] However, the resulting solution was turbid, and partial precipitation was observed, indicating that solubilization was not sufficient. This suggests that the poorly water-soluble active ingredient of the drug has precipitated, resulting in insufficient activity and failure to improve the toxicity to living organisms due to precipitation.

[0013] The method using the surfactant described in (c) above is problematic because very few surfactants are both safe for the body and exhibit effective solubility. While there have been conventional formulations of the drug Taxol dissolved using polyoxyethylated castor oil (Cremophor EL), Non-Patent Literature 1 reports that polyoxyethylated castor oil causes acute immunotoxicity, manifested as a hypersensitivity reaction (HSR) resulting from complement system activation, without involving IgE. Due to the scarcity of safe and useful surfactants, formulations exist in which paclitaxel is dissolved using toxic Cremophor.

[0014] Patent Document 3 describes the process of encapsulating poorly water-soluble active ingredients in hyaluronic acid derivatives, which requires dissolving the active ingredients in an organic solvent such as methanol. Therefore, the use of organic solvents is unavoidable. Furthermore, Patent Document 3 does not adequately solubilize the poorly water-soluble active ingredients. Additionally, there is no specific study on a formulation method for efficiently solubilizing poorly water-soluble active ingredients from a powder state, leaving room for improvement.

[0015] In Patent Document 2, etc., even when a solubilizing agent such as polyethylene glycol 300 is used for a poorly water-soluble active ingredient, only a suspension containing a relatively stable poorly water-soluble active ingredient is provided, and it is difficult to completely solubilize the poorly water-soluble active ingredient at a high concentration. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] International Publication No. 2010 / 053140 [Patent Document 2] U.S. Patent Application Publication No. 2020 / 0237859 [Patent Document 3] Special Publication No. 2014-534410 [Non-patent literature]

[0017] [Non-Patent Document 1] Toxicology 216 (2005) 106-121 [Overview of the Initiative] [Problems that the invention aims to solve]

[0018] Sustained-release drug substrates are required to maintain the concentration of the active ingredient in the body for an even longer period and to gradually release the active ingredient. In addition, they are required to be able to solubilize the active ingredient at high concentrations. The present invention has been made in view of the above circumstances, and aims to provide a hyaluronic acid derivative pharmaceutical composition and a method for producing the same that can maintain the concentration of the active ingredient in the body for a longer period of time and gradually release the active ingredient, and can solubilize the active ingredient at a high concentration. Furthermore, the present invention has been made in view of the above circumstances, and aims to provide a hyaluronic acid derivative pharmaceutical composition and a method for producing the same that can solubilize poorly water-soluble active ingredients at high concentrations without using organic solvents and while reducing the amount of highly toxic surfactants used. [Means for solving the problem]

[0019] In other words, the present invention includes the following embodiments. [1] A hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient. [2] The hyaluronic acid derivative pharmaceutical composition according to [1], wherein the association promoter (B) contains at least four ether structures (ROR) and has four or more carbon atoms. [3] The hyaluronic acid derivative pharmaceutical composition according to [1] or [2], wherein the (B) association promoter is one or more selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer, oxyethylene castor oil, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 4000, fatty acid sorbitan ester, tocopheryl polyethylene glycol succinate, and polyvinyl alcohol. [4] A hyaluronic acid derivative pharmaceutical composition according to any one of [1] to [3] that produces a precipitate at physiological salt concentration. [5] The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to [4], wherein the (C) active ingredient is at least one selected from proteins or poorly water-soluble drugs. [6] The hyaluronic acid derivative pharmaceutical composition according to [5], wherein the poorly water-soluble drug has a solubility in water of 1 mg / mL or less. [7] The hyaluronic acid derivative pharmaceutical composition according to [5] or [6], wherein the poorly water-soluble drug has a molecular weight of 200 or more. [8] The poorly water-soluble drug is a poorly water-soluble peptide. The hyaluronic acid derivative pharmaceutical composition according to any one of [5] to [7]. [9] In the poorly water-soluble peptide, at least one nitrogen atom constituting an amide bond has a methyl group. The hyaluronic acid derivative pharmaceutical composition according to [8].

[10] The poorly water-soluble peptide comprises at least one selected from the group consisting of cyclic peptides and long-chain peptides. The hyaluronic acid derivative pharmaceutical composition according to [8] or [9].

[11] The poorly water-soluble peptide is a cyclic peptide. The hyaluronic acid derivative pharmaceutical composition according to any one of [8] to

[10] .

[12] A content of the (C) active ingredient relative to 100 parts by mass of the (A) hyaluronic acid derivative introduced with a hydrophobic group is 10 parts by mass or more and 100 parts by mass or less. The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[11] .

[13] The (A) hyaluronic acid derivative introduced with a hydrophobic group has one or more repeating units represented by the following general formula (I). The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[12] .

Chemical Formula

[14] The hyaluronic acid derivative pharmaceutical composition according to

[13] , wherein the steryl group is a cholesteryl group.

[15] A hyaluronic acid derivative pharmaceutical composition according to any one of

[13] to

[14] , wherein the rate of introduction of the steryl group into the hyaluronic acid derivative is 7% or more and less than 35%.

[16] A hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[15] , wherein no precipitates are observed visually in the hyaluronic acid derivative pharmaceutical composition.

[17] A hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[16] that is filtration-sterilizable.

[18] A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient, comprising the steps of: mixing (A) the hyaluronic acid derivative into which a hydrophobic group has been introduced with (B) the association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter; and mixing (C) the active ingredient with the aqueous solution of the hyaluronic acid derivative containing the association promoter.

[19] A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient, comprising the steps of: dispersing the (C) active ingredient in the (B) association promoter to obtain a dispersion (I); preparing an aqueous solution of the hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing an association promoter to obtain an aqueous solution (II); and mixing the dispersion (I) and the aqueous solution (II).

[20] A method for producing a hyaluronic acid derivative pharmaceutical composition according to

[18] or

[19] , which does not involve a step of removing an organic solvent.

[0020] Furthermore, the present invention includes the following embodiments.

[21] A hyaluronic acid derivative pharmaceutical composition comprising (A1) a hyaluronic acid derivative into which hydrophobic groups have been introduced, (B1) a solubilizing agent, and (C1) an active ingredient, wherein the (B1) solubilizing agent contains at least four ether structures (RORs) and has four or more carbon atoms, and the content of the (B1) solubilizing agent is 0.0001 parts by mass or more and 15000 parts by mass or less per 100 parts by mass of the (A1) hyaluronic acid derivative into which hydrophobic groups have been introduced.

[22] The hyaluronic acid derivative pharmaceutical composition according to

[21] , wherein the (B1) solubilizing aid is one or more selected from the group consisting of a nonionic surfactant, polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less, and a cyclodextrin derivative.

[23] The hyaluronic acid derivative pharmaceutical composition according to

[21] or

[22] , wherein the (B1) solubilizing aid is one or more selected from the group consisting of polysorbate 80, polysorbate 65, polysorbate 60, polysorbate 40, polysorbate 20, poloxamer, polyoxyethylene hydrogenated castor oil, cyclodextrin derivatives, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 4000, and tocopheryl polyethylene glycol succinate.

[24] The hyaluronic acid derivative pharmaceutical composition according to any one of

[21] to

[23] , wherein the (B1) solubilizing aid is a nonionic surfactant, and the content of the nonionic surfactant with respect to 100 parts by mass of the (A) hydrophobic group-introduced hyaluronic acid derivative is 0.0001 parts by mass or more and 150 parts by mass or less.

[25] The hyaluronic acid derivative pharmaceutical composition according to any one of

[21] to

[24] , wherein the (B1) solubilizing agent is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less, and the content of the polyethylene glycol with respect to 100 parts by mass of the (A) hydrophobic group-introduced hyaluronic acid derivative is 25 parts by mass or more and 15000 parts by mass or less.

[26] The (C1) active ingredient is a poorly water-soluble drug having a solubility in water of 1 mg / mL or less, the hyaluronic acid derivative pharmaceutical composition according to any one of

[21] to

[25] .

[27] The hyaluronic acid derivative pharmaceutical composition according to

[26] , wherein the poorly water-soluble drug has a molecular weight of 500 or more.

[28] The hyaluronic acid derivative pharmaceutical composition according to

[26] or

[27] , wherein the poorly water-soluble drug is a poorly water-soluble peptide.

[29] The poorly water-soluble peptide is a hyaluronic acid derivative pharmaceutical composition according to

[28] , wherein at least one of the nitrogen atoms constituting the amide bond is a methyl group.

[30] The hyaluronic acid derivative pharmaceutical composition according to

[28] or

[29] , wherein the poorly water-soluble peptide comprises at least one selected from cyclic peptides and long-chain peptides.

[31] The poorly water-soluble peptide is a cyclic peptide, a hyaluronic acid derivative pharmaceutical composition according to any one of

[28] to

[30] .

[32] The hyaluronic acid derivative pharmaceutical composition according to any one of

[26] to

[31] , wherein the amount of the poorly water-soluble drug added to 100 parts by mass of the (A1) hydrophobic group-introduced hyaluronic acid derivative is 21 parts by mass or more and less than 100 parts by mass.

[33] The hyaluronic acid derivative pharmaceutical composition according to any one of

[31] to

[32] , wherein the hyaluronic acid derivative has one or more repeating units represented by the following general formula (I). [ka] (In the formula, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 Alkyl, formyl and C 1-6 This group is selected from the group consisting of alkylcarbonyl groups. Z represents a direct bond or a peptide linker consisting of any 2 to 30 amino acid residues. X 1 -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Ya -SR, -O-CO-Y b -SR, -NR b -CO-Y b It is a group selected from the group consisting of groups represented by -SR and -SSR. R a , R b and R c These are, independently, hydrogen atoms and C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. a , R b and R c The alkyl portion consists of -O- and -NR f A group selected from the group consisting of - may be inserted. R f C is a hydrogen atom. 1-12 Alkyl, amino C 2-12 Alkyl and hydroxy C 2-12 It is a group selected from the group consisting of alkyl groups. f The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a steryl group. Y is C 2-30 Alkylene, or -(CH2CH2O) m It is -CH2CH2-. Here, the alkylenes of Y are -O- and -NR g A group selected from the group consisting of - and -SS- may be inserted. R g C is a hydrogen atom. 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. g The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y a C 1-5 It is alkylene. Y b C 2-8 Alkylene or C 2-8It is alkenylene. m is an integer between 1 and 100 (inclusive).

[34] The hyaluronic acid derivative pharmaceutical composition according to

[33] , wherein the steryl group is a cholesteryl group.

[35] A hyaluronic acid derivative pharmaceutical composition according to any one of

[33] to

[34] , wherein the rate of introduction of the steryl group into the hyaluronic acid derivative is 35% or more and less than 50%.

[36] A hyaluronic acid derivative pharmaceutical composition according to any one of

[21] to

[35] , wherein the content of the hyaluronic acid derivative in the hyaluronic acid derivative pharmaceutical composition is 6 mg / mL or more and less than 45 mg / mL.

[37] The hyaluronic acid derivative pharmaceutical composition according to any one of

[21] to

[36] , wherein the content of the organic solvent in the hyaluronic acid derivative pharmaceutical composition is less than 0.8%.

[38] A method for producing a pharmaceutical composition comprising (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient, comprising the steps of: mixing (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced and (B1) a solubilizing agent to obtain an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent; and mixing (C1) an active ingredient with the aqueous solution of the hyaluronic acid derivative containing the solubilizing agent.

[39] A method for producing a pharmaceutical composition comprising (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient, comprising the steps of: dispersing the (C1) active ingredient in the (B1) solubilizing agent to obtain a dispersion (I); preparing an aqueous solution of the hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing a solubilizing agent to obtain an aqueous solution (II); and mixing the dispersion (I) and the aqueous solution (II).

[40] A method for producing a pharmaceutical composition according to

[38] or

[39] , comprising (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient, characterized in that it does not include a step of removing an organic solvent. [Effects of the Invention]

[0021] According to the hyaluronic acid derivative pharmaceutical composition of the above aspect, there can be provided a hyaluronic acid derivative pharmaceutical composition which can maintain the concentration of the active ingredient in vivo over a longer period of time, gradually release the active ingredient, and solubilize the active ingredient at a high concentration, and a method for producing the same.

[0022] According to the hyaluronic acid derivative pharmaceutical composition of the above aspect, there can be provided a hyaluronic acid derivative pharmaceutical composition which can solubilize a poorly water-soluble active ingredient at a high concentration without using an organic solvent and while reducing the usage amount of a highly toxic surfactant, and a method for producing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [Figure 1] It is a chromatogram of a hyaluronic acid derivative. [Figure 2] It is a chromatogram of a hyaluronic acid derivative pharmaceutical composition. [Figure 3] It is a graph showing the change in plasma concentration of cyclosporine. [Figure 4] It is a graph showing the change in plasma concentration of cyclosporine. MODE FOR CARRYING OUT THE INVENTION

[0024] Hereinafter, embodiments of the present invention (hereinafter referred to as "the present embodiment") will be described in detail, but the present invention is not limited thereto, and various modifications can be made without departing from the gist of the present invention.

[0025] Terms used in the present specification will be described below.

[0026] As used herein, "C 1-20 alkyl" means a linear or branched alkyl group having 1 to 20 carbon atoms, and examples thereof include "C 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, etc. C 1-20 alkyl includes C having 1 to 12 carbon atoms 1-12 alkyl, C having 1 to 6 carbon atoms 1-6 also includes alkyl groups.

[0027] As used herein, the term "C 1-6 alkylcarbonyl" refers to C wherein the alkyl moiety is the previously mentioned C 1-6 alkyl alkylcarbonyl group, and includes, for example, "C 1-4 alkylcarbonyl" such as acetyl, propionyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, iso-butylcarbonyl, tert-butylcarbonyl.

[0028] As used herein, "amino C 2-20 alkyl" refers to a linear or branched alkyl having 2 to 20 carbon atoms with an amino group as a substituent; for example, the amino group may be located on the terminal carbon atom of the alkyl group. Amino C 2-20 alkyl includes amino C having 2 to 12 carbon atoms 2-12 alkyl is also included.

[0029] As used herein, "hydroxy C 2-20 alkyl" refers to a linear or branched alkyl group having 2 to 20 carbon atoms with a hydroxy group as a substituent; for example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. Hydroxy C 2-20 alkyl includes hydroxy C having 2 to 12 carbon atoms 2-12 alkyl is also included.

[0030] As used herein, "C 2-30The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, including, for example, ethylene, propylene, etc., and having 2 to 20 carbon atoms. 2-20 Alkylene, C with 2 to 8 carbon atoms 2-8 Alkylene, base "-(CH2)" n - (where n is between 2 and 30, preferably between 2 and 20, and more preferably between 2 and 15).

[0031] The term "C" as used herein 1-5 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, including, for example, methylene, ethylene, and propylene.

[0032] The term "C" as used herein 2-8 "Alkenylene" refers to a divalent saturated hydrocarbon group having 2 to 8 carbon atoms, in a linear or branched chain, containing one or more double bonds. Examples include -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. If geometric isomerism exists, each isomer and mixtures thereof are also included.

[0033] <Hyaluronic acid derivative pharmaceutical composition 1> This embodiment is a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient. Hereafter, "(A) a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient" may be abbreviated as "pharmaceutical composition 1".

[0034] Hyaluronic acid derivatives can be formulated with active ingredients and used as pharmaceutical compositions. In the pharmaceutical composition 1 of this embodiment, the hyaluronic acid derivative forms a complex with the active ingredient (hereinafter sometimes referred to as the "active ingredient-hyaluronic acid derivative complex"). Specifically, the steryl group in the hyaluronic acid derivative and the hydrophobic moiety of the active ingredient form a complex through hydrophobic interaction, and it is presumed that the complex exhibits a cylindrical structure or a core-shell type cylindrical structure, with the active ingredient and hydrophobic moieties such as the steryl group located in the center, while hydrophilic moieties such as the hyaluronic acid-derived moiety in the hyaluronic acid derivative located at the outer edge. In other words, it is presumed that the active ingredient is encapsulated or contained within the hyaluronic acid derivative.

[0035] In the pharmaceutical composition 1 of this embodiment, the average particle size of the structure containing the active ingredient-hyaluronic acid derivative complex can be 20 nm to 220 nm, 20 nm to 150 nm, or 30 nm to 100 nm. Having the average particle size within the above numerical range allows for sterile filtration and stable aggregation and precipitation in the body, enabling the substance to exert its effect as a sustained-release substrate. The average particle size can be measured, for example, by DLS (Dynamic Light Scattering), nanotracking particle analyzers, size exclusion chromatography, high-performance liquid chromatography, and electron microscopy. More specifically, for example, it can be measured by diluting the hyaluronic acid derivative concentration at 1 mg / mL using a DLS device with 10 mM phosphate buffer containing 10 w / v% sucrose.

[0036] The (B) association promoter in the pharmaceutical composition 1 of this embodiment preferably contains at least four ether structures (RORs) and has four or more carbon atoms.

[0037] The pharmaceutical composition 1 of this embodiment contains (B) an association promoter. In this specification, (B) association promoters interact with hyaluronic acid derivatives to which hydrophobic groups have been introduced, thereby promoting the association of hyaluronic acid derivative molecules with each other. The association promoters of this embodiment form a complex with hyaluronic acid derivatives, enhancing drug complexing ability and improving precipitation performance under physiological salt concentrations. After precipitation subcutaneously, the drug in the gel is released slowly through an exchange reaction in which it is replaced by hydrophobic components such as albumin and other hydrophobic proteins in the body, or through the degradation of the gel. It is believed that the sustained release period can be controlled by controlling the sustained release of the drug through the exchange reaction, and by suppressing, shielding, and providing stealth to the influx of gel-degrading enzymes.

[0038] Next, the components of this embodiment will be described in detail below.

[0039] ≪Hyaluronic acid derivatives with added hydrophobic groups≫ The pharmaceutical composition 1 of this embodiment contains (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced. Hereafter, "(A) a hyaluronic acid derivative into which a hydrophobic group has been introduced" may be referred to as "(A) a hyaluronic acid derivative".

[0040] (A) The hyaluronic acid derivative has a steryl group as a hydrophobic group. The steryl group may be directly bonded to the hyaluronic acid or bonded via a linker. The term "linker" as used herein refers to any peptide linker or synthetic compound linker that can be introduced by genetic engineering, but in the case of the (A) hyaluronic acid derivative used in this embodiment, a peptide linker is preferred.

[0041] The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose, but a preferred length is 2 amino acids or more, and particularly preferably 15 amino acids. The upper limit of the peptide linker length is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less. (A) Peptide linkers contained in the hyaluronic acid derivative may all be of the same length, or peptide linkers of different lengths may be used.

[0042] (A) When a hyaluronic acid derivative has a steryl group as a hydrophobic group, the steryl groups in the hyaluronic acid derivative self-associate in water, and single or multiple molecules associate to form a nano-sized hydrogel.

[0043] [Steryl group] The term "steryl group" as used herein is not particularly limited as long as it is a group having a steroid skeleton. Specifically, examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmamanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simialenol, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycortisterone, etc. Examples of steryl groups include cholesteryl group, stigmasteryl group, lanosteryl group, ergosteryl group, etc., with cholesteryl group (especially cholesta-5-en-3β-yl group) being preferred.

[0044] [Steryl group introduction rate] (A) The rate of steryl group introduction into the hyaluronic acid derivative (hereinafter sometimes simply referred to as "steryl group introduction rate") is preferably 0.1% or more and less than 50%, more preferably 5% or more and less than 45%, even more preferably 10% or more and 40%, and particularly preferably 15% or more and 35%.

[0045] By keeping the steryl group introduction rate within the above range, the (A) hyaluronic acid derivative can strongly interact with poorly water-soluble drugs, the hydrophobic parts of proteins, and the hydrophobic parts of association promoters. Furthermore, by keeping the steryl group introduction rate within the above range, the hyaluronic acid derivative-drug complex, in which the (A) hyaluronic acid derivative in pharmaceutical composition 1 is compounded with a drug, exhibits improved formulation stability and enables aggregation and precipitation under physiological salt concentrations, allowing for sustained drug release.

[0046] The rate of steryl group introduction is 1It can be measured by 1H-NMR measurement. That is, the pharmaceutical composition 1 1 The integral value of the peak originating from the steryl group of (A) hyaluronic acid derivative in the 1H-NMR spectrum and the integral value of the peak originating from the acetyl group of N-acetyl-D-glucosamine contained in (A) hyaluronic acid derivative (COCH3, 1.6 ppm to 2.0 ppm, 3H) can be used to calculate the following based on the formula below. Note that in the formula, n H This represents the number of hydrogen atoms corresponding to the peak. Specifically, it can be measured according to the method described in the examples below.

[0047] [Steryl group introduction rate] (%) =[(Peak integral value derived from steryl group × 3 / n H ) / (Peak integral value derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0048] (A) The molecular weight of the hyaluronic acid derivative is not particularly limited, but from the viewpoint of improving the sustained-release function due to delayed diffusion in local administration, a hyaluronic acid derivative with a relatively large molecular weight is preferred. On the other hand, if the final dosage form is a solution formulation, a hyaluronic acid derivative with a relatively small molecular weight is preferred from the viewpoint of syringeability. The molecular weight of the hyaluronic acid derivative can be adjusted as appropriate depending on the application and dosage form.

[0049] (A) As an example of the molecular weight of the hyaluronic acid derivative, it is preferably 1,000 (1k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 300k or less, even more preferably 5k or more and 120k or less, and particularly preferably 7k or more and 100k or less. (A) The molecular weight of the hyaluronic acid derivative can generally be adjusted by using a raw material having the corresponding molecular weight.

[0050] By ensuring that the weight-average molecular weight of the hyaluronic acid derivative is above the above lower limit, the increase in viscosity can be suppressed, and a higher concentration of the hyaluronic acid derivative can be dissolved in the pharmaceutical composition 1. The weight-average molecular weight of the hyaluronic acid derivative can generally be adjusted by using a raw material having the corresponding molecular weight. More specifically, the weight-average molecular weight of the hyaluronic acid derivative is preferably 100k or less, more preferably 50k or less, and particularly preferably 40k or less, from the viewpoint of viscosity. From the standpoint of producing hyaluronic acid derivatives, a pH of 4k to 100k is preferred, and a pH of 6k to 50k is particularly preferred. From the perspective of hyaluronic acid derivatives strongly exhibiting the properties of hyaluronic acid, for example, in order to be strongly recognized by the CD44 receptor, a molecular weight of 100 kDa or higher is preferable, 200 kDa or higher is particularly preferable, and 300 kDa or higher is most preferable.

[0051] In this context, the "molecular weight of the hyaluronic acid derivative" refers to the weight-average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS).

[0052] Preferred hyaluronic acid derivatives include, for example, hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter sometimes referred to as "repeating unit (I)").

[0053] [ka]

[0054] (In the formula, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 Alkyl, formyl and C 1-6 It is a group selected from the group consisting of alkylcarbonyl groups. Z represents a direct linker or a peptide linker consisting of any 2 to 30 amino acid residues. X1 -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 It is a group selected from the group consisting of groups represented by -SR and -SSR. R a , R b and R c These are, independently, hydrogen atoms and C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. Here, R a , R b and R c The alkyl portion consists of -O- and -NR f A group selected from the group consisting of - may be inserted. R f C is a hydrogen atom. 1-12 Alkyl, amino C 2-12 Alkyl and hydroxy C 2-12 It is a group selected from the group consisting of alkyl groups. f The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a steryl group. Y is C 2-30 Alkylene, or -(CH2CH2O) m It is -CH2CH2-. Here, the alkylenes of Y are -O- and -NR g A group selected from the group consisting of - and -SS- may be inserted. R g C is a hydrogen atom. 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. gThe alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y a C 1-5 It is alkylene. Y b C 2-8 Alkylene or C 2-8 It is alkenylene. m is an integer between 1 and 100 (inclusive).

[0055] Hyaluronic acid derivatives preferably include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (Ia) (hereinafter sometimes referred to as "repeating unit (Ia)").

[0056] [ka]

[0057] (In the formula, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 Alkyl, formyl and C 1-6 It is a group selected from the group consisting of alkylcarbonyl groups. X is -NR a -Y-NR b R is a hydrophobic group represented by -COO-R. a and R b These are, independently, hydrogen atoms and C 1-6 It is a group selected from the group consisting of alkyl groups. R is a steryl group. Y is C 2-30 Alkylene, or -(CH2CH2O) m The equation is -CH2CH2-, where m is an integer between 1 and 100 (inclusive).

[0058] Here, if the hyaluronic acid derivative contains two or more repeating units (I) or repeating units (Ia), these repeating units may be the same or different.

[0059] Hyaluronic acid derivatives may be modified at positions other than the repeating unit (I) or repeating unit (Ia), for example, the hydroxyl group may be -O(C 1-6 Alkyl), -O (formyl), -O (C 1-6 The carboxyl group may be converted to an alkylcarbonyl group, and the carboxyl group may be converted to an amide or ester, and a salt may be formed.

[0060] [Repeating Unit (I)] The base "-Zn(R)" in general formula (I) a )YX 1 The formula is as follows: -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(wherein R is a hydrogen atom or a methyl group, and R and m are as already defined herein.) It includes a group selected from the group consisting of groups represented by .

[0061] The group in question is -NH-(CH2) mz -NH-COO-R;-NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz A group selected from the group consisting of -SSR (where mz, R, and m are as already defined herein) is preferred.

[0062] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 -NR b It is preferable that -COO-R. Furthermore, in another embodiment, Z is -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a It may also be a peptide linker represented by )-CO-, where n is an integer between 2 and 30, and Z a Each of these is independently H2N-CH(-Z a This represents a substituent in an α-amino acid, represented as -COOH. The peptide linker is bonded to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R) at the C-terminus. a )-YX 1 It binds to. Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as their D forms, and all α-amino acids, including synthesized amino acids, can be used. That is, Z aExamples of these include -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, etc. Also, the n Zs may be the same or different. n is an integer between 2 and 30, but is preferably between 2 and 10, and more preferably between 2 and 4. Preferred examples of peptide linkers include, for example, -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, etc.

[0063] (Y) In general formula (I), Y is -(CH2) n1 -and-(CH2CH2O) m1 A base selected from the group consisting of -CH2CH2- (where n1 is an integer between 2 and 20, preferably between 2 and 15, more preferably between 2 and 12, and even more preferably between 2 and 6; m1 is an integer between 1 and 4) is preferred. Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12 -, or -(CH2CH2O)2-CH2CH2- is preferred. Furthermore, from the viewpoint of achieving high solubility in pure water or low salt concentrations while exhibiting high precipitation ability under physiological salt concentrations, Y is preferably -(CH2)2-, -(CH2)6-, -(CH2)8- and -(CH2) 12 A group selected from the group consisting of - is preferred, and -(CH2)6- is more preferred.

[0064] Y can be, for example, -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, etc.

[0065] (Y a ) Y a -CH2- or -CH2-CH2- is preferred.

[0066] (Y b ) Y b The preferred members are -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, with -CH2-CH2- or -CH(CH3)CH2- being more preferred.

[0067] Base "-ZN(R a )YX 1 Specific examples of "" include -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-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C Examples include 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, etc. Preferred group "-Zn(R a )YX 1 " is R a , R b and R c However, it is a hydrogen atom, and Y is a linear C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2- and Y a However, linear C 1-5 It is alkylene, or Y b However, linear C 2-8 Alkylene or linear carbon 2-8 It is alkenylene.

[0068] [Repeating Unit (Ia)] In 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.

[0069] (A) In addition to the repeating unit (I), the hyaluronic acid derivative may further contain a repeating unit represented by general formula (II) (hereinafter sometimes referred to as "repeating unit (II)").

[0070] [ka]

[0071] (In the formula, R 1a , R 2a , R 3a , and R 4a These are, independently, hydrogen atoms and C 1-6 Alkyl, formyl and C 1-6 It is a group selected from the group consisting of alkylcarbonyl groups. a hydroxy and -OQ + It is a group selected from the group consisting of Q. + (This is a counter-cation.)

[0072] Here, if (A) the hyaluronic acid derivative contains two or more repeating units (II), these repeating units may be the same or different. In another embodiment, (A) the hyaluronic acid derivative may be a hyaluronic acid derivative substantially comprising repeating unit (I), repeating unit (Ia), and repeating unit (II).

[0073] [Repeating Unit (II)] In general formula (II), Q + The countercation is not particularly limited as long as it forms a salt with a carboxyl group in water, and in the case of a valency of 2 or higher, it forms a salt with multiple carboxyl groups depending on the valency. Examples of countercations include metal ions such as lithium ions, sodium ions, rubidium ions, cesium ions, magnesium ions, and calcium ions; formula: N + R j R k R l R m (In the formula, R j , R k , R l and R m These are, independently, hydrogen atoms and C 1-6 Examples include ammonium ions (selected from the group consisting of alkyl groups). Among them, Q + The preferred ions are sodium ions, potassium ions, or tetraalkylammonium ions (for example, tetra-n-butylammonium ions). j , R k , R l and R m C 1-6 It is preferable that the group be the same group selected from the group consisting of alkyl groups, and an n-butyl group is preferred.

[0074] R 1 , R 2 , R 3 , and R 4 , and R 1a , R 2a , R 3a , and R 4a It is preferable that all of them are hydrogen atoms. Also, R a and R b Preferably, all of these are hydrogen atoms.

[0075] In particular, (A) the hyaluronic acid derivative is preferably a hyaluronic acid derivative substantially composed of repeating unit (I) and repeating unit (II). In (A) the hyaluronic acid derivative, of the repeating units of the disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative, for example, 80% or more, preferably 90% or more, and more preferably 95% or more are repeating units (I) and repeating unit (II). (A) the hyaluronic acid derivative may consist only of repeating unit (I) and repeating unit (II).

[0076] The content of (A) hyaluronic acid derivative relative to the total amount of the pharmaceutical composition is preferably 1 mg / mL or more and less than 50 mg / mL, more preferably 3 mg / mL or more and 45 mg / mL or less, and even more preferably 5 mg / mL or more and 40 mg / mL or less.

[0077] (A) A method for producing hyaluronic acid derivatives will be described later.

[0078] (B) Association promoter In the present invention, the association promoter can be measured more specifically by gel permeation chromatography, and it can be confirmed that it is an association promoter as defined in this embodiment by the ratio of areas A1 to A2, A2 / A1, shown below.

[0079] Figure 1 is a chromatogram of a hyaluronic acid derivative. An example of area A1 shown in Figure 1 is 1814. Figure 2 is a chromatogram of hyaluronic acid derivative pharmaceutical composition 1. An example of area A2 shown in Figure 2 is 2813.

[0080] Figure 2 shows the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment. Here, the refractive index intensity at the start of measurement is set to zero, and the line drawn horizontally from this point is taken as the baseline. For example, before starting the measurement, the refractive index intensity is adjusted so that the increase or decrease is within ±0.5 mV, and the refractive index intensity is adjusted so that the increase or decrease over 5 minutes is 0.5 mV or less. For example, the first point where the increase in refractive index intensity exceeds an amount equivalent to 5 times the noise value three times is taken as the "starting point" of the chromatogram, and the elution time is set to 0 minutes.

[0081] For example, the point where the refractive index intensity becomes 1 / 1000th of the maximum refractive index intensity is defined as the "endpoint" of the chromatogram. If the refractive index intensity does not reach 1 / 1000th of the maximum refractive index intensity, "Tlim" is defined as the "endpoint". "Tlim" is defined as the elution time at which the maximum refractive index intensity is observed when measuring 2 kDa polyacrylic acid.

[0082] In this device, the refractive index intensity is calculated every 0.00167 minutes. Here, each area value is calculated using the analysis application on the GPC workstation EcoSEC Elite-WS.

[0083] When calculating each area value, peaks caused by the developing solvent used in gel permeation chromatography, as well as false peaks caused by baseline fluctuations due to the column or instrument used, are excluded.

[0084] A ratio of area A2 to area A1, A2 / A1, of 1.2 or higher indicates that association between hyaluronic acid derivative molecules or between hyaluronic acid derivatives and association promoters is being promoted. If association between hyaluronic acid derivative molecules or between hyaluronic acid derivatives and association promoters is not being promoted, the ratio A2 / A1 will be in the range of 0.95 to 1.19.

[0085] The ratio A2 / A1 is preferably 1.20 or higher, more preferably 1.30 or higher, even more preferably 1.40 or higher, and particularly preferably 1.50 or higher. When the ratio of area A2 to A1, A2 / A1, is above the lower limit, a relatively large amount of the association-promoted hyaluronic acid derivative can be contained, and when formulated with (C) the active ingredient, a large amount of (C) the active ingredient can be retained, and (C) the active ingredient can be solubilized at a high concentration. In addition, (B) the association promoter controls the release of (C) the active ingredient, and the sustained-release period of the drug in the body can be maintained over a long period.

[0086] On the other hand, the upper limit of A2 / A1 is preferable as the ratio of area A2 to area A1 is higher. Therefore, the upper limit of A2 / A1 is not particularly limited, but may be 4.0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.1 or less, 2.0 or less, 1.9 or less, or 1.5 or less.

[0087] (B) The association promoter is not particularly limited as long as it is a surfactant that can be commonly used in pharmaceutical applications. (B) The association promoter is preferably a component that contains at least four ether structures (RORs) and has four or more carbon atoms.

[0088] (B) Examples of association promoters include polysorbates (having 5 or more ether structures and 10 or more carbon atoms), polyoxyethylene fatty acid esters, sorbitan fatty acid esters, and polyoxyethylene castor oil. (B) Examples of association promoters include polysorbate 80 (having 20 or more ether structures and 64 or more carbon atoms), polysorbate 65 (having 20 or more ether structures and 100 or more carbon atoms), polysorbate 60 (having 20 or more ether structures and 64 or more carbon atoms), polysorbate 40 (having 20 or more ether structures and 62 or more carbon atoms), polysorbate 20 (having 20 or more ether structures and 57 or more carbon atoms), polyethylene glycerides, etc. Examples include chol monolaurate (having 7 or more ether structures and 28 or more carbon atoms), polyoxyl stearate 40 (having 39 or more ether structures and 98 or more carbon atoms), polyoxyl stearate 45 (having 44 or more ether structures and 108 or more carbon atoms), polyoxyl stearate 55 (having 54 or more ether structures and 128 or more carbon atoms), and cremophor EL (having 35 or more ether structures and 127 or more carbon atoms). Among these, polysorbate 20, polysorbate 80, and polyexyethylene castor oil are particularly preferred.

[0089] (B) The association promoter is not limited to the surfactants mentioned above, but includes, for example, poloxamer 188 (having 98 or more ether structures and 225 or more carbon atoms), poloxamer 124 (having 26 or more ether structures and 74 or more carbon atoms), poloxamer 237 (having 93 or more ether structures and 225 or more carbon atoms), poloxamer 338 (having 177 or more ether structures and 400 or more carbon atoms), poloxamer 407 (having 147 or more ether structures and 352 or more carbon atoms), polyethylene glycol 300 (having 5 or more ether structures and 12 or more carbon atoms), polyethylene glycol 400 (having 7 or more ether structures and 16 or more carbon atoms), polyethylene glycol 4000 (having 59 or more ether structures and 120 or more carbon atoms), polyvinyl alcohol (average degree of polymerization: 500, having 4 or more ether structures and 4 or more carbon atoms), etc.

[0090] The amount of (B) association promoter added to pharmaceutical composition 1 is preferably 0.001 parts by mass or more and 15,000 parts by mass or less, more preferably 0.05 parts by mass or more and 5,000 parts by mass or less, and even more preferably 1 part by mass or more and 4,000 parts by mass or less, per 100 parts by mass of hyaluronic acid derivative.

[0091] (B) When the association promoter is at least one selected from the group of surfactants, the amount of association promoter (B) added to the pharmaceutical composition 1 is preferably 0.01 parts by mass or more and 150 parts by mass or less, and more preferably 0.05 parts by mass or more and 100 parts by mass or less, per 100 parts by mass of the hyaluronic acid derivative.

[0092] (B) If the association promoter is at least one selected from the group other than the surfactant, the amount of association promoter (B) added to the pharmaceutical composition 1 is preferably 10 parts by mass or more and 15,000 parts by mass or less, more preferably 100 parts by mass or more and 10,000 parts by mass or less, even more preferably 1,000 parts by mass or more and 5,000 parts by mass or less, particularly preferably 1,500 parts by mass or more and 4,000 parts by mass or less, and most preferably 2,000 parts by mass or more and 3,500 parts by mass or less, per 100 parts by mass of the hyaluronic acid derivative.

[0093] (B) The less association-promoting agent there is, the better. However, if it is below the lower limit, the active ingredient cannot be sufficiently solubilized. If it exceeds the upper limit, the toxicity tends to increase due to too much (B) solubilizing aid, and the viscosity of the formulation also increases, so it is preferable to keep it within the above range.

[0094] (B) There are no particular restrictions on the means of dissolving the association promoter, and examples include stirring, shaking, and ultrasonic treatment.

[0095] The pharmaceutical composition 1 of this embodiment, by containing (B) an association promoter, can retain a large amount of the active ingredient, and furthermore, by preventing the inflow of hydrophobic biocomponents into the hyaluronic acid derivative composition that has gelled subcutaneously, the release rate of the active ingredient can be controlled, and the active ingredient can be released slowly over a long period of time. It should be noted that the desired effect may be obtained by a mechanism different from the above mechanism.

[0096] (C) Active Ingredients The active ingredients are not particularly limited, but include pharmaceutically active peptides or proteins, nucleic acids, low-molecular-weight compounds, medium-molecular-weight compounds, antigens (cancer antigens, infectious disease-derived antigens, autoantigens in immune diseases, etc.). Among these, low-molecular-weight compounds, medium-molecular-weight compounds, and peptides are more preferred.

[0097] The diseases to which the pharmaceutical composition 1 of this embodiment can be applied are not particularly limited, and it can be widely used for the prevention or treatment of diseases currently known or diseases that may be discovered in the future. These may be chronic or acute diseases. Examples of diseases currently known include cancer, infectious diseases, immune diseases, inflammatory bowel disorders, allergic diseases, skin diseases, hypertension, diabetes, neurological diseases, genetic diseases, cardiovascular diseases, cerebrovascular diseases, respiratory diseases, eye diseases, ear diseases, bone and joint diseases, and pain disorders.

[0098] [Medicinal active peptides or proteins] A pharmaceutically active peptide or protein means a substance that, when administered in a therapeutically effective dose to a subject, has a positive or beneficial effect on the subject's condition or disease. Preferred pharmaceutically active peptides or proteins are those that have curative or symptomatic properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Pharmacoactive peptides or proteins may also have prophylactic properties and can be used to delay the onset of a disease or to reduce the severity of such a disease or condition. The term "pharmaceutically active peptide or protein" includes full-length proteins or polypeptides, and may also refer to pharmaceutically active fragments thereof. This term also includes pharmaceutically active analogs of peptides or proteins.

[0099] Examples of pharmacoactive proteins include, but are not limited to, cytokines and immune system proteins such as immunoactive compounds (e.g., interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factors (G-CSF), granulocyte-macrophage colony-stimulating factors (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, adresin, seletin, homing receptors, T cell receptors, immunoglobulins, antibodies, hormones (insulin, thyroid hormones, catecholamines, gonadotropins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic enzymes or degrading enzymes, steroid-producing enzymes, etc.) Examples include kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylyl cyclase or guanylate cyclase, neuramidases, etc., receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone-binding proteins or growth factor-binding proteins, etc.), transcription factors and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin and myosin, etc.), and blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, modified factor VIII, anticoagulant factors).

[0100] [Nucleic acid] Nucleic acids include DNA and RNA, and include, for example, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and nucleic acid aptamers.

[0101] [Low molecular compounds] Examples of low molecular weight compounds include compounds with a molecular weight of less than approximately 500, such as anticancer agents (e.g., alkylating agents, antimetabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroids, nonsteroidal anti-inflammatory drugs, etc.), antirheumatic agents, and antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, novel quinolone antibiotics, sulfonamides, etc.).

[0102] The active ingredient may be a Rho kinase inhibitor, an endothelin A receptor inhibitor, a transmembrane conductance regulator (CFTR) modulator, a TRPV1 inhibitor, an NK1 receptor inhibitor, a purine receptor inhibitor, angiotensin receptor inhibitor, a peroxisome proliferator-responsive receptor, a P2Y receptor inhibitor, a VEGF inhibitor, or an active ingredient that simultaneously inhibits two of these.

[0103] Furthermore, as an active ingredient, highly hydrophobic, i.e., poorly water-soluble, hyaluronic acid derivatives can be preferably used because they can fully exhibit interaction with the steryl group of the hyaluronic acid derivatives mentioned above. Note that "poorly water-soluble" in the 17th edition of the Japanese Pharmacopoeia refers to a substance that requires 30 mL or more of water to dissolve 1 g of solute.

[0104] Examples of poorly water-soluble, solid active ingredients include acetaminophen, ibuprofen, benzoic acid, ethenzamide, caffeine, camphor, quinine, calcium gluconate, dimethylcaprol, sulfamine, theophylline, theobromine, riboflavin, mephenesin, phenobervital, aminophylline, thioacetazone, quercetin, rutin, salicylic acid, sodium theophylline, pyrapital, quinine hydrochloride, irgapyrin, digitoxin, griseofulvin, phenacetin, and other antipyretic analgesics, nervous system drugs, sedatives and hypnotics, muscle relaxants, antihypertensives, antihistamines, etc.; acetylspiramycin, ampicillin, erythromycin, xatamycin, chloramphenicol, triacetate Examples of active pharmaceutical ingredients listed in the Japanese Pharmacopoeia, including antibiotics such as tiloleandmycin, nystatin, and colistin sulfate; steroid hormones such as methyltestosterone, methylandrostethrondiol, progesterone, estradiol benzoate, ethinirestradiol, deoxycorticosterone acetate, cortisone acetate, hydrocortisone, hydrocortisone acetate, and prednisolone; nonsteroidal yolk hormones such as dienstrol, hexasastrol, diethylstilbesterol, diethylstilbesterol dibrohyonate, and chlorotrianicene; and other fat-soluble vitamins. One of these active ingredients may be used, or two or more may be used in combination.

[0105] The active ingredient may be a poorly water-soluble oil or liquid. Examples of poorly water-soluble oil or liquid active ingredients include vitamins such as teprenone, indomethacin farnesyl, menatetrenone, phytonadione, vitamin A oil, phenipentol, vitamin D, and vitamin E; higher unsaturated fatty acids such as DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), and liver oil; coenzyme Q; and oil-soluble flavorings such as orange oil, lemon oil, and peppermint oil, as listed in the Japanese Pharmacopoeia,

[0106] The active ingredient may be a semi-solid active ingredient that is poorly water-soluble. Examples of semi-solid active ingredients that are poorly water-soluble include earthworm, licorice, cinnamon, peony, peony bark, valerian, Japanese pepper, ginger, citrus peel, ephedra, nandina fruit, oyster shell, fennel, phellodendron bark, balloon flower, plantago radish, plantago japonica, garlic, seneca, fritillary, fennel, phellodendron bark, cow's roe, turmeric, chamomile, gentian, ox gall, animal bile, ginseng, ginger, lanceolata, clove, citrus peel, atractylodes, ginseng, ginseng, kakkonto, keishito, kososan, shihokeitoshito, shoshihoto, shoseiryuto, maimondoto, hanato, hanago, ginseng, kakkonto, keishito, kososan, shihokeitoshito, shoshihoto, shoseiryuto, maimondoto, hanago, hanago, maoto, etc., as well as herbal medicines or herbal extracts; oyster meat extract, propolis and propolis extract, coenzyme Q, etc. You may use one of these active ingredients, or you may use two or more in combination.

[0107] [Medium molecule compounds] In this specification, "medium-sized molecules" refer to peptides, macrolide compounds, nucleic acids, natural products, or derivatives thereof with a molecular weight of approximately 500 to 5000, which are neither low-molecular-weight (organic compounds with a molecular weight of up to approximately 500) nor high-molecular-weight (proteins with a molecular weight of 10,000 or more). Preferably, peptides with a molecular weight of approximately 500 to 2000, i.e., linear or cyclic peptides with approximately 5 to 20 amino acid residues. Cyclic peptides are preferred as peptides, and their details will be described later. Macrolide compounds are macrocyclic lactones, and are a general term for compounds with 12 or more members in the ring. Examples of medium-sized molecules include FK506 and rapamycin.

[0108] [antigen] (Cancer antigen) Cancer antigens are antigens that are expressed in large quantities on cancer cells, and in some cases, only on cancer cells. Cancer antigens can be expressed inside cancer cells or on the surface of cancer cells.

[0109] The antigen proteins that can be used in the pharmaceutical composition 1 of this embodiment are not limited to ERK1, ERK2, WT1, MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophyllin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, and prostate-specific membrane antigen (PSM). A), T cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE- 1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, large Examples include enteric adenomatous neoplasm (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, cerebral glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, etc.

[0110] The antigen protein described above may be used in its entirety, or it may be a sequence with a portion of it deleted.

[0111] The antigen peptide that can be used in the pharmaceutical composition 1 of this embodiment is an antigen peptide that contains one or more epitopes selected from the group consisting of CD8-positive cytotoxic T cell recognition epitopes and CD4-positive helper T cell recognition epitopes from the sequence of an antigen protein. In one embodiment, from the viewpoint of being loaded onto an MHC class I molecule or an MHC class II molecule after degradation by an antigen-presenting cell, the antigen peptide is preferably an antigen peptide that contains two or more epitopes. Specifically, the antigen peptide is an antigen peptide that contains an epitope of the antigen protein of a tumor cell.

[0112] In one embodiment, the antigen peptide has, for example, 8 to 120 amino acids, preferably 8 to 80 amino acids, more preferably 15 to 80 amino acids, even more preferably 16 to 80 amino acids, even more preferably 23 to 80 amino acids, even more preferably 23 to 60 amino acids, and particularly preferably 23 to 50 amino acids.

[0113] In one embodiment, from the viewpoint of inducing the activation of cytotoxic T cells (CTLs) by helper T cells, the antigen peptide is an antigen peptide containing one or more CD8-positive cytotoxic T cell-recognizing epitopes and one or more CD4-positive helper T cell-recognizing epitopes.

[0114] In one embodiment, if there are two or more epitopes, amino acid linkers may be placed between the epitopes. The linker has, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, and more preferably 4 to 8 amino acids. Examples of amino acids used for the linker include glycine (G), tyrosine (Y), leucine (L), and tryptophan (W). Preferably, tyrosine (Y), leucine (L), and tryptophan (W). Specific examples of amino acid linkers include a linker consisting of four consecutive tyrosine (Y) molecules (4Y), a linker consisting of four consecutive leucine (L) molecules (4L), a linker consisting of four consecutive tryptophan (W) molecules (4W), a linker consisting of six consecutive glycine (G) molecules (6G), a linker consisting of six consecutive tyrosine (Y) molecules (6Y), a linker consisting of six consecutive leucine (L) molecules (6L), a linker consisting of six consecutive tryptophan (W) molecules (6W), a linker consisting of eight consecutive tyrosine (Y) molecules (8Y), a linker consisting of six consecutive leucine (L) molecules (8L), and a linker consisting of eight consecutive tryptophan (W) molecules (8W), with 6Y, 6L, or 6W being preferred.

[0115] (Infection-derived antigens) Infectious disease-derived antigens are not particularly limited as long as they are infectious pathogens or antigens derived from infectious pathogens. Examples of infectious pathogens include viruses, bacteria, fungi, and nematodes. Infectious disease-derived antigens may be either antigenic proteins or antigenic peptides.

[0116] The diseases caused by the infectious pathogens listed above are not particularly limited and include, for example, adenoviruses, herpesviruses (e.g., HSV-I, HSV-II, CMV, VZV), poxviruses (e.g., orthopoxviruses such as smallpox or vaccinia, molluscum contagiosum), picornaviruses (e.g., rhinovirus, enterovirus), orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)), coronaviruses (e.g., SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS-CoV-2), papovaviruses (e.g., papillomaviruses such as those causing genital warts, common cysts, and plantar warts), hepadnaviruses (e.g., hepatitis B virus), and flaviviruses (e.g., hepatitis C virus, dengue virus). Viral diseases such as diseases caused by viral infections, including retroviruses (e.g., lentiviruses like HIV); Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, HIV Bacterial diseases such as those caused by bacterial infections including Lio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, and Bordetella; fungal diseases including, but not limited to, Chlamydia, candidiasis, aspergillosis, histoplasmosis, and cryptococcal meningitis; and malaria, Pneumocystis carinii pneumonia, leshmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis infections.

[0117] The structure of an antigen that can be used in the pharmaceutical composition 1 of the present embodiment is not particularly limited as long as it is at least a part of various components constituting a pathogen, and examples thereof include live vaccines, inactivated whole particles, parts thereof, protein subunits, proteins, peptides, and the like. Among these, protein subunits, proteins, or peptides are preferable from the viewpoint of complexation with a hyaluronic acid derivative.

[0118] Here, the influenza virus described above is an RNA enveloped virus belonging to the Orthomyxoviridae family having a particle size of about 100 nm in diameter, and is classified into types A, B and C based on the antigenicity of internal proteins. The influenza virus consists of a ribonucleic acid (RNA) core associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope having a lipid bilayer structure, and an external glycoprotein. The inner layer of the viral envelope is mainly composed of matrix proteins, and the outer layer is mostly composed of host-derived lipid substances. Further, the RNA of the influenza virus has a segmented structure. Influenza that causes pandemics worldwide is caused by influenza A virus, which has two types of envelope glycoproteins: hemagglutinin (HA) and neuraminidase (NA), and is divided into 16 subtypes for HA and 9 subtypes for NA based on differences in antigenicity. As the antigen derived from an infectious disease described above, antigens derived from influenza A virus and influenza B virus are preferably used. The subtypes of influenza A virus and influenza B virus described above are not particularly limited, and may be any subtype isolated so far or any subtype isolated in the future.

[0119] Further, the influenza virus-derived antigen is not particularly limited as long as it is at least a part of various components constituting the influenza virus. Examples include whole inactivated virus particles obtained by inactivating purified virus particles with an organic solvent / surfactant or other reagents, and viral subunits prepared by removing impurities from whole virus particles and purifying HA and / or NA. From the viewpoint of immunogenicity, HA subunits or whole virus particles are preferable. The above-mentioned whole virus particles are more preferably those inactivated by formalin or the like. The present invention is particularly effective for HA subunits (split viruses), which have few impurities and require an adjuvant such as an immunostimulant.

[0120] The method for preparing the above-mentioned influenza virus antigen is not particularly limited, and any known method can be used without limitation. For example, a method may be mentioned in which a virus strain isolated from an influenza-infected animal or an influenza patient is inoculated into chicken eggs or the like, cultured by a conventional method, and the antigen is prepared from a purified virus stock. Alternatively, a virus-derived antigen prepared in cultured cells by genetic engineering may be used.

[0121] (Antigen for immune diseases) The antigen for immune diseases is not particularly limited as long as it contains an epitope of a target protein for an immune disease. The immune disease is not particularly limited, and examples include psoriasis vulgaris, ankylosing spondylitis, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, bronchial asthma, chronic urticaria, hay fever, atopic dermatitis, and the like. The target protein is not particularly limited, and examples include IL-17A, DPP4, S100A9, PCSK9, IL-23, IgE, TNFα, IL-12 / 23p40, IL-6, α4β7 integrin, IL-4 / 13, IL-5, BLyS, IL-13, and the like. Reference 1 (International Publication No. WO2017 / 164409) describes a peptide derived from IL-17A.

[0122] When the active ingredient (C) in the present invention is a poorly water-soluble drug, the present invention can be used more effectively. Poorly water-soluble drugs refer to drugs that, in the 17th edition of the Japanese Pharmacopoeia, are classified as either slightly soluble, slightly soluble, poorly soluble, extremely poorly soluble, or almost insoluble, among those classified as very soluble, easily soluble, slightly soluble, slightly poorly soluble, poorly soluble, very poorly soluble, or almost insoluble in terms of solubility.

[0123] Specifically, (C) the active ingredient is a poorly water-soluble drug with a solubility in water of 1 mg / mL or less. The pharmaceutical composition 1 of the present invention can solubilize even poorly water-soluble drugs at high concentrations without using organic solvents and while reducing the amount of highly toxic surfactants used.

[0124] (C) The active ingredient preferably has a molecular weight of 200 or more, more preferably 300 or more, more preferably 400 or more, more preferably 500 or more, even more preferably 600 or more, even more preferably 700 or more, even more preferably 800 or more, even more preferably 900 or more, particularly preferably 1000 or more, particularly preferably 1100 or more, and most preferably 1200 or more.

[0125] (C) The molecular weight of the active ingredient is a value calculated from the molecular formula of the compound.

[0126] (C) The active ingredient, a poorly water-soluble drug, is preferably a poorly water-soluble peptide. The poorly water-soluble peptide may contain acidic amino acids, basic amino acids, or neutral amino acids, with basic or neutral amino acids being preferred. When compounding, the pH of the solution may be appropriately selected considering the isoelectric point. The amino acids may also include natural or unnatural amino acids.

[0127] It is preferable that poorly water-soluble peptides have at least one methyl group among the nitrogen atoms constituting the amide bond. The imparting of hydrophobicity through methylation enhances the interaction with the hydrophobic portion of the hyaluronic acid derivative, leading to greater solubilization. Furthermore, this enhanced interaction is presumed to improve the stability of the formulation.

[0128] The poorly water-soluble peptides preferably include at least one selected from cyclic peptides, long-chain peptides, hydrophobic peptides, membrane-damaging peptides, and peptide-drug conjugates.

[0129] Poorly water-soluble peptides are preferably cyclic peptides. A cyclic and rigid skeleton maximizes interaction with the hydrophobic portion of hyaluronic acid, forming a stable structure with hyaluronic acid derivatives.

[0130] In terms of cyclic size, cyclic peptides consisting of 4 to 49 amino acids are preferred, 6 to 30 are more preferred, and 8 to 25 are most preferred. The number of rings in the molecule is not particularly limited, but it is preferable that there be one to eight rings.

[0131] Examples of hydrophobic peptides include alkylated polypeptides. Insulin detemir, marketed by Novo Nordisk Pharma, is an insulin analog designed to exhibit affinity for albumin by attaching a C14 fatty acid side chain to the lysine at position 29 of the human insulin B chain. This fatty acid side chain promotes self-association between insulin detemir hexamers and stabilizes the drug by binding to albumin at the subcutaneous injection site, thereby reducing the absorption rate from the injection site. By using the hyaluronic acid derivative pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later, the hydrophobic portion of the complex of the hyaluronic acid derivative and the association promoter or solubilizing agent strongly interacts with the hydrophobic portion of the peptide in vivo, which can extend the sustained-release period.

[0132] Furthermore, the hyaluronic acid derivative pharmaceutical composition 1 or the pharmaceutical composition 2 described later, which contains the above peptide, can be expected to exhibit longer-term sustained release because the hyaluronic acid derivative effectively suppresses degradation by peptide-degrading enzymes in the body.

[0133] In this embodiment, the amount of (C) active ingredient blended with 100 parts by mass of (A) hyaluronic acid derivative is preferably 10 parts by mass or more and 100 parts by mass or less, more preferably 15 parts by mass or more and 50 parts by mass or less, and even more preferably 20 parts by mass or more and 40 parts by mass or less.

[0134] (C) The active ingredient, which is not particularly limited, may be a single poorly water-soluble drug or may be a combination of two or more.

[0135] The pharmaceutical composition 1 of this embodiment can solubilize the active ingredient (C), which is a poorly water-soluble drug, without using organic solvents during formulation, thereby reducing the use of conventional highly toxic solubilizing agents. Furthermore, the solubilizing aid (B) can promote the hydrophobic interaction between the steryl groups of the hyaluronic acid derivative (A). This is presumed to enable the solubilization of the powdered active ingredient (C) at a high concentration. It should be noted that the desired effect may be obtained by a mechanism different from the one described above.

[0136] In other words, the pharmaceutical composition 1 of this embodiment can also be called an organic solvent-free composition or a composition for solubilizing powdered drugs.

[0137] <<Other additives>> The pharmaceutical composition 1 of this embodiment may be administered alone or in accordance with conventional methods with a pharmacologically acceptable carrier. When used in combination with a pharmacologically acceptable carrier, for example, the hyaluronic acid derivative and the active ingredient, as well as an adjuvant as needed, may be mixed with water or other physiologically acceptable liquid (e.g., physiological saline, phosphate-buffered saline (PBS)), and may also contain physiologically acceptable buffers, excipients, vehicles, preservatives, stabilizers, binders, lyophilization aids, etc.

[0138] Examples of buffer solutions include Tris, sodium phosphate, potassium phosphate, histidine, or citrate.

[0139] The hyaluronic acid derivative is used after being dissolved in a pharmaceutically acceptable medium at any concentration. For example, buffer solutions such as water for injection and phosphate buffer are preferred. The buffering agent is not particularly limited as long as it is a compound having a buffering capacity that maintains the pH of the composition in the range of 4 to 10. Examples of buffering agents include acetates such as sodium acetate; phosphates such as sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; amino acid salts such as ε-aminocaproic acid and sodium glutamate; boric acid and salts thereof; and mixtures of the foregoing. Among these, water for injection, phosphate buffer, sucrose-containing water for injection, sucrose-containing phosphate buffer, and glycerol are particularly preferred.

[0140] The pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later may contain a pH adjuster. Examples of the pH adjuster include hydrochloric acid, citric acid, phosphoric acid, acetic acid, tartaric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate.

[0141] If necessary, an acid such as an organic acid or an inorganic acid may also be added. Further, these pH adjusters may be used alone in one kind, or may be used in any combination of two or more kinds.

[0142] The pH of the pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later is not particularly limited as long as it is within a pharmaceutically acceptable range. For example, it is in the range of 4.0 to 9.0, preferably 4.0 to 8.8, and more preferably 6.5 to 8.8.

[0143] Examples of preservatives for the pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later include benzalkonium chloride, methyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, sorbic acid, and alkylpolyaminoethylglycine. If necessary, one or more preservatives may be further blended, and there is no particular limitation as long as they are pharmaceutically acceptable. Examples of preservatives used in the present invention include benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, parahydroxybenzoic acid esters such as ethyl parahydroxybenzoate, benzyl alcohol, m-cresol, phenol, phenethyl alcohol, sorbic acid or its salts, and thimerosal.

[0144] The pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later may further contain one or more viscosifying agents as needed, and are not particularly limited as long as they are permissible as pharmaceuticals. Examples of thickening agents for pharmaceutical composition 1 or pharmaceutical composition 2 described later in the present invention include cellulosic polymers (methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, etc.), vinyl polymers (polyvinylpyrrolidone, polyvinyl alcohol, etc.), sugars (mucopolysaccharides such as hyaluronic acid and its salts, gellan gum, sodium alginate, dextran, cyclodextrin, and other polysaccharides), and oxyalkylene polymers (polyoxyethylene polyoxypropylene block copolymer). The molecular weight of the thickening agent in the present invention is, for example, a number average molecular weight of 0.5 × 10⁻⁶. 4 ~100×10 4 You can choose from a range of degrees.

[0145] Examples of stabilizers include sodium edetate hydrate and polyvinylpyrrolidone (povidone).

[0146] The pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later may contain a chelating agent. Examples of chelating agents include disodium edetate, trisodium edetate, tetrasodium edetate, diethyleneamine pentaacetic acid, and mixtures thereof, and are used to stabilize drugs or formulations.

[0147] Examples of isotonic agents include sucrose, glucose, dextrose, lactose, mannitol, and calcium or magnesium compounds, such as CaCl2. According to Japanese Patent Publication No. 4758893, the antimicrobial and preservative effects can be improved by incorporating glycerol at a concentration (2-2.5% (v / v)) that substantially achieves isotonic pressure. Therefore, in addition to its function as an isotonic agent, it is also expected to have antimicrobial and preservative effects. The glycerol content is, for example, 0.01-10% (w / v), preferably 0.05-5% (w / v), more preferably 0.1-3.0% (w / v), even more preferably 0.3-3.0% (w / v), and particularly preferably 0.3-2.5% (w / v).

[0148] A base may be added to pharmaceutical composition 1 of this embodiment or pharmaceutical composition 2 described later, as needed. While there are no particular limitations as long as the base is pharmaceutically acceptable, examples include sodium hydroxide, potassium hydroxide, monoethanolamine, diethanolamine, triethanolamine, trometamol, and meglumine.

[0149] The pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later may contain inorganic salts as needed. While there are no particular limitations as long as the base is pharmaceutically acceptable, examples include zinc chloride and zinc acetate.

[0150] The pharmaceutical composition 1 of this embodiment, or the pharmaceutical composition 2 described later, may be a formulated product. The formulation can be in the form of a solid, semi-solid, or liquid. In the case of a solid, examples of forms include powder, granules, pills, pellets, tablets, and capsules. Among these, freeze-dried powder is preferred as the solid. Examples of semi-solid forms include gels and similar structures. In the case of liquids, examples include suspensions obtained by diluting or suspending the powder in water or a buffer such as phosphate buffer (PB) or phosphate-buffered saline (PBS).

[0151] [Physical properties of pharmaceutical compositions] The pharmaceutical composition of this embodiment is preferably one that produces a precipitate under physiological salt concentration. Specifically, it is preferable that the pharmaceutical composition has a precipitation rate in vitro of 20%, preferably 50% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, and most preferably 92% or more, under the following test conditions.

[0152] (Test conditions) • Preparation of precipitate samples 200 μL of concentrated buffer (40 mM PB, 600 mM NaCl aqueous solution) is placed in a microcentrifuge tube (1.5 mL), and 600 μL of a formulation consisting of a hyaluronic acid derivative pharmaceutical composition is added. Then, the mixture is voltexed for 30 seconds, incubated at 37°C for 20 minutes, and the precipitate is allowed to settle using a centrifuge (2000 G, 5 min). Subsequently, the hyaluronic acid derivative in the supernatant is subjected to GPC measurement.

[0153] • Preparation of blank samples 200 μL of sterile water for injection is placed in a microcentrifuge tube (1.5 mL), and 600 μL of a preparation consisting of a hyaluronic acid derivative pharmaceutical composition (theoretical concentration of hyaluronic acid derivative is X mg / mL, X > 1.33) is added. Then, the mixture is voltexed for 30 seconds, incubated at 37°C for 20 minutes, and then subjected to centrifugation (2000 G, 5 min). Subsequently, the supernatant is diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative and subjected to GPC measurement.

[0154] GPC measurement conditions Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0155] • Confirmation of sedimentation In this specification, a hyaluronic acid derivative pharmaceutical composition satisfying a precipitation rate of 20% or more, as expressed by the following formula, is evaluated as precipitation occurring under physiological salt concentrations. Sedimentation rate (%) = {1 - (Area value of hyaluronic acid derivative in precipitated sample) ÷ ((Area value of hyaluronic acid derivative in blank sample) × (0.75X))} × 100

[0156] In the above test, the centrifugation conditions and incubation time may be arbitrarily set when preparing the precipitated sample. For example, if the precipitate rate is 20% or more when subjected to the above conditions after standing at 37°C for 2 weeks, it will be judged that precipitate will form under physiological salt concentration.

[0157] It is preferable that the pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later does not show any visible precipitates in an environment of 20°C. If precipitates are observed, they can be detected using a light-shielding automatic particle analyzer (liquid particle counter "KL-05"), which is used in the first method, "light-shielding particle counting method," of the insoluble particulate matter test method for injectable drugs specified in the Japanese Pharmacopoeia. Micrometer-sized drug crystals may precipitate from supersaturated solutions in which amorphous drugs are completely dissolved. If no precipitates are present, no particles will be observed by the aforementioned light-shielding automatic particle analyzer. Alternatively, this can be determined by using a DLS device to verify the presence of particles larger than 1 μm.

[0158] It is preferable that the pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later is filterable. The ability to sterilize by filtration can be measured by evaluating the fluid flow rate using the following syringe filter. Specifically, the evaluation will determine whether a sterile filtration filter with a pore size of 0.45 μm or 0.22 μm can pass through. More specifically, when a 3 mL sterile syringe filter made of polyethersulfone (PES) with a pore size of 0.45 μm and a diameter of 13 mmΦ is used as the filtration membrane, it will be determined whether 50% or more, preferably 60%, more preferably 70% or more, even more preferably 80% or more, and most preferably 90% or more of the hyaluronic acid derivative that passes through the filtration membrane of a 2 mL hyaluronic acid derivative composition can pass through.

[0159] <Hyaluronic acid derivative pharmaceutical composition 2> This embodiment is a hyaluronic acid derivative pharmaceutical composition comprising (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient. Hereafter, "(A1) a hyaluronic acid derivative pharmaceutical composition comprising (B1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, and (C1) an active ingredient" may be abbreviated as "pharmaceutical composition 2".

[0160] In the pharmaceutical composition 2 of this embodiment, the average particle size of the spherical structure containing the active ingredient-hyaluronic acid derivative complex can be 20 nm to 220 nm, 20 nm to 150 nm, or 30 nm to 100 nm. Having an average particle size within the above numerical range allows for a stable structure to exist in the body and facilitates passage through lymph nodes. The average particle size can be measured, for example, by DLS (Dynamic Light Scattering), nanotracking particle analyzers, size exclusion chromatography, high-performance liquid chromatography, and electron microscopy. More specifically, for example, the hyaluronic acid derivative concentration can be diluted to 1 mg / mL using a DLS device with 10 mM phosphate buffer or 10 mM phosphate buffer containing 10 w / v% sucrose before measurement.

[0161] The (B1) solubilizing aid in the pharmaceutical composition 2 of this embodiment contains at least four ether structures (ROR) and satisfies the requirement of having four or more carbon atoms. The pharmaceutical composition 2 of this embodiment has a content of (B1) solubilizing agent of 0.0001 parts by mass or more and 15000 parts by mass or less per 100 parts by mass of (A1) hyaluronic acid derivative into which hydrophobic groups have been introduced.

[0162] The pharmaceutical composition 2 of this embodiment contains a specific amount of a specific solubilizing agent. The pharmaceutical composition of this embodiment, containing the above-mentioned (B1) solubilizing aid, can interact with the hyaluronic acid derivative into which the above-mentioned (A1) hydrophobic group has been introduced, due to the appropriate polarity provided by the ether structure and the appropriate hydrophobicity provided by the alkyl skeleton, thereby promoting the interaction between hydrophobic groups in the hyaluronic acid derivative into which the above-mentioned (A1) hydrophobic group has been introduced. This makes it possible to provide a pharmaceutical composition that can solubilize poorly water-soluble active ingredients at high concentrations.

[0163] If the amount of the solubilizing agent (B1) described above is equal to or greater than the lower limit value, the active ingredient can be sufficiently solubilized in the pharmaceutical composition. If the amount of the solubilizing agent (B1) described above is below the upper limit, the viscosity of the pharmaceutical composition will not become too high, and the active ingredient can be sufficiently solubilized in the pharmaceutical composition without affecting the living body.

[0164] (A1) Hyaluronic acid derivative with introduced hydrophobic groups The description of the hyaluronic acid derivative with the hydrophobic group (A1) introduced in pharmaceutical composition 2 is generally the same as the description of the hyaluronic acid derivative with the hydrophobic group (A) introduced in pharmaceutical composition 1.

[0165] [Steryl group introduction rate] (A1) The rate of introduction of steryl groups into the hyaluronic acid derivative is preferably 0.1% or more and less than 50%, more preferably 5% or more and less than 48%, even more preferably 35% or more and 47%, and particularly preferably 37% or more and 45%.

[0166] By keeping the steryl group introduction rate within the above range, the (A1) hyaluronic acid derivative can strongly interact with poorly water-soluble drugs and the hydrophobic parts of surfactants. Furthermore, by keeping the steryl group introduction rate within the above range, the hyaluronic acid derivative-drug complex, in which the (A1) hyaluronic acid derivative in pharmaceutical composition 2 is compounded with a drug, can have improved formulation stability.

[0167] (A1) As an example of the molecular weight of the hyaluronic acid derivative, it is preferably 1,000 (1k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 300k or less, even more preferably 5k or more and 120k or less, and particularly preferably 7k or more and 100k or less. (A1) The molecular weight of the hyaluronic acid derivative can generally be adjusted by using a raw material having the corresponding molecular weight.

[0168] When the weight-average molecular weight of the hyaluronic acid derivative is above the lower limit, molecular entanglement is increased, and retention in the bloodstream can be further enhanced. On the other hand, when the weight-average molecular weight of the hyaluronic acid derivative is below the upper limit, the increase in viscosity can be suppressed, and a higher concentration of the hyaluronic acid derivative can be dissolved in the pharmaceutical composition. The weight-average molecular weight of the hyaluronic acid derivative can generally be adjusted by using a raw material having a corresponding molecular weight.

[0169] More specifically, the weight-average molecular weight of the hyaluronic acid derivative is preferably 100k or less, more preferably 50k or less, particularly preferably 20k or less, and most preferably 15k or less, from the viewpoint of viscosity and dispersibility. The unit of molecular weight is Da. From the standpoint of producing hyaluronic acid derivatives, a kJ of 4k to 20k is preferred, 6k to 16k is particularly preferred, and 8k to 12k is most preferred.

[0170] From the perspective of hyaluronic acid derivatives strongly exhibiting the properties of hyaluronic acid, for example, in order to be strongly recognized by the CD44 receptor, a value of 100k or higher is preferable, 200k or higher is particularly preferable, and 300k or higher is most preferable.

[0171] The content of (A1) hyaluronic acid derivative relative to the total amount of pharmaceutical composition 2 is preferably 6 mg / mL or more and less than 65 mg / mL, more preferably 8 mg / mL or more and 50 mg / mL or less, and even more preferably 10 mg / mL or more and 30 mg / mL or less.

[0172] ≪(B1) Solubilization aid≫ In the present invention, "solubilization" means making the (C1) active ingredient, described later, soluble in water until it becomes visibly clear. A "solubilizing agent" is an agent that, when added to water together with (C1) the active ingredient and (A1) the hyaluronic acid derivative, has the effect of increasing the solubility of (C) the active ingredient.

[0173] (B1) There are no particular restrictions on the solubilizing aid as long as it has the effect of dissolving (solubilizing) the (C1) active ingredient in the aqueous phase, and can be appropriately selected according to the purpose, for example, nonionic surfactants. The (B1) solubilizing aid used in the present invention is an agent that can be commonly used in pharmaceutical applications and contains at least four ether structures (ROR) and satisfies the requirement of having four or more carbon atoms.

[0174] (B1) The solubilizing agent is preferably one or more selected from the group consisting of a nonionic surfactant, polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less, and cyclodextrin derivatives.

[0175] (B1) Examples of solubilizing agents include polysorbate, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, and polyoxyethylene castor oil.

[0176] (B1) As solubilizing aids, polysorbate 80 (having 20 or more ether structures and 64 or more carbon atoms), polysorbate 65 (having 20 or more ether structures and 100 or more carbon atoms), polysorbate 60 (having 20 or more ether structures and 64 or more carbon atoms), polysorbate 40 (having 20 or more ether structures and 62 or more carbon atoms), polysorbate 20 (having 20 or more ether structures and 57 or more carbon atoms), poloxamer (having 28 or more ether structures and 74 or more carbon atoms), and polyoxyethylene hydrogenated castor oil (having 35 ether structures) are used. Preferably, one or more selected from the group consisting of (having the above characteristics and having 57 or more carbon atoms), cyclodextrin derivatives (having 12 or more ether structures and having 36 or more carbon atoms), polyethylene glycol 300 (having 5 or more ether structures and having 12 or more carbon atoms), polyethylene glycol 400 (having 7 or more ether structures and having 16 or more carbon atoms), polyethylene glycol 4000 (having 59 or more ether structures and having 120 or more carbon atoms), and tocopheryl polyethylene glycol succinate (having 21 or more ether structures and having 35 or more carbon atoms).

[0177] Other examples include polyethylene glycol monolaurate (containing 7 or more ether structures and 28 or more carbon atoms), polyoxyl stearate 40 (containing 39 or more ether structures and 98 or more carbon atoms), polyoxyl stearate 45 (containing 44 or more ether structures and 108 or more carbon atoms), polyoxyl stearate 55 (containing 54 or more ether structures and 128 or more carbon atoms), and cremophor EL (containing 35 or more ether structures and 127 or more carbon atoms).

[0178] (B1) Examples of solubilizing aids include poloxamer 188 (having 98 or more ether structures and 225 or more carbon atoms), poloxamer 124 (having 26 or more ether structures and 74 or more carbon atoms), poloxamer 237 (having 93 or more ether structures and 225 or more carbon atoms), poloxamer 338 (having 177 or more ether structures and 400 or more carbon atoms), poloxamer 407 (having 147 or more ether structures and 352 or more carbon atoms), polyethylene glycol 300 (having 5 or more ether structures and 12 or more carbon atoms), polyethylene glycol 400 (having 7 or more ether structures and 16 or more carbon atoms), polyethylene glycol 4000 (having 59 or more ether structures and 120 or more carbon atoms), and polyvinyl alcohol (average degree of polymerization: 500, having 4 or more ether structures and 4 or more carbon atoms).

[0179] The content of the solubilizing agent (B1) per 100 parts by mass of the hyaluronic acid derivative (A1) is 0.0001 parts by mass or more and 15000 parts by mass or less, preferably 0.01 parts by mass or more and 150 parts by mass or less, more preferably 0.05 parts by mass or more and 100 parts by mass or less, even more preferably 1 part by mass or more and 50 parts by mass or less, particularly preferably 5 parts by mass or more and 30 parts by mass or less, and most preferably 10 parts by mass or more and 20 parts by mass or less.

[0180] (B1) When the solubilizing agent is a nonionic surfactant, the content of the nonionic surfactant per 100 parts by mass of (A1) hyaluronic acid derivative is preferably 0.0001 parts by mass or more and 150 parts by mass or less, more preferably 0.001 parts by mass or more and 100 parts by mass or less, and even more preferably 0.005 parts by mass or more and 50 parts by mass or less.

[0181] (B1) When the solubilizing agent is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less, the polyethylene glycol content per 100 parts by mass of (A1) hyaluronic acid derivative is preferably 25 parts by mass or more and 15000 parts by mass or less, more preferably 250 parts by mass or more and 10000 parts by mass or less, and even more preferably 500 parts by mass or more and 5000 parts by mass or less.

[0182] (B1) The less the solubilizing agent, the better. However, if it is below the lower limit, the active ingredient cannot be sufficiently solubilized. If it exceeds the upper limit, the toxicity tends to increase due to the excessive amount of (B1) solubilizing agent, and the viscosity of the formulation also increases. Therefore, it is preferable to keep it within the above range.

[0183] (B1) There are no particular restrictions on the means of dissolving the solubilizing agent, and examples include stirring, shaking, and ultrasonic treatment.

[0184] (C1) Active Ingredient The description of the active ingredient (C1) contained in pharmaceutical composition 2 is generally the same as the description of the active ingredient (C) contained in pharmaceutical composition 1.

[0185] In pharmaceutical composition 2, the amount of (C1) a poorly water-soluble drug, which is the active ingredient, relative to 100 parts by mass of (A1) a hyaluronic acid derivative is preferably 21 parts by mass or more and less than 100 parts by mass, more preferably 22 parts by mass or more and 70 parts by mass or less, and even more preferably 23 parts by mass or more and 50 parts by mass or less.

[0186] (C1) The active ingredient, which is not particularly limited but is a poorly water-soluble drug, may be formulated as a single ingredient or as a combination of two or more ingredients.

[0187] The pharmaceutical composition 2 of this embodiment can solubilize the active ingredient (C1), which is a poorly water-soluble drug, without using organic solvents during formulation, thereby reducing the amount of conventional highly toxic solubilizing agents used. Furthermore, by including (B1) a solubilizing agent, the hydrophobic interactions between the steryl groups of the (A1) hyaluronic acid derivative can be promoted. This is thought to expand the hydrophobic region of the hyaluronic acid derivative pharmaceutical composition, thereby increasing the amount of hydrophobic drug that can be carried. In addition, since the solubilizing agent can act as a highly mobile hydrophobic region within the hyaluronic acid derivative pharmaceutical composition, in addition to the hydrophobic region chemically bound to the hyaluronic acid polymer, it is presumed that the powdered (C1) active ingredient can be solubilized at a high concentration. It should be noted that the desired effect may be obtained by a mechanism different from the one described above.

[0188] In other words, the pharmaceutical composition 2 of this embodiment can also be called an organic solvent-free composition or a composition for solubilizing powdered drugs.

[0189] The content of the organic solvent in the pharmaceutical composition 2 is preferably less than 0.8%. Here, the organic solvent contained in pharmaceutical composition 2 is a solvent that falls under classes 1 to 3 as defined in the Pharmaceutical Residual Solvent Guidelines.

[0190] Specifically, examples of Class 1 organic solvents include benzene, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethene, and 1,1,1-trichloroethane.

[0191] Examples of Class 2 organic solvents include acetonitrile, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, formamide, hexane, methanol, 2-methoxyethanol, methyl butyl ketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetralin, toluene, 1,1,2-trichloroethene, and xylene.

[0192] Examples of Class 3 organic solvents include acetic acid, acetone, anisole, 1-butanol, 2-butanol, n-butyl acetate, t-butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.

[0193] While it is preferable that the above-mentioned Class 1 to 3 organic solvents are not included in the pharmaceutical composition 2, they are components that inevitably remain in the manufacturing process of hyaluronic acid derivatives. Even if the organic solvents are Class 1 to 3, they can be used safely as long as the content of the organic solvent in the pharmaceutical composition 2 is less than 0.8%.

[0194] <<Other additives>> The pharmaceutical composition 2 of this embodiment may be administered alone or in accordance with conventional methods with a pharmacologically acceptable carrier. When used in combination with a pharmacologically acceptable carrier, for example, the hyaluronic acid derivative and the active ingredient, as well as an adjuvant as needed, may be mixed with water or other physiologically acceptable liquid (e.g., physiological saline, phosphate-buffered saline (PBS)), and may also include physiologically acceptable buffers, excipients, vehicles, preservatives, stabilizers, binders, lyophilization aids, etc. The description of additives is the same as the description of "other additives" in the pharmaceutical composition 1 described above.

[0195] ≪Method for producing hyaluronic acid derivatives≫ Hyaluronic acid derivatives can be obtained, for example, by converting the carboxyl group of glucuronic acid to an amide and introducing a steryl group. Furthermore, the rate of steryl group introduction can be controlled by adjusting the amount of the compound containing steryl groups that reacts with the raw material hyaluronic acid or its derivative.

[0196] Specifically, as a method for converting the carboxyl group of glucuronic acid to an amide and introducing a steryl group, for example, a raw material hyaluronic acid or its derivative, preferably hyaluronic acid or its derivative composed only of repeating units (II), is ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and in the presence of a suitable condensing agent, the hyaluronic acid salt is mixed with the esteryl group in a solvent, and the formula is "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 One method involves reacting an amine to which a steryl group (in particular a cholesteryl group) represented by ", where Z and R are as already defined herein" has been introduced with .

[0197] The coupling agent that can be used in the above reaction is not particularly limited and includes, for example, 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorphorium (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).

[0198] In particular, although not limited to, DMT-MM is preferred because the reaction proceeds with high efficiency even in a mixed solvent of water and an organic solvent. Furthermore, by using DMT-MM as a condensing agent, it is possible to selectively form amide bonds between amino groups and carboxyl groups while suppressing ester bond formation in systems where many hydroxyl groups coexist. By using this condensing agent, it is possible to prevent, for example, the reaction of the solvent alcohol with the carboxyl groups of the hyaluronic acid portion, or the formation of unwanted crosslinks by intramolecular or intermolecular bonding between carboxyl groups and hydroxyl groups simultaneously present in the hyaluronic acid portion.

[0199] Solvents used in the steryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyhydric alcohols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixtures thereof. The polyhydric alcohol may be a dihydric alcohol or a trihydric alcohol. Examples of dihydric alcohols include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of trihydric alcohols include glycerin and trimethylolpropane.

[0200] Alternatively, the raw material hyaluronic acid or its derivative may be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and the hyaluronic acid salt and the spacer portion may be reacted in a solvent in the presence of a suitable condensing agent (protection and deprotection reactions may be performed as needed) to convert the carboxyl group (-COOH) of the raw material hyaluronic acid or its derivative, and then reacted with a suitable reagent. Examples of combinations of groups derived from the carboxyl group 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; -CONR a -Y-OCO-Hal + HO-R; -CONR a -Y-SH + Hal-R; -CONR a -Y-Hal + HS-R; -CONR a -Y-CO-Y a -Hal + HS-R; -CONR a -Y-CO-Y a -SH + Hal-R; -CONR a -Y-O-CO-CH=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 (wherein R a , R b , R c , Y, Y a , Y b(wherein Z is as already defined herein, and Hal represents a halogen atom selected from the group consisting of fluorine, chlorine, bromine, and iodine).

[0201] Reaction modes include dehalogenation reactions, condensation reactions, dehydration reactions, nucleophilic addition reactions such as Michael addition, and oxidative disulfide formation reactions. These are well-known reactions and can be appropriately selected by those skilled in the art, and carried out under preferred reaction conditions. If the converted product or reactant has a carboxyl group, it may be reacted with an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester.

[0202] Another method involves reacting the carboxyl group of hyaluronic acid or its derivative with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative into which a spacer having a mercapto group modified with a leaving group at its terminal end is introduced, and then forming a disulfide bond by nucleophilic substitution of thiocholesterol with this derivative.

[0203] Furthermore, a method can be described in which a portion of the spacer is introduced into the carboxyl group of hyaluronic acid or its derivative, and a portion of the spacer is introduced into the steryl group, and these are reacted. Some specific examples have been described above, but further, if -SS- is inserted into Y, a method can be described in which a hyaluronic acid derivative in which a spacer having a mercapto group at the terminal is introduced into the carboxyl group of hyaluronic acid, and a steryl group in which a spacer having a mercapto group at the terminal is introduced, and these are reacted oxidatively to form a disulfide bond. In this case, one mercapto group can be reacted with 2-mercaptopyridine to form a disulfide, and then substituted with the other mercapto group.

[0204] Furthermore, other substituents may be introduced after the preparation of the hyaluronic acid derivative. For example, 0.1% to 99.5%, preferably 40% to 65%, of the carboxyl groups in the hyaluronic acid derivative substantially composed of repeating unit (I) and repeating unit (II) may be added as -CO-X z, [wherein 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(CO2H)-(CH2)2-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 By converting to an alkyl group (selected from the group consisting of p1 being an integer between 2 and 10, q being an integer between 1 and 200, and r being an integer between 1 and 3), it is also possible to chemically crosslink molecules within the molecule or between molecules including other molecules to form a gel.

[0205] The obtained hyaluronic acid derivative may be dried. Examples of drying methods include forced-air drying, drying in a constant-temperature bath, reduced-pressure drying, hot-air circulation drying, and freeze-drying. Freeze-drying is preferred among these. When freeze-drying is performed, it is preferable that the hyaluronic acid derivative further contains a cryoprotectant from the viewpoint of more effectively suppressing the increase in particle size of the fine particles formed by the hyaluronic acid derivative.

[0206] The cryoprotectant is not particularly limited as long as it is known as a "freeze-protectant" or "freeze-drying protectant," and examples include disaccharides, sorbitol, dextran, propylene glycol, glycerin, glycerol, polyvinylpyrrolidone, dimethyl sulfoxide, etc.

[0207] The disaccharides are not particularly limited, and examples include sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, genthiobiose, turanose, maltulose, palatinose, genthiobiulose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, sylabiose, neohesperidose, rutinose, rutinulose, bicyanose, xylobiose, and primevelose. Among these, sucrose, trehalose, maltose, or lactose are preferred because they are widely used as cryoprotective agents. Furthermore, sucrose is more preferred from the viewpoint of its track record as a pharmaceutical additive and its ability to more effectively suppress the increase in particle size of the fine particles formed by the hyaluronic acid derivative during freeze-drying.

[0208] The cryoprotectant may be added in solid form or dissolved in a solvent such as water.

[0209] The amount of cryoprotectant added is not particularly limited, but 20 parts by mass or more per 100 parts by mass of hyaluronic acid derivative is preferred. A more sufficient particle size increase suppression effect can be obtained by adding an amount of cryoprotectant above the lower limit. On the other hand, the upper limit of the amount of cryoprotectant added is not particularly limited, but for example, it can be 100,000 parts by mass.

[0210] The equipment used in freeze-drying is not particularly limited; for example, a commercially available freeze-dryer can be used. Among these, a freeze-dryer that can monitor the vacuum level inside the device during freeze-drying is preferred from the viewpoint of controlling the vacuum level, and a shelf-type freeze-dryer is preferred from the viewpoint of controlling the product temperature.

[0211] <Method for producing pharmaceutical composition 1> The pharmaceutical composition 1 of this embodiment can be manufactured by the following manufacturing method 1 or manufacturing method 2 of the pharmaceutical composition 1. The following describes each manufacturing method.

[0212] ≪Method for producing pharmaceutical composition 1≫ Manufacturing method 1 is a method for producing a pharmaceutical composition 1, comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient. Manufacturing method 1 includes the steps of (A) mixing a hyaluronic acid derivative into which hydrophobic groups have been introduced with (B) an association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter, and (C) mixing the active ingredient with the aqueous solution of the hyaluronic acid derivative containing the association promoter.

[0213] ≪Method for producing pharmaceutical composition 1≫ Manufacturing method 2 is a method for producing pharmaceutical composition 1, comprising (A) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B) an association promoter, and (C) an active ingredient. Manufacturing method 2 includes the steps of: (C) dispersing an active ingredient in (B) an association promoter to obtain a dispersion (I); preparing an aqueous solution of a hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing an association promoter to obtain an aqueous solution (II); and mixing the dispersion (I) and the aqueous solution (II). In manufacturing methods 1 and 2, depending on the structure of the drug, it is permissible to use appropriate pH and buffering materials as needed during manufacturing.

[0214] In manufacturing methods 1 and 2, it is preferable that the process does not include a step to remove the organic solvent.

[0215] <Method for producing pharmaceutical composition 2> The pharmaceutical composition 2 of this embodiment can be manufactured by the following manufacturing method 1 or manufacturing method 2 of the pharmaceutical composition 2. The following describes each manufacturing method.

[0216] ≪Method 1 for producing pharmaceutical composition 2≫ Method 1 for producing pharmaceutical composition 2 is a method for producing pharmaceutical composition 2 that contains (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient. Method 1 for producing the pharmaceutical composition 2 includes the steps of: (A1) mixing a hyaluronic acid derivative into which hydrophobic groups have been introduced with (B1) a solubilizing agent to obtain an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent; and (C1) mixing the active ingredient with the aqueous solution of the hyaluronic acid derivative containing the solubilizing agent.

[0217] ≪Method for producing pharmaceutical composition 2≫ The second method for producing the pharmaceutical composition 2 is a method for producing the pharmaceutical composition 2 that contains (A1) a hyaluronic acid derivative into which a hydrophobic group has been introduced, (B1) a solubilizing agent, and (C1) an active ingredient. The second method for producing the pharmaceutical composition 2 includes the steps of: dispersing (C1) an active ingredient in (B1) a solubilizing agent to obtain a dispersion (I); preparing an aqueous solution of a hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing a solubilizing agent to obtain an aqueous solution (II); and mixing the dispersion (I) and the aqueous solution (II).

[0218] In the manufacturing methods 1 and 2 for the pharmaceutical composition 2, it is preferable that the process does not include a step to remove the organic solvent.

[0219] <Administration Method> The target animals for administering pharmaceutical composition 1 or pharmaceutical composition 2 of this embodiment include animals classified as mammals, including humans (such as monkeys, marmosets, mice, rats, cattle, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.).

[0220] The administration route of pharmaceutical composition 1 or pharmaceutical composition 2 of this embodiment is not particularly limited, and any currently known administration route can be used as appropriate depending on the intended use, the location of the tissue to be treated, etc. For example, possible routes of administration include subcutaneous, intramuscular, intravenous, intra-arterial, intrathecal, intracerebral, intra-articular, intraperitoneal, vaginal, sacral, rectal, intravitreous, periorbital, intradermal, intraperitoneal, intranasal, transbronchial, transpulmonary, transdermal, sublingual, oral, oral administration, and ophthalmic administration. Among these, subcutaneous, intramuscular, intravitreous, intrathecal, and intra-articular administration are preferred. It may also be administered topically as a powder, cream, ointment, or eye drops. Administration by injection includes subcutaneous injection, intramuscular injection, intravenous injection, intra-arterial injection, intrathecal injection, intra-articular injection, intraperitoneal injection, intravitreous injection, periorbital injection, intradermal injection, and intratumor injection.

[0221] In eye drops, using hyaluronic acid derivatives can reduce the initial burst of drug release, suppress aggregation that may occur between drugs during storage and after administration, reduce variability in efficacy, and allow the drug to precipitate and adhere to and remain on the corneal epithelium under physiological conditions, thereby allowing for a longer-term sustained release and reducing the number of eye drops. Furthermore, it is possible to promote drug uptake by corneal epithelial cells and conjunctival epithelial cells. In addition, it possesses high moisturizing properties, viscosity, and biocompatibility derived from hyaluronic acid, which can alleviate inflammation, pain, stress, and damage upon contact with the formulation, thus providing a highly safe formulation. As a result, by efficiently extracting the effects of the drug, we provide hyaluronic acid derivative pharmaceutical composition 1 or pharmaceutical composition 2 that is excellent in tear film stabilization, corneal epithelial disorder treatment, meibomian gland dysfunction treatment, and pain suppression.

[0222] In the pharmaceutical composition 1 or pharmaceutical composition 2 of this embodiment, when administered parenterally, the dosage can be appropriately selected considering the type of recipient (including age and sex), but generally, for example, in a human (assuming a body weight of 60 kg), the amount of the active ingredient (preferably protein, peptide, or low molecular weight compound) per dose can be 0.01 μg to 20 mg, 0.1 μg to 15 mg, or 1 μg to 10 mg.

[0223] The number of administrations may be a single dose of the above-mentioned dosage, or it may be administered two or more times, such as once every 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or every six months. Alternatively, it may be administered to two or more sites in a single administration.

[0224] <<Other Embodiments>> In one embodiment, the present invention provides a method for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases and immune diseases, and chronic diseases, comprising administering an effective amount of the above-mentioned pharmaceutical composition 1 or pharmaceutical composition 2 to a patient or animal. Examples of infectious diseases include those listed under "Infectious Disease-Derived Antigens" in the "Antigens" section above. Furthermore, the term "effective dose" as used here includes a dose that is effective for prevention or treatment, that is, a dose suitable for preventing or treating the onset of the aforementioned diseases.

[0225] In one embodiment, the present invention provides a composition for the prevention or treatment of one or more diseases selected from the group consisting of cancer, infectious diseases, immune diseases, inflammatory bowel disorders, allergic diseases, skin diseases, hypertension, diabetes, neurological diseases, genetic diseases, cardiovascular diseases, cerebrovascular diseases, respiratory diseases, eye diseases, ear diseases, and bone and joint diseases, the composition comprising the above-mentioned active ingredient-hyaluronic acid derivative complex.

[0226] In one embodiment, the present invention provides the use of the above-mentioned active ingredient-hyaluronic acid derivative complex for producing pharmaceutical composition 1 or pharmaceutical composition 2. [Examples]

[0227] The present invention will be described in detail below with reference to examples, but these are not intended to limit the scope of the present invention to these examples.

[0228] <Synthesis of hyaluronic acid derivatives> [Synthesis Example 1] Hyaluronic acid derivatives were prepared according to the following steps 1-A, 2-A, and 3-A.

[0229] [Process 1-A] (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1-A and then 1-2-A.

[0230] [Process 1-1-A] To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred under ice cooling for 30 minutes. The temperature was then raised to room temperature (approximately 25°C), and the mixture was stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed 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 removed under reduced pressure.

[0231] [Process 1-2-A] The obtained residue was dissolved in ethyl acetate (40 mL), and 40 mL of 4N hydrochloric acid / ethyl acetate solution was added and stirred overnight at room temperature (approximately 25°C). The resulting precipitate was collected by centrifugation. The obtained solid was washed four times with ethyl acetate and dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0232] [Process 2-A] (Preparation of tetrabutylammonium (TBA) salt of hyaluronic acid) Hyaluronic acid TBA salt (HA-TBA) was prepared according to the following steps 2-1-A and then 2-2-A.

[0233] [Process 2-1-A] DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed approximately three times with ultrapure water by decantation. Approximately 1.5 times the molar equivalent of 40% by mass of tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added relative to the cation exchange capacity of the resin, and the mixture was stirred for 30 minutes. After removing the excess TBA-OH solution by decantation, the mixture was further washed with excess ultrapure water to obtain a TBA-chlorinated cation exchange resin.

[0234] [Process 2-2-A] Sodium hyaluronate salt (HA-Na) with a molecular weight of 35,000 (35 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of cation exchange resin, which had been TBA-chlorinated in [Step 2-1-A], was added in an amount equivalent to 5 times the molar amount of HA units (unit molecular weight 401.3) in terms of the resin's ion exchange capacity. After stirring for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA) as a white solid.

[0235] [Process 3-A] An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in [Step 2-2-A] above was prepared. Then, Chol hydrochloride was added in a molar ratio of 19 / 100 relative to the disaccharide repeating units (HA units) present in the HA-TBA synthesized in [Step 1-A] above. Next, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added in a molar ratio of 24 / 100 relative to the HA units, and the mixture was stirred overnight at room temperature (approximately 25°C). The reaction solution was dialyzed in the following order: 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water (Spectrapore 7, molecular weight cutoff (MWCO): 3,500). The resulting dialysate was freeze-dried to obtain the target product (HA-C6-Chol) as a white solid.

[0236] Products 1 In the 1H-NMR spectrum, peaks originating from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks originating from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were observed, and the cholesterol introduction rate was 19%.

[0237] [Synthesis Example 2] Hyaluronic acid derivatives were prepared according to the following steps 1-B, 2-B, and 3-B.

[0238] [Process 1-B] (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1-B and then 1-2-B.

[0239] [Process 1-1-B] To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred under ice cooling for 30 minutes. The temperature was then raised to room temperature (approximately 25°C), and the mixture was stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed 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 removed under reduced pressure.

[0240] [Process 1-2-B] The obtained residue was dissolved in ethyl acetate (40 mL), and 40 mL of 4N hydrochloric acid / ethyl acetate solution was added and stirred overnight at room temperature (approximately 25°C). The resulting precipitate was collected by centrifugation. The obtained solid was washed four times with ethyl acetate and dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0241] [Process 2-B] (Preparation of tetrabutylammonium (TBA) salt of hyaluronic acid) Hyaluronic acid TBA salt (HA-TBA) was prepared according to the following steps 2-1-B and then 2-2-B.

[0242] [Process 2-1-B] DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed approximately three times with ultrapure water by decantation. Approximately 1.5 times the molar equivalent of 40% by mass of tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added relative to the cation exchange capacity of the resin, and the mixture was stirred for 30 minutes. After removing the excess TBA-OH solution by decantation, the mixture was further washed with excess ultrapure water to obtain a TBA-chlorinated cation exchange resin.

[0243] [Process 2-2-B] Sodium hyaluronate salt (HA-Na) with a molecular weight of 35,000 (35 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of cation exchange resin, which had been TBA-chlorinated in "(1) Step 2-1-B", was added in an amount equivalent to 5 times the molar amount of HA units (unit molecular weight 401.3) in terms of the resin's ion exchange capacity. After stirring for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA) as a white solid.

[0244] [Process 3-B] An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in [Step 2-2-B] was prepared. Then, Chol hydrochloride was added in a molar ratio of 31 / 100 relative to the disaccharide repeating units (HA units) present in the HA-TBA synthesized in [Step 1-B].

[0245] Next, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added to the HA units in a molar ratio of 37 / 100, and the mixture was stirred overnight at room temperature (approximately 25°C). The reaction solution was dialyzed in the following order: 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water (Spectrapore 7, molecular weight cutoff (MWCO): 3,500).

[0246] The resulting dialysate was freeze-dried to obtain the target product (HA-C6-Chol) as a white solid. 1In the 1H-NMR spectrum, peaks originating from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks originating from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were observed, and the cholesterol introduction rate was 30%.

[0247] <Test Example 1> As Test Example 1, we formulated cyclosporine (CyA), a poorly water-soluble peptide.

[0248] [Example 1-1] A CyA formulation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing a synthesis promoter (polysorbate 80). The following procedures were performed at 20°C and room temperature. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. In a separate vial, polysorbate 80 (Fujifilm Wako, product number: 164-21591) was diluted with sterile water for injection to a concentration of 2 mg / mL.

[0249] Next, 200.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed into another vial, and then 1.0 mL of ethanol was added to uniformly dissolve the CyA. After this, 7.2 mL of a 12 mg / mL aqueous solution of hyaluronic acid derivative and 1.2 mL of 2 mg / mL polysorbate 80 were added and mixed. After stirring for 1 hour, 0.046 mL of a 200 mg / mL ethanol solution containing CyA was added dropwise to the hyaluronic acid derivative aqueous solution containing polysorbate 80 to compound the drugs.

[0250] Furthermore, after all the CyA compounds were combined, the concentration was adjusted with sterile water for injection. Then, sucrose (Fujifilm Wako, for manufacturing only) was added in powder form at a ratio of 10% by mass to obtain a 10% sucrose solution, and the mixture was stirred for 3 hours to homogenize it, thus preparing the hyaluronic acid derivative pharmaceutical composition containing the association promoter. The final solution was visually clear. In addition, no precipitates were observed during 2 weeks of refrigerated storage. The above operations were performed at 20°C. The final formulation composition is shown in Table 1.

[0251] [Examples 1-2] A CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing an association promoter (polyethylene glycol 400). The following operations were performed at 20°C, under room temperature conditions. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. 88.0 mg of powdered CyA (Tokyo Chemical Industries, product number: C2408) was weighed into a separate vial, and then 10.0 mL of polyethylene glycol 400 (Fujifilm Wako, product number: 161-09065) was added to uniformly dissolve the CyA.

[0252] Next, 2.0 mL of an 8.8 mg / mL CyA-containing polyethylene glycol 400 solution was added to a vial containing 6 mL of a 12 mg / mL hyaluronic acid derivative aqueous solution while stirring to perform drug compounding. After all of the CyA had been compounded, sucrose (Fujifilm Wako, for manufacturing use only) was added in powder form at a ratio of 10% by mass to a sucrose solution, and the mixture was stirred for 3 hours to homogenize it, thus preparing the hyaluronic acid derivative pharmaceutical composition containing the association promoter. Finally, the solution was visually clear. Furthermore, no precipitates were observed during 2 weeks of refrigerated storage. The final formulation composition is shown in Table 1.

[0253] [Examples 1-3] A CyA formulation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing association promoters (polyethylene glycol 400 and polysorbate 80). The following procedures were performed at 20°C and room temperature.

[0254] The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. 92.6 mg of powdered CyA (Tokyo Chemical Industries, product number: C2408) was weighed into a separate vial, and then 10.0 mL of polyethylene glycol 400 (Fujifilm Wako, product number: 161-09065) was added to uniformly dissolve the CyA. Subsequently, 1.9 mL of a 9.26 mg / mL CyA-containing polyethylene glycol 400 solution was added to a vial containing 6 mL of a 12 mg / mL aqueous solution of the hyaluronic acid derivative while stirring to perform drug compounding.

[0255] In addition, after all of the CyA had been compounded, 0.10 mL of 100 mg / mL polysorbate 80 was added. Sucrose (Fujifilm Wako, for manufacturing purposes only) was added in powder form to obtain a 10% by mass sucrose solution, and the mixture was stirred for 3 hours to homogenize it, thus preparing the hyaluronic acid derivative pharmaceutical composition containing the association promoter. Finally, the solution was visually clear. Furthermore, no precipitates were observed during 2 weeks of refrigerated storage. The final formulation composition is shown in Table 1.

[0256] [Comparative Example 1-1] A CyA preparation was prepared using α-cyclodextrin. The following operations were performed at 20°C, under room temperature conditions. CyA powder was dissolved in a 10% sucrose solution containing 10% α-cyclodextrin (Aldrich) at a concentration of 10% by mass, at a concentration of 0.5 mg / mL, to prepare the solution. The solution was visually clear at the end. No precipitates were observed during 2 weeks of refrigerated storage. The final formulation composition is shown in Table 1.

[0257] [Comparative Example 1-2] A CyA formulation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%). The following operations were performed at 20°C, under room temperature conditions. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours.

[0258] Next, 200.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed into another vial, and then 1.0 mL of ethanol was added to uniformly dissolve the CyA. After this, 0.046 mL of the 200 mg / mL CyA-containing ethanol solution was added dropwise to a 12 mg / mL aqueous solution of hyaluronic acid derivative to compound the drugs. After all of the CyA had compounded, the concentration was adjusted with water for injection, and then sucrose (Fujifilm Wako, for manufacturing use only) was added in powder form at a rate that resulted in a 10% by mass sucrose solution, and the mixture was stirred for 3 hours to homogenize it. Finally, the solution was visually clear. The above operations were performed at 20°C. The final formulation composition is shown in Table 1.

[0259] [Table 1]

[0260] <Test Example 2> Pharmacokinetic studies of CyA were conducted in rats. Solution formulations consisting of the hyaluronic acid derivative pharmaceutical compositions prepared in Examples 1-1, 1-2, 1-3 and Comparative Example 1-2, as well as the solution formulation of Comparative Example 1-1, were administered subcutaneously to normal rats (SD, 6 weeks old, male) using a 25G needle at the doses (mg / kg) shown in Table 2.

[0261] After administration, jugular vein blood was collected at time using heparinized syringes, and aprotinin was added as a protease inhibitor. The obtained blood was separated into plasma and measured by LC-MS / MS. Figure 3 shows the plasma concentration profiles of CyA after administration of various CyA preparations, as well as the plasma concentration profiles of CyA in Comparative Example 1. In Figure 3, Example 2-1 is the case when a solution preparation consisting of the hyaluronic acid derivative pharmaceutical composition of Example 1-1 was administered. Examples 2-2 to 2-3 and Comparative Examples 2-1 to 2-2 are the cases when a solution preparation consisting of the hyaluronic acid derivative pharmaceutical compositions of Examples 1-2 to 1-3 and Comparative Example 1-2, and the solution preparation of Comparative Example 1-1, respectively, were administered.

[0262] Also, pharmacokinetic parameters (drug half-life (T) 1 / 2 ) and mean residence time (MRT), serum drug concentration - (0 to infinity) time area (AUC) inf The values ​​were analyzed using WinNonlin Ver.8.3 (manufactured by Pharsight), and the results are shown in Table 2.

[0263] [Table 2]

[0264] In this specification, sustained-release properties are evaluated by the mean residual time (MRT), and a drug composition containing a hyaluronic acid derivative containing an association promoter is evaluated as having sustained-release properties if its MRT is greater than that of the hyaluronic acid derivative drug composition alone.

[0265] More specifically, if the following formula (A) is satisfied, it is evaluated as being able to "maintain the concentration of the active ingredient in the body over a long period of time." (MRT of the hyaluronic acid derivative pharmaceutical composition containing the association promoter) ÷ (MRT of the hyaluronic acid derivative pharmaceutical composition) ≥ 1.1 ... Formula (A)

[0266] In Examples 2-1, 2-2, and 2-3, which are formulations containing a hyaluronic acid derivative association promoter, the blood concentration half-life was longer and the MRT value was higher compared to Comparative Example 2-2, which did not contain the association promoter. When the sustained-release properties were evaluated using formula (A), the values ​​were 1.35 for Example 2-1, 1.42 for Example 2-2, and 1.49 for Example 2-3 compared to Comparative Example 2-2. In other words, it was found that the drug remained in the blood for a long period of time. From the above, it was confirmed that the hyaluronic acid derivative composition of the present invention has superior sustained-release properties for a longer period of time.

[0267] <Test Example 3> We conducted an evaluation of the degree of meeting facilitation using GPC.

[0268] [Example 3-1] The degree to which GPC promoted the association of hyaluronic acid derivatives in Example 1-1, prepared in Test Example 1, was evaluated.

[0269] [Evaluation of the degree of association promotion of hyaluronic acid derivatives: Area ratio A2 / A1] Figure 1 shows the chromatogram of the hyaluronic acid derivative used in Synthesis Example 1. The hyaluronic acid derivative was dissolved in sterile water for injection at a concentration of 1 mg / mL by stirring for 24 hours, and then subjected to GPC measurement. At this time, the area value enclosed by the chromatogram and baseline was defined as A1. Figure 2 is a chromatogram of the hyaluronic acid derivative composition of Example 1-1. The hyaluronic acid derivative was diluted with sterile water for injection to a concentration of 1 mg / mL and measured. At this time, the area value enclosed by the chromatogram and baseline was defined as A2.

[0270] (GPC measurement conditions) Equipment: HLC8320-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (Differential Refractive Index) Temperature: 30℃

[0271] [Examples 3-2, 3-3, and Comparative Examples 3-1, 3-2] Examples 1-2, 1-3, and Comparative Example 1-2 were measured in the same manner as in Example 1-1, after diluting the hyaluronic acid derivative with sterile water for injection to a concentration of 1 mg / mL. Comparative Example 2-1 was subjected to GPC measurement without dilution. At this time, the area value A2, which is the area enclosed by each chromatogram and baseline, was calculated, and the area ratio A2 / A1 is shown in Table 3.

[0272] [Comparative Examples 3-3 to 3-6] Aqueous solutions were prepared in proportions such that the concentration of the additive was the same as in Examples 3-1 to 3-3 and Comparative Example 3-2, and were subjected to GPC measurement in the same manner.

[0273] [Table 3]

[0274] These results suggest that the association of hyaluronic acid derivatives in Examples 3-1 to 3-3 is promoted by polysorbate 80 and polyethylene glycol 400. On the other hand, with ethanol, there was almost no change in the area value of the hyaluronic acid derivative peak in GPC regardless of whether ethanol was added or not, confirming that the association of hyaluronic acid derivatives was not promoted.

[0275] <Test Example 4> We evaluated the association promoters using GPC. [Examples 4-1, 4-2, 4-3 and Comparative Examples 4-1, 4-2] Using the same method as in Test Example 1, hyaluronic acid derivative aqueous solutions were prepared according to Table 4, with the same composition except that they did not contain the active ingredient CyA and the isotonic agent sucrose. Each prepared sample was diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative and subjected to GPC measurement.

[0276] [Table 4]

[0277] <Test Example 5> We confirmed the precipitation behavior under physiological salt concentrations in vitro.

[0278] [Examples 5-1 to 5-3, Comparative Examples 5-1, 5-2] 150 μL of each formulation obtained in Example 1 was sampled into a 1.5 mL microcentrifuge tube. Then, concentrated buffer (40 mM PB (pH 7.4), 600 mM NaCl) was added in proportion to the final buffer composition of 10 mM PB (pH 7.4), 150 mM NaCl, and the hyaluronic acid derivative was precipitated. After incubation at 37°C for 20 minutes, the mixture was centrifuged at 2000 G for 5 minutes, 50 μL of the supernatant was sampled, diluted 2-fold with HP-β-CD aqueous solution (300 mM), incubated for 1 hour, and then diluted again with 350 μL of HP-β-CD aqueous solution (10 mM) before being subjected to GPC measurement. The remaining percentage of the hyaluronic acid derivative in the solution relative to the initial amount used was calculated from the peak area of ​​the detected hyaluronic acid derivative, and the percentage of precipitated hyaluronic acid derivative (precipitation rate) was calculated. The results of the precipitation evaluation method described above are consistent with those of the precipitation evaluation method described in [Physical Properties of Pharmaceutical Composition] (Test Conditions) and Preparation of Precipitated Samples.

[0279] (GPC measurement conditions) Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10mM HP-β-CD / phosphate buffer (pH7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (Differential Refractive Index) Temperature: 30℃

[0280] [Table 5]

[0281] These results suggest that the hyaluronic acid derivatives of Examples 5-1 to 5-3 not only have their association promoted by polysorbate 80 and polyethylene glycol 400, but also improve their precipitation performance under physiological salt concentrations in vitro.

[0282] <Test Example 6> We examined the types of association promoters. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 6-1 to 6-11 and Comparative Examples 6-1 to 6-6.

[0283] [Example 6-1] The hyaluronic acid derivative aqueous solution prepared above was diluted with sterile water for injection to a concentration of 2 mg / mL. In a separate vial, polyethylene glycol 300 was diluted with sterile water for injection to a concentration of 10 mg / mL. The hyaluronic acid derivative aqueous solution and the polyethylene glycol 300 aqueous solution were mixed in a 1:1 volume ratio to prepare the compositions shown in Table 6 below. The mixture was then incubated at 20°C for 24 hours before being subjected to GPC measurement.

[0284] (GPC measurement conditions) Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (Differential Refractive Index) Temperature: 30℃

[0285] [Examples 6-2 to 6-11, Comparative Examples 6-1 to 6-6] Solutions were prepared in the same manner as in Example 6-1, with the compositions shown in Table 6 below, and subjected to GPC measurement.

[0286] [Table 6]

[0287] These results suggest that the association of hyaluronic acid derivatives in Examples 6-1 to 6-11 was promoted by polyethylene glycol 300, poloxamer 188, and cremofol EL, in addition to polysorbate 80 and polyethylene glycol 400. On the other hand, in Comparative Examples 6-1 to 6-4, tetrahydrofuran (having one ether structure and 4 carbon atoms), ethanol (not having an ether structure and 2 carbon atoms), and ethylene glycol (not having an ether structure and 2 carbon atoms) showed almost no change in the area value of the hyaluronic acid derivative peak in GPC regardless of the presence or absence of ethanol, etc., confirming that the association of hyaluronic acid derivatives was not promoted. Here, similar to polysorbate 80 and polyethylene glycol 400, no GPC peaks of the association promoters alone were detected at the peak position of the hyaluronic acid derivatives for poloxamer 188 and cremofol EL.

[0288] <Test Example 7> A hyaluronic acid derivative pharmaceutical composition was manufactured. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 7-1 to 7-10 and Comparative Examples 7-1 to 7-6.

[0289] [Example 7-1] A CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing a synthesis promoter (polysorbate 80). The following operations were performed at 20°C, under room temperature conditions. 1.8 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed into a vial, and then diluted in another vial by adding water for injection to a concentration of 0.5 mg / mL of polysorbate 80. The weighed CyA was suspended in 18 μL of 0.5 mg / mL aqueous solution of polysorbate 80. Subsequently, 450 μL of 12 mg / mL aqueous solution of hyaluronic acid derivative was added to the CyA-containing polysorbate 80 aqueous solution while stirring, and 432 μL of water for injection was added to compound the drugs so that the formulation composition would be CyA concentration of 2 mg / mL, hyaluronic acid derivative concentration of 6 mg / mL, and polysorbate 80 concentration of 0.01 mg / mL. After stirring at 20°C for 24 hours, the precipitate was filtered through a 0.45 μm sterile filter to remove the precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement as described later. The final composition is shown in Table 7.

[0290] [Examples 7-2 to 7-10] A hyaluronic acid derivative pharmaceutical composition containing an association promoter was prepared using the same method as in Example 7-1, such that the final composition's association promoter concentration was as shown in Table 7 below. Furthermore, the CyA concentration in the formulation after filtration with a 0.45 μm sterile filter was quantified by HPLC, as in Example 7-1, and the results are summarized in Table 7.

[0291] [HPLC quantitative analysis conditions: Reverse-phase chromatography analysis conditions] Column: InertSustain C18, particle size 5μm × inner diameter 4.6mm × length 150mm Column temperature: 40℃ Mobile phase: Eluent A 0.1% TFA / acetonitrile Eluent B 0.1%TFA / water Mobile phase ratio: Eluent A / Eluent B=9 / 1 Flow rate: 1mL / min Injection volume: 30μL Detector: UV (210nm)

[0292] [Precipitation evaluation conditions] The precipitation behavior of the various pharmaceutical compositions prepared in Test Example 7 under physiological salt concentrations in vitro was evaluated using the following method.

[0293] [Preparation of precipitated samples] 200 μL of concentrated buffer (40 mM PB, 150 mM NaCl aqueous solution) is placed in a microcentrifuge tube (1.5 mL), and 600 μL of a formulation consisting of a hyaluronic acid derivative pharmaceutical composition is added. Then, the mixture is voltexed for 30 seconds, incubated at 37°C for 20 minutes, and the precipitate is allowed to settle using a centrifuge (2000 G, 5 min). Subsequently, the hyaluronic acid derivative in the supernatant is subjected to GPC measurement.

[0294] [Preparation of blank samples] 200 μL of sterile water for injection is placed in a microcentrifuge tube (1.5 mL), and 600 μL of a preparation consisting of a hyaluronic acid derivative pharmaceutical composition (theoretical concentration of hyaluronic acid derivative is X mg / mL, X > 1.33) is added. Then, the mixture is voltexed for 30 seconds, incubated at 37°C for 20 minutes, and then subjected to centrifugation (2000 G, 5 min). Subsequently, the supernatant is diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative and subjected to GPC measurement.

[0295] [GPC measurement conditions] Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0296] [Checking for sedimentation] In this specification, a hyaluronic acid derivative pharmaceutical composition satisfying a precipitation rate of 20% or more, as expressed by the following formula, is to be precipitated under physiological salt concentration. Sedimentation rate (%) = {1 - (Area value of hyaluronic acid derivative in precipitated sample) ÷ ((Area value of hyaluronic acid derivative in blank sample) × (0.75X))} × 100

[0297] [Table 7]

[0298] These results demonstrate that even with the addition of small amounts of various association promoters such as polysorbate 80, polysorbate 20, poloxamer 188, and cremofol EL, poorly water-soluble active ingredients can be solubilized at high concentrations from powder without the use of organic solvents. Furthermore, it was suggested that the association promoters also improve precipitation performance. Therefore, it is expected that association promoters can promote the association of hyaluronic acid derivatives, resulting in pharmaceutical compositions with high active ingredient content and long-term sustained release capabilities.

[0299] <Test Example 8> We investigated the different types of hyaluronic acid derivatives. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in sterile water for injection at a concentration of 2.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 8-1 to 8-11 and Comparative Examples 8-1 to 8-6.

[0300] [Example 8-1] Polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL in a glass vial. A 6 mL clean vial was prepared, and 400 μL of the hyaluronic acid derivative aqueous solution and 10 μL of the polysorbate 80 aqueous solution were added. After mixing for 30 seconds using a voltex, 390 μL of sterile water for injection was added to adjust the total volume to 800 μL. The solutions were prepared according to the proportions shown in Table 8 below. After incubation at 20°C for 24 hours, the solutions were subjected to GPC measurement.

[0301] (GPC measurement conditions) Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (Differential Refractive Index) Temperature: 30℃

[0302] [Examples 8-2 to 8-4, Comparative Examples 8-1 to 8-6] Solutions were prepared in the same manner as in Example 8-1, with the compositions shown in Table 8 below, and subjected to GPC measurement.

[0303] [Table 8]

[0304] These results suggest that the association of hyaluronic acid derivatives in Examples 8-1 to 8-4 was promoted by polysorbate 80, polysorbate 20, and cremofol EL. On the other hand, in Comparative Examples 8-1 to 8-6, tetrahydrofuran (having one ether structure and 4 carbon atoms), ethanol (not having an ether structure and 2 carbon atoms), and acetonitrile (not having an ether structure and 2 carbon atoms) showed almost no change in the area value of the hyaluronic acid derivative peak in GPC regardless of the presence or absence of the addition of tetrahydrofuran, etc., confirming that the association of hyaluronic acid derivatives was not promoted. Therefore, it is expected that even in hyaluronic acid derivatives with a small molecular weight and a high rate of hydrophobic group introduction (10k HA-C6-Chol-30%), the association of hyaluronic acid derivatives can be similarly promoted by association promoters, resulting in a pharmaceutical composition with a high content of active ingredients and capable of long-term sustained release.

[0305] <Test Example 9> We prepared a hyaluronic acid derivative pharmaceutical composition using human growth hormone (protein).

[0306] [Example 9-1] The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved at a concentration of 4.0 mg / mL in sterile water for injection by stirring at 20°C for 24 hours, and this solution was used in Examples 9-1 to 9-2 and Comparative Example 9-1.

[0307] [Examples 9-1 to 9-2] The association promoter and sterile water for injection were added to the above hyaluronic acid derivative aqueous solution. Furthermore, human growth hormone (hGH: Genotropin® for injection) powder was dissolved in sterile water for injection in a separate vial to a concentration of 2 mg / mL. After preparation, 250 μL of this solution was added to the aqueous solution of the hyaluronic acid derivative containing the association promoter, adjusting the total volume to 1 mL. Subsequently, the complexation with hGH was promoted by incubation at 37°C for 24 hours. The composition of the final formulation was adjusted to the concentrations shown in Table 9.

[0308] [Comparative Example 9-1] Tetrahydrofuran (THF) and sterile water for injection were added to the hyaluronic acid derivative aqueous solution prepared at a concentration of 4.0 mg / mL. In addition, 250 μL of 2.0 mg / mL hGH aqueous solution was added to the THF-containing hyaluronic acid derivative aqueous solution to adjust the total volume to 1 mL. Subsequently, the mixture was incubated at 37°C for 24 hours to promote complexation with hGH. As in Example 9-1, the composition of the final formulation was adjusted to the concentrations shown in Table 9.

[0309] Examples 9-1 to 9-2 and Comparative Example 9-1 were all prepared to have a concentration of 1.0 mg / mL of hyaluronic acid derivative and 0.5 mg / mL of hGH. After 24 hours, clear solutions were obtained for Examples 9-1 to 9-2. However, only Comparative Example 9-1 became cloudy after incubation at 37°C for 24 hours, making it difficult to obtain a complex solution of hyaluronic acid derivative and hGH.

[0310] [Table 9]

[0311] Next, the hGH compounding rate for Examples 9-1 to 9-2 was evaluated using the GPC method shown below.

[0312] [GPC measurement conditions] Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: UV (280nm) Temperature: 30℃

[0313] Samples of hGH alone at concentrations of 0.50 mg / mL and 0.25 mg / mL were prepared and subjected to GPC measurement. At this time, a peak for hGH alone was confirmed at 11.5 minutes. When hGH was compounded and incorporated into the hyaluronic acid derivative composition, the peak area value of hGH alone at 11.5 minutes decreased. The concentration of hGH encapsulated in the hyaluronic acid derivative composition was calculated as the hGH compounding rate based on the rate of decrease in peak area. It was also confirmed that the peak area value derived from the hyaluronic acid derivative composition increased in the hyaluronic acid derivative composition containing hGH. From the hGH compounding rate calculated above, the actual parts by mass of (C) active ingredient (hGH) per 100 parts by mass of hyaluronic acid derivative are shown in Table 9.

[0314] These results show that the hyaluronic acid derivative composition containing polyethylene glycol 400, an association promoter, efficiently conjugated the protein drug hGH. Long-term sustained-release functionality was expected not only for poorly soluble drugs but also for water-soluble drugs. Furthermore, it was inferred that long-term sustained-release would be possible for many modalities by applying this composition to protein drugs in addition to peptide drugs such as CyA.

[0315] On the other hand, the hGH-hyaluronic acid derivative pharmaceutical composition prepared in Comparative Example 9-1 yielded a cloudy sample, possibly because THF inhibited the compounding process. It is presumed that the hGH did not compound well with the hyaluronic acid derivative. Based on the above, it is expected that a protein-hyaluronic acid derivative pharmaceutical composition can be obtained in which complexation with hGH is efficiently carried out in the presence of an association promoter, and which can be released sustained over a long period in vivo.

[0316] <Test Example 10> [Synthesis Example 3] Based on Test Example 6, the degree of association promotion of hyaluronic acid derivatives (10k HA-C6-Chol-40%) obtained by the same method as in Synthesis Example 2 was evaluated using GPC.

[0317] (GPC measurement conditions) Equipment: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (Differential Refractive Index) Temperature: 30℃

[0318] Using a method similar to that in Test Example 3, the area ratio A2 / A1 used to evaluate the degree of association promotion of hyaluronic acid derivatives was calculated.

[0319] The hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was completely dissolved by adding 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1.0 mg / mL and stirring overnight. The area value calculated by GPC evaluation of this sample was designated as A1.

[0320] [Example 10-1] The hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was dissolved in sterile water for injection at a concentration of 36.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 10-1 to 10-9. In a separate vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 10 μL of 10 mg / mL polysorbate 80 aqueous solution while stirring, and finally 390 μL of sterile water for injection was added. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL, and then subjected to GPC measurement.

[0321] [Example 10-2] First, a 100 mM PB (pH 7.4) phosphate buffer solution was prepared using phosphate buffer powder (Fujifilm Wako Co., Ltd.: 167-14491 for Biochemistry).

[0322] Next, the following solution was prepared using the solution prepared in Example 10-1. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, then 50 μL of 10 mg / mL polysorbate 80 aqueous solution was added and mixed while stirring, 100 μL of 100 mM PB phosphate buffer was added, and finally 250 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution to a final concentration of 10 mM PB. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0323] [Example 10-3] The following solution preparations were made using the solution prepared in Example 10-1. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 50 μL of 10 mg / mL polysorbate 80 aqueous solution while stirring. Finally, 350 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0324] [Example 10-4] Next, the following solutions were prepared using the solutions prepared in Examples 10-1 and 10-2. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 100 μL of polyethylene glycol 400 (Nacalai Tesque) while stirring, then 100 μL of 100 mM PB phosphate buffer, and finally 200 μL of sterile water for injection to prepare the hyaluronic acid derivative aqueous solution to a final concentration of 10 mM PB. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0325] [Example 10-5] Next, the solution prepared in Example 10-1 was used to prepare the following solution. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, then 100 μL of polyethylene glycol 400 (Nacalai Tesque) was added and mixed while stirring, and finally 300 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0326] [Example 10-6] Next, the solution prepared in Example 10-1 was used to prepare the following solution. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 300 μL of polyethylene glycol 400 (Nacalai Tesque) while stirring, and finally 100 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0327] [Example 10-7] First, Poloxamer 338 (Aldrich) was dissolved in sterile water for injection to a concentration of 150 mg / mL. Next, the solution prepared in Example 10-1 was used to prepare the following solution. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 100 μL of 150 mg / mL Poloxamer 338 aqueous solution while stirring, and finally 300 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL and then subjected to GPC measurement.

[0328] [Example 10-8] The following solutions were prepared using the solutions prepared in Examples 10-1, 10-2, and 10-7. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 300 μL of Poloxamer 338 aqueous solution while stirring. 100 μL of 100 mM PB phosphate buffer was then added to prepare the hyaluronic acid derivative aqueous solution to a final concentration of 10 mM PB. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL before being subjected to GPC measurement.

[0329] [Example 10-9] The following solutions were prepared using the solutions prepared in Examples 10-1 and 10-7. 600 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 300 μL of 150 mg / mL Poloxamer 338 aqueous solution while stirring. Finally, 100 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative, and then subjected to GPC measurement.

[0330] [Example 10-10] First, Chol-PEG600 (Avanti) was dissolved in sterile water for injection at a concentration of 50 mg / mL. Next, the hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was dissolved in sterile water for injection at a concentration of 10.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 10-10 to 10-12. 750 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 2.8 μL of 50 mg / mL Chol-PEG600 aqueous solution while stirring, and finally, 747.2 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL, and then subjected to GPC measurement.

[0331] [Examples 10-11] The following solutions were prepared using the solution prepared in Example 10-10. 750 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 11.2 μL of 50 mg / mL Chol-PEG600 aqueous solution while stirring. Finally, 738.8 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0332] [Examples 10-12] The following solutions were prepared using the solution prepared in Example 10-10. 750 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 28 μL of 50 mg / mL Chol-PEG600 aqueous solution while stirring, and finally 722 μL of sterile water for injection was added to prepare the hyaluronic acid derivative aqueous solution. After incubation at 20°C for 24 hours, the solution was diluted with 10 mM PB (phosphate buffer, pH 7.4) to a concentration of 1 mg / mL of hyaluronic acid derivative and then subjected to GPC measurement.

[0333] Using the same method as in Test Example 3, the area value A2, which is the area enclosed by the chromatograms and baselines of Examples 10-1 to 10-12, was calculated, and the area ratio A2 / A1 is shown in Table 10.

[0334] [Table 10]

[0335] These results suggest that the association of hyaluronic acid derivatives in Examples 10-1 to 10-12 was promoted by polysorbate 80, polyethylene glycol 400, poloxamer 338, and chol-PEG600. Similar to polysorbate 80 and polyethylene glycol 400, no GPC peaks were detected for poloxamer 338 or chol-PEG600 alone at the peak position of the hyaluronic acid derivatives.

[0336] <Test Example 11> As Test Example 11, we formulated cyclosporine (CyA), a poorly water-soluble peptide.

[0337] [Example 11-1] A CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing a synthesis promoter (polysorbate 80). The following operations were performed at 20°C, under room temperature conditions. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in a 10% sucrose aqueous solution at a concentration of 12.0 mg / mL.

[0338] Afterward, the mixture was stirred at 20°C for 24 hours to dissolve. For the 10% sucrose solution, sucrose powder (manufactured by Fujifilm Wako Co., Ltd., for manufacturing purposes only) was thoroughly dissolved in sterile water for injection, and then the solution was sterile filtered through a 0.22 μm filter (membrane material: PTFE). In a separate vial, 10.0 mg of powdered CyA (Tokyo Chemical Industry Co., Ltd., product number: C2408) was weighed, and then 0.50 mL of a polysorbate 80 aqueous solution dissolved in sterile water for injection at a concentration of 100 mg / mL was added, and the mixture was stirred with a stirrer tip to disperse the CyA.

[0339] Next, 3.75 mL of a 12 mg / mL aqueous solution of hyaluronic acid derivative was added to the vial containing the CyA suspension while stirring, followed by the addition of 0.75 mL of a 10% aqueous solution of sucrose, after which the drugs were compounded. The solution of the hyaluronic acid derivative pharmaceutical composition containing the association promoter, in which all CyA was compounded, was visually clear. Furthermore, no precipitates were observed during 2 weeks of refrigerated storage. The final formulation composition is shown in Table 11.

[0340] [Comparative Example 11-1] A CyA preparation was prepared using α-cyclodextrin. The following procedures were performed at 20°C and room temperature. First, 2.0 g of sucrose and 2.0 g of α-Cyclodextrin (manufactured by Tokyo Chemical Industry Co., Ltd.) were weighed into a beaker and dissolved in approximately 15 mL of sterile water for injection. The solution was transferred to a graduated cylinder and made up to 20 mL. Next, the solution was filtered through a 0.45 μm filter to obtain a 10% sucrose / 10% α-Cyclodextrin aqueous solution. 2.0 mg of Cyclosporin A (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed into a conical tube, and 10 mL of the 10% sucrose / 10% α-Cyclodextrin aqueous solution was added and dissolved by stirring. Finally, the solution was filtered through a 0.2 μm filter to obtain the administration solution. The final formulation composition is shown in Table 11.

[0341] [Comparative Example 11-2] A CyA formulation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%). The following operations were performed at 20°C, under room temperature conditions. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours.

[0342] Next, 200.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed into another vial, and then 1.0 mL of ethanol was added to homogeneously dissolve the CyA. After this, 0.0225 mL of the 200 mg / mL CyA-containing ethanol solution was added dropwise to 3.75 mL of a 12 mg / mL hyaluronic acid derivative aqueous solution, followed by the addition of 1.2275 mL of a 10% sucrose aqueous solution to compound the drugs. The mixture was then slowly stirred at room temperature for more than 12 hours to homogenize it. The solution was visually clear, and it was confirmed that the powder was completely dissolved before it was used as the administration solution. The final formulation composition is shown in Table 11.

[0343] [Table 11]

[0344] <Test Example 12> Pharmacokinetic studies of CyA were conducted in rats. Solution formulations consisting of the hyaluronic acid derivative pharmaceutical compositions prepared in Example 11-1 and Comparative Example 11-2, as well as the solution formulation of Comparative Example 11-1, were administered subcutaneously to normal rats (SD, 6 weeks old, male) using a 25G needle at the doses (mg / kg) shown in Table 12.

[0345] Blood samples were collected from the subclavian vein at time points after administration. Equal volumes of 0.1% ZnSO4 aqueous solution were added to the whole blood for hemolysis, followed by pretreatment using a Micro Volume QuEChERS Kit. The supernatant after centrifugation was filtered and measured by LC-MS / MS. Figure 4 shows the blood concentration profiles of CyA after administration of various CyA preparations, as well as the blood concentration profiles of CyA in Comparative Example 1. In Figure 4, Example 12-1 is the case where the solution preparation consisting of the hyaluronic acid derivative pharmaceutical composition of Example 11-1 was administered. Comparative Example 12-2 is the case where the solution preparation consisting of the hyaluronic acid derivative pharmaceutical composition of Comparative Example 11-2 was administered, and Comparative Example 12-1 is the case where the solution preparation of Comparative Example 11-1 was administered.

[0346] Also, the highest plasma concentration (C max The values ​​used were measured values. Area under the blood concentration-time curve (AUC) 0→∞ ) and area under the blood concentration-first moment curve (AUMC 0→∞ The area up to the detectable measurement time is calculated using the linear trapezoidal method, and the area thereafter up to infinity is calculated using the vanishing rate constant (k) approximately calculated from the three points before the detection limit. e The calculation was performed using the following formula. Finally, the average dwell time (MRT) was calculated using the following formula, and its value is shown in Table 12.

[0347]

number

[0348] [Table 12]

[0349] In Example 12-1, a formulation containing a hyaluronic acid derivative association promoter, the Cmax / Dose value, an indicator of abnormal initial release, was smaller and the MRT value was larger compared to Comparative Example 12-2, which did not contain the association promoter. When the sustained-release properties were evaluated using formula (A), Example 12-1 was 2.49 compared to Comparative Example 12-2. This indicates that the drug remained in the bloodstream for a longer period. From the above, it was confirmed that the hyaluronic acid derivative composition of the present invention has superior sustained-release properties for a longer period. <Test Example 13> A hyaluronic acid derivative pharmaceutical composition was manufactured. The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours, and this solution was used in Examples 13-1 to 13-2 and Comparative Example 13-1. The following operations were performed at 20°C, under room temperature conditions.

[0350] [Example 13-1] First, polysorbate 80 (Thermo Scientific) was dissolved in sterile water for injection to a concentration of 10 mg / mL. Next, glycerin (Nacalai Tesque, product code: 17045-94) was dissolved in sterile water for injection to a concentration of 170 mg / mL. Furthermore, 1 mL of 0.5 mol / L EDTA solution (pH 8.0) (Nacalai Tesque, product code: 06894-14) was added to 28.240 mL of sterile water for injection to prepare a 5.0 mg / mL EDTA buffer solution.

[0351] Here, 416.7 μL of the above hyaluronic acid derivative aqueous solution was added to a 6 ml sterile vial, followed by the addition of 100 μL of 10 mg / mL polysorbate 80 aqueous solution while stirring, then 318.3 μL of sterile water for injection was added, and finally 15 μL of 5.0 mg / mL EDTA buffer was added to prepare the hyaluronic acid derivative aqueous solution. At this time, the EDTA concentration was 0.075 mg / mL, the glycerin concentration was 25.5 mg / mL, the polysorbate 80 concentration was 1 mg / mL, and the hyaluronic acid derivative concentration was 5 mg / mL. After stirring at 20°C for 24 hours, the solution was diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative and subjected to GPC measurement. A hyaluronic acid derivative solution was prepared with the same composition except that it did not contain an association promoter, and the obtained area value was taken as A1. The association promotion degree A2 / A1 due to the association promoter was calculated and is shown in Table 13.

[0352] Next, a CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing a synthesis promoter (polysorbate 80). First, 2.5 mg of CyA was weighed into a 6 mL sterile vial. Then, 450 μL of the hyaluronic acid derivative solution prepared in Example 13-1 was added and stirred at 20°C for 24 hours. Undissolved CyA powder was observed, and the process proceeded to the next step when the mixture was saturated. The CyA-containing hyaluronic acid derivative pharmaceutical composition prepared above was sterile filtered through a 13 mmφ 0.22 μm PES filter to remove any drug that had precipitated and could not be encapsulated in the hyaluronic acid derivative. The CyA concentration in the resulting filtrate was quantified by reverse-phase HPLC. This experiment was performed with n=2, yielding 1.50 mg / mL and 1.41 mg / mL. The average values ​​of the CyA concentrations in the formulations are summarized in Table 13. In addition, the precipitation status was verified using the same method as in Test Example 7. The results are also summarized in Table 13.

[0353] [Example 13-2] An aqueous solution of a hyaluronic acid derivative containing an association promoter was prepared in the same manner as in Example 13-1, with all other concentrations being the same except for the polysorbate concentration of 3 mg / mL. At this time, the EDTA concentration was 0.075 mg / mL, the glycerin concentration was 25.5 mg / mL, the polysorbate 80 concentration was 3 mg / mL, and the hyaluronic acid derivative concentration was 5 mg / mL. Subsequently, the mixture was stirred at 20°C for 24 hours and diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative, and then subjected to GPC measurement. A hyaluronic acid derivative solution was prepared with the same composition except that it did not contain an association promoter, and the obtained area value was set as A1. The degree of association promotion by the association promoter was calculated in the same manner as in Example 13-1 and is shown in Table 13.

[0354] Next, a CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing a synthesis promoter (polysorbate 80). First, 2.5 mg of CyA was weighed into a 6 mL sterile vial. Then, 450 μL of the hyaluronic acid derivative solution prepared in Example 13-2 was added and stirred at 20°C for 24 hours. Undissolved CyA powder was observed, and the process proceeded to the next step when the mixture was saturated. The CyA-containing hyaluronic acid derivative pharmaceutical composition prepared above was sterile filtered through a 13 mmφ 0.22 μm PES filter to remove any drug that had precipitated and could not be encapsulated in the hyaluronic acid derivative. The CyA concentration in the resulting filtrate was quantified by reverse-phase HPLC. This experiment was performed with n=2, yielding 1.89 mg / mL and 1.87 mg / mL. The average values ​​of the CyA concentrations in the formulations are summarized in Table 13. In addition, the precipitation status was verified using the same method as in Test Example 7. The results are also summarized in Table 13.

[0355] [Comparative Example 13-1] An aqueous solution of hyaluronic acid derivative was prepared in the same manner as in Example 13-1, with all concentrations being the same except for the polysorbate concentration being 0 mg / mL. At this time, the EDTA concentration was 0.075 mg / mL, the glycerin concentration was 25.5 mg / mL, the polysorbate 80 concentration was 0 mg / mL, and the hyaluronic acid derivative concentration was 5 mg / mL. Subsequently, the mixture was stirred at 20°C for 24 hours and diluted with sterile water for injection to a concentration of 1 mg / mL of hyaluronic acid derivative before being subjected to GPC measurement.

[0356] Next, a CyA preparation was prepared using a hyaluronic acid derivative (35k HA-C6-Chol-19%). First, 2.5 mg of CyA was weighed into a 6 mL sterile vial. Then, 450 μL of the hyaluronic acid derivative solution prepared in Comparative Example 13-1 was added and stirred at 20°C for 24 hours. Undissolved CyA powder was observed, and the process proceeded to the next step when the solution was saturated. Sterile filtration of the CyA-containing hyaluronic acid derivative pharmaceutical composition prepared above was attempted using a 13 mmφ 0.22 μm PES filter, but filtration proved difficult.

[0357] Here, we also examined the GPC peak of the association promoter alone under conditions that did not contain the hyaluronic acid derivative, but no peaks originating from the association promoter were detected at the peak position of the hyaluronic acid derivative. The final composition is shown in Table 13.

[0358] [Table 13]

[0359] The results showed that polysorbate 80 promoted the association of hyaluronic acid derivatives (A2 / A1 = 1.90~2.10) even in a glycerin-containing EDTA solution used as an isotonic agent. Furthermore, the results suggested that the enhanced association improved the sterile filterability of the hyaluronic acid derivative pharmaceutical composition.

[0360] Furthermore, the amount of CyA, the active ingredient, was 29.1 or 37.6 parts by mass per 100 parts by mass of hyaluronic acid derivative, confirming that it could be solubilized at a high concentration. This suggests that longer-term sustained release can be expected through ophthalmic, intramuscular, or subcutaneous administration.

[0361] <Test Example 14> Using a method similar to that in Test Example 3, the degree of association promotion was evaluated for another association promoter using GPC.

[0362] [Example 14-1] The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. 416.7 μL of the 12.0 mg / mL aqueous solution of the hyaluronic acid derivative was added to a 1.5 mL Eppendorf tube, followed by 50 μL of polyvinyl alcohol (degree of polymerization 500, Nacalai Tesque, product number: 11738-62) dissolved in sterile water for injection at a concentration of 100 mg / mL. Then, sterile water for injection was added to adjust the total volume to 1000 μL (Table 14). After incubation at 20°C for 2 hours, the solution was diluted 5-fold with sterile water for injection to a concentration of 1 mg / mL, and then subjected to GPC measurement.

[0363] [Example 14-2] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was dissolved in sterile water for injection at a concentration of 62.6 mg / mL by stirring at 20°C for 24 hours. 79.9 μL of the 62.6 mg / mL aqueous solution of the hyaluronic acid derivative was added to a 1.5 mL Eppendorf tube, followed by 50 μL of polyvinyl alcohol (degree of polymerization 500, Nacalai Tesque, product number: 11738-62) dissolved in sterile water for injection at a concentration of 100 mg / mL. Then, sterile water for injection was added to adjust the total volume to 1000 μL (Table 14). After incubation at 20°C for 2 hours, the solution was diluted 5-fold with sterile water for injection to a concentration of 1 mg / mL, and then subjected to GPC measurement.

[0364] [Example 14-3] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in sterile water for injection at a concentration of 40.0 mg / mL by stirring at 20°C for 24 hours. 125.0 μL of the 40.0 mg / mL aqueous solution of the hyaluronic acid derivative was added to a 1.5 mL Eppendorf tube, followed by 50 μL of polyvinyl alcohol (degree of polymerization 500, Nacalai Tesque, product number: 11738-62) dissolved in sterile water for injection at a concentration of 100 mg / mL. Then, sterile water for injection was added to adjust the total volume to 1000 μL (Table 14). After incubation at 20°C for 2 hours, the solution was diluted 5-fold with sterile water for injection to a concentration of 1 mg / mL, and then subjected to GPC measurement.

[0365] Here, we also examined the GPC peak of the association promoter alone under conditions that did not contain hyaluronic acid derivatives, but no peaks derived from the association promoter PVA were detected at the same peak position as the hyaluronic acid derivatives.

[0366] [Examples 14-4 to 14-6] The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. Furthermore, aqueous solutions of the hyaluronic acid derivative containing the association promoter were prepared using the 10 mg / mL polysorbate 80 aqueous solution, 170 mg / mL glycerol aqueous solution, sterile water for injection, and 5.0 mg / mL LEDTA solution prepared in Example 13-1, in proportions to the concentrations shown in Table 14. The samples were then incubated at 20°C for 24 hours before being subjected to GPC measurement.

[0367] [Examples 14-7 to 14-8] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was dissolved in sterile water for injection at a concentration of 62.6 mg / mL by stirring at 20°C for 24 hours. Furthermore, aqueous solutions of the hyaluronic acid derivative containing the association promoter were prepared using the 10 mg / mL polysorbate 80 aqueous solution, 170 mg / mL glycerol aqueous solution, sterile water for injection, and 5.0 mg / mL LEDTA solution prepared in Example 13-1, in proportions to the concentrations shown in Table 14. The samples were then incubated at 20°C for 24 hours before being subjected to GPC measurement.

[0368] [Examples 14-9 to 14-11] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in sterile water for injection at a concentration of 40.0 mg / mL by stirring at 20°C for 24 hours. Furthermore, aqueous solutions of the hyaluronic acid derivative containing the association promoter were prepared using the 10 mg / mL polysorbate 80 aqueous solution, 170 mg / mL glycerol aqueous solution, sterile water for injection, and 5.0 mg / mL LEDTA solution prepared in Example 13-1, in proportions to the concentrations shown in Table 14. The samples were then incubated at 20°C for 24 hours before being subjected to GPC measurement.

[0369] [Examples 14-4 to 14-11] In all cases, the hyaluronic acid derivative was diluted five-fold with sterile water for injection to a concentration of 1 mg / mL before being subjected to GPC measurement.

[0370] Here, we also examined the GPC peak of the association promoter alone under conditions that did not contain hyaluronic acid derivatives, but no peaks derived from polysorbate 80, the association promoter, were detected at the peak position of the hyaluronic acid derivative. The final composition and degree of association promotion are shown in Table 14.

[0371] [Table 14]

[0372] These results show that PVA-mediated aggregation promotion was observed even for hyaluronic acid derivatives with different molecular weights and cholesteryl group introduction rates, as seen in Examples 14-1 to 14-3. Therefore, it can be expected that expanding the hydrophobic portion within the nanoparticles will increase the amount of poorly soluble drugs solubilized and extend the sustained-release period after in vivo administration.

[0373] Furthermore, the results from Examples 14-4 to 14-11 showed that hyaluronic acid derivatives with different molecular weights and cholesteryl group introduction rates, as well as the inclusion of other additives such as glycerol and EDTA, did not inhibit the association promotion. JP6271672B2 mentions that EDTA plays a role as a protease inhibitor and can be included as an API stabilizer, and Japanese Patent Publication No. 4758893 describes how the inclusion of glycerol improves antimicrobial and preservative efficacy. This demonstrates that even in the presence of such antimicrobial and preservative agents and API stabilizers, the association promoter interacts effectively to promote the association of hyaluronic acid derivatives. The association promotion effect is expected to be present in various other buffers, antimicrobial and preservative agents, and stabilizers, and by expanding the hydrophobic portion within the nanoparticles, it is expected to increase the solubilization of poorly soluble drugs and extend the sustained-release period after in vivo administration.

[0374] <Test Example 15> A hyaluronic acid derivative pharmaceutical composition was prepared using semaglutide (peptide).

[0375] [Example 15-1] The lyophilized hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in sterile water for injection at a concentration of 12.0 mg / mL by stirring at 20°C for 24 hours. Then, it was dissolved in sterile water for injection to a glycerol concentration of 170 mg / mL in another vial. In yet another vial, 0.5 mol / l-EDTA solution (pH 8.0) (Nacalai Tesque) was used to dilute with sterile water for injection to an EDTA concentration of 5 mg / mL. In yet another vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL. Using the solutions prepared above, aqueous solutions of the hyaluronic acid derivative containing the association promoter were prepared.

[0376] Specifically, 416.7 μL of an aqueous solution of 12.0 mg / mL hyaluronic acid derivative (35k HA-C6-Chol-19%) was added, followed by 100 μL of an aqueous solution of 10 mg / mL polysorbate 80, which was added while stirring with a stirrer, and then 150 μL of a glycerol solution of 170 mg / mL. After that, 318.3 μL of sterile water for injection and 15 μL of a 5 mg / mL EDTA solution were added and the mixture was stirred overnight. In addition, semaglutide powder (manufactured by Funakoshi, product code: AG-CP3-0032) was dissolved in 10 mM phosphate buffer in an Eppendorf tube to a concentration of 1 mg / mL, and after preparation, 150 μL of the aqueous solution of the hyaluronic acid derivative containing the association promoter and 150 μL of the 1 mg / mL semaglutide solution were mixed, and then the mixture was incubated at 20°C for 1 hour to promote complexation with semaglutide. The final formulation was adjusted to have the concentrations listed in Table 15.

[0377] [Example 15-2] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in sterile water for injection at a concentration of 40.0 mg / mL by stirring at 20°C for 24 hours. Then, it was dissolved in sterile water for injection to a glycerol concentration of 170 mg / mL in another vial. In yet another vial, 0.5 mol / l-EDTA solution (pH 8.0) (Nacalai Tesque) was used to dilute with sterile water for injection to an EDTA concentration of 5 mg / mL. In yet another vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL. Using the solutions prepared above, aqueous solutions of the hyaluronic acid derivative containing the association promoter were prepared.

[0378] Specifically, 125 μL of an aqueous solution of 40.0 mg / mL hyaluronic acid derivative 10k HA-C6-Chol-30%) was added, followed by 100 μL of an aqueous solution of 10 mg / mL polysorbate 80 while stirring with a stirrer, and then 150 μL of a 170 mg / mL glycerol solution was added. After that, 610 μL of sterile water for injection and 15 μL of a 5 mg / mL EDTA solution were added and stirred overnight. In addition, semaglutide powder (manufactured by Funakoshi, product code: AG-CP3-0032) was dissolved in 10 mM phosphate buffer in an Eppendorf tube to a concentration of 1 mg / mL, and after preparation, 150 μL of the aqueous solution of the hyaluronic acid derivative containing the association promoter and 150 μL of the 1 mg / mL semaglutide solution were mixed, and then the mixture was incubated at 20°C for 1 hour to promote complexation with semaglutide. The final formulation was adjusted to have the concentrations listed in Table 15.

[0379] In all of Examples 15-1 to 15-2, the hyaluronic acid derivative was adjusted to a concentration of 2.5 mg / mL and the semaglutide to 0.5 mg / mL. For Examples 15-1 to 15-2, the semaglutide compounding rate was evaluated using the GPC method shown below.

[0380] [GPC measurement conditions] Equipment: HLC8320-GPC (manufactured by Tosoh Corporation) Column: G3000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, part number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: UV (220nm) Temperature: 30℃

[0381] Samples of semaglutide alone at concentrations of 0.50 mg / mL and 0.25 mg / mL were prepared and subjected to GPC measurement. At this time, a peak for semaglutide alone was confirmed at 9.0 min. When combined with the hyaluronic acid derivative composition and incorporated, the peak area value of semaglutide alone at 9.0 min decreased. The concentration of semaglutide encapsulated in the hyaluronic acid derivative composition was calculated as the semaglutide complexation rate based on the rate of decrease in peak area. From the semaglutide complexation rate calculated above, the actual parts by mass of (C) active ingredient (semaglutide) per 100 parts by mass of hyaluronic acid derivative are shown in Table 15.

[0382] These results show that the hyaluronic acid derivative composition containing polysorbate 80, an association promoter, efficiently conjugated the peptide drug semaglutide. JP6271672B2 mentions that EDTA can act as a protease inhibitor and be included as an API stabilizer. This suggests that even with such stabilizers, association promoters can interact properly and be encapsulated in the hyaluronic acid derivative. Based on these results, it is inferred that long-term sustained release will be possible for many modalities by applying this to long-chain peptide drugs in addition to cyclic peptide drugs like CyA. Furthermore, it is thought that other stabilizers, preservatives such as m-cresol and phenol, and other isotonic agents such as D-mannitol and propylene glycol can also be utilized. Based on the above, it is expected that compounding with semaglutide can be efficiently carried out in the presence of an association promoter, and that a long-chain peptide-hyaluronic acid derivative pharmaceutical composition capable of long-term sustained release in vivo can be obtained.

[0383] [Table 15]

[0384] <Synthesis of hyaluronic acid derivatives> [Synthesis example 1B] Hyaluronic acid derivatives were prepared according to steps 1B to 3B.

[0385] [Process 1B] (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1B-1 and then 1B-2.

[0386] (Process 1B-1) To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred under ice cooling for 30 minutes. The temperature was then raised to room temperature (approximately 25°C), and the mixture was stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed 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 removed under reduced pressure.

[0387] (Process 1B-2) The obtained residue was dissolved in ethyl acetate (40 mL), and 40 mL of 4N hydrochloric acid / ethyl acetate solution was added and stirred overnight at room temperature (approximately 25°C). The resulting precipitate was collected by centrifugation. The obtained solid was washed four times with ethyl acetate and dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0388] [Process 2B] (Preparation of tetrabutylammonium (TBA) salt of hyaluronic acid) Hyaluronic acid TBA salt (HA-TBA) was prepared according to the following steps 2B-1 and then 2B-2.

[0389] (Process 2B-1) DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed approximately three times with ultrapure water by decantation. Approximately 1.5 times the molar equivalent of 40% by mass of tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added relative to the cation exchange capacity of the resin, and the mixture was stirred for 30 minutes. After removing the excess TBA-OH solution by decantation, the mixture was further washed with excess ultrapure water to obtain a TBA-chlorinated cation exchange resin.

[0390] (Process 2B-2) Sodium hyaluronate salt (HA-Na) with a molecular weight of 10,000 (10 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of cation exchange resin, which had been TBA-chlorinated in "Step 2B-1," was added in an amount equivalent to 5 times the molar amount of HA units (unit molecular weight 401.3) in terms of the resin's ion exchange capacity. After stirring for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA) as a white solid.

[0391] [Process 3B] An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in "Step 2B-2" was prepared. Then, Chol hydrochloride was added in a molar ratio of 44 / 100 relative to the disaccharide repeating units (HA units) present in the HA-TBA synthesized in "Step 1B". Next, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added in a molar ratio of 48 / 100 relative to the HA units, and the mixture was stirred overnight at room temperature (approximately 25°C). The reaction solution was dialyzed in the following order: 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water (Spectrapore 7, molecular weight cutoff (MWCO): 3,500). The resulting dialysate was freeze-dried to obtain the target product (HA-C6-Chol) as a white solid. 1 In the 1H-NMR spectrum, peaks originating from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and peaks originating from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were observed, and the cholesterol introduction rate was 44%.

[0392] <Test Example 1B> [Example 1B-1] Using a hyaluronic acid derivative (10k HA-C6-Chol-44%), cyclosporine (CyA), a poorly water-soluble peptide, was formulated from powder.

[0393] [Step to obtain aqueous solution (II)] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. This yielded aqueous solution (II) of the hyaluronic acid derivative.

[0394] [Step to obtain dispersion (I)] In a separate vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL. Next, 4.0 mg of powdered CyA (manufactured by Tokyo Chemical Industry Co., Ltd., product number: C2408) was weighed into another vial, and then 80 μL of 10 mg / mL polysorbate 80 was added. This yielded a CyA dispersion (I) in which CyA was dispersed in polysorbate 80.

[0395] [Mixing process] A stirrer tip was placed in the vial and 0.30 mL of aqueous solution (II), an aqueous solution of hyaluronic acid derivative, was added while stirring. Then, CyA dispersion was added in a proportion that brought the total volume to 0.650 mL. After this, the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 16.

[0396] [Table 16]

[0397] Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement, as described later. The CyA concentrations in the filtered formulation are shown in Table 17.

[0398] [HPLC quantitative analysis conditions: Reverse-phase chromatography analysis conditions] Column: InertSustain C18, particle size 5μm × inner diameter 4.6mm × length 150mm Column temperature: 40℃ Mobile phase: Eluent A 0.1% TFA / acetonitrile Eluent B 0.1%TFA / water Mobile phase ratio: Eluent A: Eluent B=9:1 Flow rate: 1mL / min Injection volume: 30μL Detector: UV (210nm)

[0399] [Examples 1B-2 to 1B-11] CyA formulations were prepared using hyaluronic acid derivatives and solubilizers, following the same procedure as in Example 1B-1, except for changes in the type and amount of solubilizer added. For each formulation, the theoretical concentration in the formulation calculated from the amount added and the actual drug concentration in the formulation quantified by HPLC measurement are shown in Tables 17-18.

[0400] [Comparative example 1B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. 4.0 mg of powdered CyA was weighed into a separate vial, and then 0.30 mL of the 36.0 mg / mL aqueous solution of the hyaluronic acid derivative was added. A stirrer tip was placed in the vial, and sterile water for injection was added to bring the total volume to 0.650 mL. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. It was confirmed that all of the filtrate was clear. Table 4 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0401] [Comparative example 1B-2] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. 4.0 mg of powdered CyA was weighed into a separate vial, and then 0.1783 mL of the 36.0 mg / mL aqueous solution of the hyaluronic acid derivative was added. A stirrer tip was placed in the vial, and sterile water for injection was added to bring the liquid volume to 0.650 mL. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. It was confirmed that all of the filtrate was clear. Table 19 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0402] [Comparative example 1B-3] The sodium hyaluronate salt (HA-Na) raw material with a molecular weight of 10,000 (10 kDa) used in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 55 mg / mL. Then, polysorbate 20 and sodium hyaluronate were appropriately diluted with sterile water for injection to prepare aqueous solutions at the concentrations shown in Table 4. 4.0 mg of powdered CyA was weighed into a separate vial, and then 0.65 mL of the HA-Na aqueous solution containing polysorbate 20 was added. A stirrer tip was placed in the vial and the mixture was stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC. Table 19 shows the theoretical concentration in the formulation calculated from the amount added and the actual drug concentration in the formulation quantified by HPLC.

[0403] [Comparative example 1B-4] Polysorbate 80 was diluted with sterile water for injection to a concentration of 100 mg / mL. 4.0 mg of powdered CyA was weighed into a separate vial, and then 0.65 mL of the above-mentioned 100.0 mg / mL polysorbate 80 was added. A stirrer tip was placed in the vial and the mixture was stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove the precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 19 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0404] [Comparative Examples 1B-5~1B~12] The concentrations of the solubilizing agent were as shown in the table below. Otherwise, formulation and quantification of the drug concentration were carried out using the same procedure as in Comparative Example 1B-4 or Example 1B-1. The results are shown in Tables 19-20.

[0405] The Span83 used in Comparative Example 1B-9 was manufactured by Tokyo Chemical Industry Co., Ltd., and the sucrose stearate ester used in Comparative Examples 1B-10 to 1B-12 was manufactured by Mitsubishi Chemical Corporation.

[0406] [Table 17]

[0407] [Table 18]

[0408] [Table 19]

[0409] [Table 20]

[0410] As shown in Tables 17-18 above, when formulating poorly water-soluble drugs, solubilization can be achieved without using organic solvents, reducing the amount of conventional highly toxic solubilizers such as polysorbate 80 and cremofol EL used. Furthermore, the solubilizing aid promotes hydrophobic interactions between the steryl groups of the hyaluronic acid derivative, thereby increasing the amount of hydrophobic drug that can be carried by expanding the hydrophobic region of the hyaluronic acid derivative. Moreover, it is presumed that the solubilizing aid within the hyaluronic acid derivative composition acts as a highly mobile hydrophobic region, not only in the hydrophobic region chemically bound to the hyaluronic acid polymer, but also in other areas, enabling the solubilization of powdered drugs at high concentrations. As a result, while generally the amount of poorly water-soluble drugs solubilized increases in proportion to the amount of solubilizer added, in this invention, a high-concentration formulation of the active ingredient can be prepared despite a reduction in the total mass of the solubilizer.

[0411] In Comparative Examples 1B-1 and 1B-2, when the hyaluronic acid derivative concentration was 9.88 mg / mL, the CyA concentration in the formulation was 0.68 mg / mL. Furthermore, even when the hyaluronic acid derivative concentration was increased to 36 mg / mL, only a slight improvement was observed, and the CyA concentration in the formulation was 3.57 mg / mL.

[0412] In Comparative Example 1B-3, no synergistic effect from the solubilizing agent was observed in hyaluronic acid that was not modified with hydrophobic groups.

[0413] Comparative Examples 1B-4 to 1B-8 represent the solubilizing ability of the solubilizing agent alone. However, when compared to the drug concentration obtained by adding the solubilizing abilities of the hyaluronic acid derivatives alone, the examples in the present invention can significantly improve the solubility of poorly water-soluble drugs.

[0414] Furthermore, upon intensive investigation of the structure of the solubilizing aids, comparative examples 1B-9 to 1B-12 all satisfy the requirement of having 4 or more carbon atoms, but are solubilizing aids that have only one or three ether structures. In all of these cases, the drug and the solubilizing aid could not be stably dissolved in the hyaluronic acid derivative, making filtration extremely difficult.

[0415] Based on these results, a composition comprising a hyaluronic acid derivative and a solubilizing agent containing at least four ether structures (RORs) and satisfying the requirement of having four or more carbon atoms can significantly improve the solubilization ability of poorly water-soluble drugs even when using a powder without using any organic solvents and when the concentration of the solubilizer is reduced, thus providing a simple formulation method and a pharmaceutical composition with low toxicity.

[0416] <Test Example 2B> [Examples 2B-1 to 2B-12] The amount of solubilizing agent added was examined in detail. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection to a concentration of 5 mg / mL. 1.0 mg of powdered CyA was weighed into another vial, and 0.2 mL was added to achieve the solubilizing agent concentration in the final composition of the formulation as shown in Table 21. A stirrer tip was then added and the mixture was stirred.

[0417] Next, 0.8 mL of the 5 mg / mL aqueous solution of the hyaluronic acid derivative was added while stirring, and the mixture was stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 21 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0418] [Comparative Example 2B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection to a concentration of 5 mg / mL. In a separate vial, 1.0 mg of powdered CyA was weighed out, and 0.1 mL of a 10 mg / mL aqueous solution of sodium stearoyl lactylate was added so that the concentration of the solubilizing agent in the final composition of the formulation was as shown in Table 21. A stirrer tip was then added and the mixture was stirred.

[0419] Next, 0.8 mL of the 5 mg / mL aqueous solution of the hyaluronic acid derivative was added while stirring, followed by the addition of 0.1 mL of sterile water for injection. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 21 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0420] [Comparative Examples 2B-2 to 2B-6] 1.0 mg of powdered CyA was weighed into a vial, and 0.2 mL was added to ensure that the final concentration of the solubilizing agent in the formulation was as shown in Table 21. A stirrer tip was then added and the mixture was stirred. Subsequently, 0.8 mL of sterile water for injection was added while stirring, and the mixture was stirred for 24 hours to solubilize the drug using only the solubilizing agent. The mixture was then filtered through a 0.45 μm sterile filter to remove the precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 21 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0421] [Table 21]

[0422] From the results above, solubilizing aids containing at least four ether structures (RORs) and having four or more carbon atoms improved the drug concentration in the formulation, as shown in Examples 2B-1 to 2B-13. On the other hand, sodium stearoyl lactylate, a solubilizing aid that does not contain at least four ether structures (RORs), showed almost no solubilizing effect.

[0423] Furthermore, in Comparative Examples 2B-2 to 2B-6, it was confirmed that the solubilizing agent itself had almost no solubilizing ability. These results suggest that the solubilizing ability can be significantly improved by adding a small amount of solubilizing agent to a hyaluronic acid derivative. As shown in Table 21 above, the solubilizing agent improves the solubilization ability from powder without using organic solvents simply by adding 0.003 parts by mass or more per 100 parts by mass of hyaluronic acid derivative.

[0424] <Test Example 3B> [Example 3B-1 to 3B-14] Next, we examined the available solubilizing agents in detail. Specifically, we investigated whether polyethylene glycol 300 and polyethylene glycol 400, which contain at least 5 ether structures (ROR) and have 12 or more carbon atoms, can be used as solubilizing agents.

[0425] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection to a concentration of 8 mg / mL. In a separate vial, 1.6 mg of powdered CyA was weighed, and the solubilizing agent and sterile water for injection were added to a total volume of 0.5 mL. A stirrer tip was then added and the mixture was stirred. At this time, the concentration of the solubilizing agent in the final hyaluronic acid derivative pharmaceutical composition was adjusted to match the concentration shown in Table 7 below.

[0426] Next, 0.5 mL of the 8 mg / mL aqueous solution of the hyaluronic acid derivative was added while stirring, and the mixture was stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 22 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0427] [Comparative Examples 3B-1 to 3B-6] 1.0 mg of powdered CyA was weighed into a vial, and 0.5 mL of solubilizing agent and sterile water for injection were added. A stirrer tip was then inserted and the mixture was stirred. At this time, the concentration of the solubilizing agent in the final pharmaceutical composition containing the solubilizing agent was adjusted to match the levels shown in Table 22 below.

[0428] Next, 0.5 mL of sterile water for injection was added while stirring, and the mixture was stirred for 24 hours to solubilize the drug using only the solubilizing agent. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 22 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0429] [Table 22]

[0430] Based on the results above, in polyethylene glycol 300 and polyethylene glycol 400, solubilizing aids containing at least four ether structures (ROR) and having four or more carbon atoms improved the drug concentration in the formulation, as shown in Examples 3B-1 to 3B-14. The active ingredient could be solubilized at a higher concentration than the sum of the solubilization amounts of the hyaluronic acid derivative alone and the solubilizing aid alone, demonstrating a synergistic effect.

[0431] <Test Example 4B> [Example 4B-1] Next, a detailed verification of the manufacturing method for hyaluronic acid derivative pharmaceutical compositions was conducted. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL.

[0432] Next, 4.0 mg of powdered CyA was weighed into another vial, and then 160 μL of 10 mg / mL polysorbate 80 was added. While stirring with a stirrer tip in the vial, 0.30 mL of the above 36.0 mg / mL hyaluronic acid derivative aqueous solution was added, and then sterile water for injection was added to bring the total volume to 0.650 mL. After this, the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 23. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1.

[0433] Table 23 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0434] [Example 4B-2] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, polysorbate 80 was diluted with sterile water for injection to a concentration of 10 mg / mL. Subsequently, 320 μL of 10 mg / mL polysorbate 80 was added to another vial. While stirring with a stirrer tip in the vial, 0.60 mL of the above 36.0 mg / mL hyaluronic acid derivative aqueous solution was added, followed by the addition of sterile water for injection to bring the total volume to 1.30 mL. In yet another vial, 4.0 mg of powdered CyA was weighed, and 0.65 mL of the above solubilizing agent-containing hyaluronic acid derivative was added. The mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 20. Next, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 23 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0435] [Table 23]

[0436] It has been found that a hyaluronic acid derivative pharmaceutical composition excellent in increasing the solubility of poorly water-soluble active ingredients in water can be obtained from powder by either a formulation method comprising the steps of (I) dispersing the active ingredient in a solubilizing agent and (II) preparing an aqueous solution of the hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing the solubilizing agent, and mixing (I) and (II); or a formulation method comprising mixing the active ingredient with an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent. Furthermore, it has been found that this manufacturing method is similar even when the solubilizing agent is not polysorbate 80, and high solubilizing ability can be expected regardless of the mixing method.

[0437] <Test Example 5B> [Example 5B-1] Next, we conducted an investigation into the types of poorly water-soluble drugs. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL.

[0438] Next, 1.1 mg of powdered paclitaxel (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed into another vial, and then 0.40 mL of the 36.0 mg / mL aqueous solution of the hyaluronic acid derivative was added. A stirrer tip was placed in the vial and while stirring, 0.40 mL of sterile water for injection was added, followed by 200 μL of 10 mg / mL polysorbate 80. After this, the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 24. Subsequently, the mixture was filtered through a 0.22 μm sterile filter to remove the precipitated paclitaxel, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1.

[0439] Table 24 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0440] [Example 5B-2] The formulation was prepared using the same procedure as in Example 5B-1, except that the poorly water-soluble drug was changed from paclitaxel to fluticasone propionate (manufactured by Tokyo Chemical Industry Co., Ltd.).

[0441] [Comparative Example 5B-1] The formulation was prepared using the same procedure as in Example 5B-1, except that a solubilizing agent was not added.

[0442] [Comparative Example 5B-2] The formulation was prepared using the same procedure as in Example 5B-2, except that a solubilizing agent was not added.

[0443] [Comparative Example 5B-3] The formulation was prepared using the same procedure as in Example 5B-1, except that a hyaluronic acid derivative was not added.

[0444] [Comparative Example 5B-4] The formulation was prepared using the same procedure as in Example 5B-2, except that a hyaluronic acid derivative was not added.

[0445] Table 24 shows the theoretical concentration in the formulation calculated from the amount added and the actual drug concentration in the formulation quantified by HPLC measurement for Examples 5B-1 to 5B-2 and Comparative Examples 5B-1 to 5B-4.

[0446] [Table 24]

[0447] From the results above, solubilizing aids containing at least four ether structures (RORs) and having four or more carbon atoms improved the drug concentration in the formulation, as shown in Examples 5B-1 to 5B-2. On the other hand, Comparative Examples 5B-3 to 5B-4 confirmed that solubilizing aids alone had almost no solubilizing ability for poorly water-soluble drugs. These results indicate that even for low-molecular-weight, poorly water-soluble drugs, adding a small amount of solubilizing aid to a hyaluronic acid derivative can significantly improve its solubilizing ability.

[0448] [Example 6B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, cholesterol-PEG600 (Aldrich: C1145-250MG) was diluted with sterile water for injection to a concentration of 10 mg / mL. Subsequently, 400 μL of 10 mg / mL cholesterol-PEG600 was added to another vial, and while stirring with a stirrer tip in the vial, 0.60 mL of the above 36.0 mg / mL hyaluronic acid derivative aqueous solution was added. Furthermore, 5.0 mg of powdered temsirolimus (manufactured by Tokyo Chemical Industry Co., Ltd., product number: T3574) was weighed into yet another vial, and 0.5 mL of the above hyaluronic acid derivative containing the solubilizing agent was added, and the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table X1. Next, the mixture was filtered through a 0.45 μm sterile filter to remove the precipitated temsirolimus, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 25 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0449] [Comparative Example 6B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. 5.0 mg of temsirolimus (manufactured by Tokyo Chemical Industry Co., Ltd., product number: T3574) was weighed into a separate vial, and then 0.30 mL of the above 36.0 mg / mL aqueous solution of hyaluronic acid derivative was added. A stirrer tip was placed in the vial, and sterile water for injection was added to bring the liquid volume to 0.50 mL. After this, the mixture was stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove the precipitated temsirolimus, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. It was confirmed that all of the filtrate was clear. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0450] [Comparative Example 6B-2] 5.0 mg of temsirolimus (manufactured by Tokyo Chemical Industry Co., Ltd., product number: T3574) was weighed into a vial. Then, a stirrer tip was placed in the vial, and 200 μL of 10 mg / mL cholesterol-PEG600 was added. Water for injection was then added to bring the total volume to 0.50 mL. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated temsirolimus, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. It was confirmed that all filtrates were clear. Table 25 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0451] [Table 25]

[0452] Based on the results above, in the case of cholesterol-PEG600, solubilizing aids containing at least four ether structures (RORs) and having four or more carbon atoms improved the drug concentration in the formulation, as shown in Example 6B-1. The active ingredient could be solubilized at a higher concentration than the sum of the solubilization amounts of the hyaluronic acid derivative alone and the solubilizing aid alone, demonstrating a synergistic effect.

[0453] [Example 7B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, cholesterol-PEG600 (Aldrich: C1145-250MG) was diluted with sterile water for injection to a concentration of 10 mg / mL. Subsequently, 400 μL of 10 mg / mL cholesterol-PEG600 was added to another vial, and while stirring with a stirrer tip in the vial, 0.60 mL of the above 36.0 mg / mL hyaluronic acid derivative aqueous solution was added. In yet another vial, 4.0 mg of powdered CyA was weighed out, 5.0 mg of another substance was weighed out, and 0.5 mL of the above hyaluronic acid derivative containing the solubilizing agent was added, and the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table Y1. Next, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0454] [Example 7B-2] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, cholesterol-PEG600 (Aldrich: C1145-250MG) was diluted with sterile water for injection to a concentration of 10 mg / mL. Subsequently, 10.0 mg of powdered CyA was weighed, followed by the addition of 0.60 mL of the 36.0 mg / mL hyaluronic acid derivative aqueous solution. Then, while stirring with a stirrer tip in the vial, 40 μL of 10 mg / mL cholesterol-PEG600 aqueous solution was added, followed by the addition of 360 μL of sterile water for injection. The mixture was then stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 26. Next, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0455] [Example 7B-3] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. In a separate vial, HP-β-CD (manufactured by Tokyo Chemical Industry Co., Ltd.: H0979) was diluted with sterile water for injection to a concentration of 10 mg / mL. Subsequently, 10.0 mg of powdered CyA was weighed, followed by the addition of 0.60 mL of the 36.0 mg / mL hyaluronic acid derivative aqueous solution. Then, while stirring with a stirrer tip in the vial, 272.2 μL of 10 mg / mL HP-β-CD aqueous solution was added. After that, 127.8 μL of sterile water for injection was added, and the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table Y1. Next, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0456] [Comparative Example 7B-1] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in sterile water for injection at a concentration of 36.0 mg / mL. 10.0 mg of powdered CyA was weighed into a separate vial, and then 0.60 mL of the 36.0 mg / mL aqueous solution of the hyaluronic acid derivative was added. A stirrer tip was placed in the vial, and sterile water for injection was added until the liquid volume reached 1.00 mL. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. It was confirmed that all of the filtrate was clear. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0457] [Comparative Example 7B-2] 10.0 mg of powdered CyA was weighed into a vial. Then, a stirrer tip was placed in the vial, and 400 μL of 10 mg / mL cholesterol-PEG600 was added. Water for injection was then added to bring the total volume to 1.0 mL. The mixture was then stirred for 24 hours to solubilize the drug. Subsequently, the mixture was filtered through a 0.45 μm sterile filter to remove precipitated CyA, and the drug concentration in the formulation was quantified by HPLC measurement in the same manner as in Example 1B-1. All filtrates were confirmed to be clear. Table 26 shows the theoretical concentration in the formulation calculated from the amount added, and the actual drug concentration in the formulation quantified by HPLC measurement.

[0458] [Table 26]

[0459] Based on the results above, in the case of cholesterol-PEG600 and HP-β-CD, solubilizing aids containing at least four ether structures (ROR) and having four or more carbon atoms improved the drug concentration in the formulation, as shown in Examples 7B-1 to 7B-3. The active ingredient could be solubilized at a higher concentration than the sum of the solubilization amounts of the hyaluronic acid derivative alone and the solubilizing aid alone, demonstrating a synergistic effect. [Industrial applicability]

[0460] The hyaluronic acid derivative pharmaceutical composition of this embodiment allows for the solubilization of a large amount of drug from powder, maximizing the amount of solubilization, and controlling the release rate of the active ingredient from the hyaluronic acid derivative composition that has gelled subcutaneously, thereby providing a pharmaceutical composition using a hyaluronic acid derivative that is excellent at sustained release of the active ingredient over a long period of time.

Claims

1. (A) Hyaluronic acid derivatives into which steryl groups have been introduced, (B) Association promoter and (C) Contains the active ingredient, The aforementioned (B) association promoter is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A hyaluronic acid derivative pharmaceutical composition wherein the content of (B) association promoter is 0.0001 parts by mass or more and 15000 parts by mass or less per 100 parts by mass of the (A) hyaluronic acid derivative into which a steryl group has been introduced.

2. A hyaluronic acid derivative pharmaceutical composition according to claim 1, wherein a precipitate is formed under physiological salt concentration.

3. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein the (C) active ingredient is at least one selected from proteins or poorly water-soluble drugs.

4. The hyaluronic acid derivative pharmaceutical composition according to claim 3, wherein the poorly water-soluble drug has a solubility in water of 1 mg / mL or less.

5. The hyaluronic acid derivative pharmaceutical composition according to claim 3, wherein the poorly water-soluble drug has a molecular weight of 200 or more.

6. The hyaluronic acid derivative pharmaceutical composition according to claim 3, wherein the poorly water-soluble drug is a poorly water-soluble peptide.

7. The hyaluronic acid derivative pharmaceutical composition according to claim 6, wherein the poorly water-soluble peptide has at least one nitrogen atom constituting the amide bond having a methyl group.

8. The hyaluronic acid derivative pharmaceutical composition according to claim 6, wherein the poorly water-soluble peptide comprises at least one selected from cyclic peptides and long-chain peptides.

9. The hyaluronic acid derivative pharmaceutical composition according to claim 6, wherein the poorly water-soluble peptide is a cyclic peptide.

10. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein the content of the active ingredient (C) in 100 parts by mass of the hyaluronic acid derivative into which the steryl group (A) is introduced is 10 parts by mass or more and 100 parts by mass or less.

11. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein the hyaluronic acid derivative into which the (A) steryl group is introduced has one or more repeating units represented by the following general formula (I). 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ , and R ４ These are, independently, hydrogen atoms and C １－６ Alkyl, formyl and C １－６ It is a group selected from the group consisting of alkylcarbonyl groups. Z represents a direct bond or a peptide linker consisting of any 2 to 30 amino acid residues. X １ is -NR ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NR ｃ -R, -COO-R, -O-COO-R, -S-R, -CO-Y ａ -S-R, -O-CO-Y ｂ -S-R, -NR ｂ -CO-Y ｂ -S-R, and -S-S-R, a group selected from the group consisting of groups represented by. R ａ , R ｂ and R ｃ These are, independently, hydrogen atoms and C １－２０ Alkyl, amino C ２－２０ Alkyl and hydroxy C ２－２０ A group selected from the group consisting of alkyl groups. ａ , R ｂ and R ｃ The alkyl portion consists of -O- and -NR ｆ A group selected from the group consisting of - may be inserted. R ｆ C is a hydrogen atom. １－１２ Alkyl, amino C ２－１２ Alkyl and hydroxy C ２－１２ A group selected from the group consisting of alkyl groups. ｆ The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a steryl group. Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -. Here, the alkylenes of Y are -O- and -NR ｇ A group selected from the group consisting of - and -S-S- may be inserted. R ｇ C is a hydrogen atom. １－２０ Alkyl, amino C ２－２０ Alkyl and hydroxy C ２－２０ A group selected from the group consisting of alkyl groups. ｇ The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y ａ C １－５ It is alkylene. Y ｂ C ２－８ Alkylene or C ２－８ It is alkenylene. m is an integer between 1 and 100 (inclusive).

12. The hyaluronic acid derivative pharmaceutical composition according to claim 11, wherein the steryl group is a cholesteryl group.

13. The hyaluronic acid derivative pharmaceutical composition according to claim 11, wherein the rate of introduction of the steryl group into the hyaluronic acid derivative is 7% or more and less than 35%.

14. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein no precipitates are observed visually in the hyaluronic acid derivative pharmaceutical composition.

15. A hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, which is filterable for sterilization.

16. A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a steryl group has been introduced, (B) an association promoter, and (C) an active ingredient, The steps include: mixing (A) a hyaluronic acid derivative into which a steryl group has been introduced with (B) an association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter; The process includes a mixing step of mixing the (C) active ingredient with an aqueous solution of the hyaluronic acid derivative containing the association promoter, The aforementioned (B) association promoter is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A method for producing a hyaluronic acid derivative pharmaceutical composition, wherein the content of (B) association promoter in 100 parts by mass of (A) hyaluronic acid derivative into which a steryl group has been introduced is 0.0001 parts by mass or more and 15000 parts by mass or less.

17. A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a steryl group has been introduced, (B) an association promoter, and (C) an active ingredient, The process includes the steps of: dispersing the active ingredient (C) in the association promoter (B) to obtain a dispersion (I); and preparing an aqueous solution of hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing an association promoter to obtain an aqueous solution (II). The step includes mixing the dispersion (I) and the aqueous solution (II), The aforementioned (B) association promoter is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A method for producing a hyaluronic acid derivative pharmaceutical composition, wherein the content of (B) association promoter in 100 parts by mass of (A) hyaluronic acid derivative into which a steryl group has been introduced is 0.0001 parts by mass or more and 15000 parts by mass or less.

18. A method for producing a hyaluronic acid derivative pharmaceutical composition according to claim 16 or 17, which does not include a step of removing an organic solvent.

19. (A) Hyaluronic acid derivatives into which steryl groups have been introduced, (B) Solubilizing agent, (C) Contains the active ingredient, The aforementioned (B) solubilizing aid is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A hyaluronic acid derivative pharmaceutical composition wherein the content of (B) solubilizing aid is 0.0001 parts by mass or more and 15000 parts by mass or less per 100 parts by mass of the (A) hyaluronic acid derivative into which a steryl group has been introduced.

20. The aforementioned (B) solubilizing aid is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. The hyaluronic acid derivative pharmaceutical composition according to claim 19, wherein the content of polyethylene glycol in 100 parts by mass of the (A) hyaluronic acid derivative into which the steryl group has been introduced is 25 parts by mass or more and 15,000 parts by mass or less.

21. The hyaluronic acid derivative pharmaceutical composition according to claim 19, wherein the (C) active ingredient is a poorly water-soluble drug having a solubility in water of 1 mg / mL or less.

22. The hyaluronic acid derivative pharmaceutical composition according to claim 21, wherein the poorly water-soluble drug has a molecular weight of 200 or more.

23. The hyaluronic acid derivative pharmaceutical composition according to claim 21, wherein the poorly water-soluble drug is a poorly water-soluble peptide.

24. The hyaluronic acid derivative pharmaceutical composition according to claim 23, wherein the poorly water-soluble peptide has at least one nitrogen atom constituting the amide bond having a methyl group.

25. The hyaluronic acid derivative pharmaceutical composition according to claim 23, wherein the poorly water-soluble peptide comprises at least one selected from cyclic peptides and long-chain peptides.

26. The hyaluronic acid derivative pharmaceutical composition according to claim 23, wherein the poorly water-soluble peptide is a cyclic peptide.

27. The hyaluronic acid derivative pharmaceutical composition according to claim 21, wherein the amount of the poorly water-soluble drug added to 100 parts by mass of the (A) hyaluronic acid derivative into which the steryl group has been introduced is 21 parts by mass or more and less than 100 parts by mass.

28. The hyaluronic acid derivative pharmaceutical composition according to claim 19, wherein the hyaluronic acid derivative has one or more repeating units represented by the following general formula (I). 【Chemistry 2】 (In the formula, R １ , R ２ , R ３ , and R ４ These are, independently, hydrogen atoms and C １－６ Alkyl, formyl and C １－６ It is a group selected from the group consisting of alkylcarbonyl groups. Z represents a direct bond or a peptide linker consisting of any 2 to 30 amino acid residues. X １ -NR ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NR ｃ -R, -COO-R, -O-COO-R, -SR, -CO-Y ａ -SR, -O-CO-Y ｂ -S-R, -NR ｂ -CO-Y ｂ It is a group selected from the group consisting of groups represented by -S-R and -S-S-R. R ａ , R ｂ and R ｃ These are, independently, hydrogen atoms and C １－２０ Alkyl, amino C ２－２０ Alkyl and hydroxy C ２－２０ A group selected from the group consisting of alkyl groups. ａ , R ｂ and R ｃ The alkyl portion consists of -O- and -NR ｆ A group selected from the group consisting of - may be inserted. R ｆ C is a hydrogen atom. １－１２ Alkyl, amino C ２－１２ Alkyl and hydroxy C ２－１２ A group selected from the group consisting of alkyl groups. ｆ The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a steryl group. Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -. Here, the alkylenes of Y are -O- and -NR ｇ A group selected from the group consisting of - 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 a group selected from the group consisting of alkyl. R ｇ a group selected from the group consisting of -O- and -NH- may be inserted into the alkyl moiety of. Y ａ C １－５ It is alkylene. Y ｂ C ２－８ Alkylene or C ２－８ It is alkenylene. m is an integer between 1 and 100 (inclusive).

29. The hyaluronic acid derivative pharmaceutical composition according to claim 28, wherein the steryl group is a cholesteryl group.

30. The hyaluronic acid derivative pharmaceutical composition according to claim 28, wherein the rate of introduction of the steryl group into the hyaluronic acid derivative is 35% or more and less than 50%.

31. The hyaluronic acid derivative pharmaceutical composition according to claim 19, wherein the content of the hyaluronic acid derivative in the hyaluronic acid derivative pharmaceutical composition is 6 mg / mL or more and less than 45 mg / mL.

32. The hyaluronic acid derivative pharmaceutical composition according to claim 19, wherein the content of the organic solvent in the hyaluronic acid derivative pharmaceutical composition is less than 0.8%.

33. A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a steryl group has been introduced, (B) a solubilizing agent, and (C) an active ingredient, (A) A hyaluronic acid derivative into which a steryl group has been introduced, and (B) a solubilizing agent are mixed to obtain an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent. The process includes a mixing step of mixing the (C) active ingredient with an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent, The aforementioned (B) solubilizing aid is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A method for producing the pharmaceutical composition, wherein the content of (B) solubilizing aid relative to 100 parts by mass of (A) hyaluronic acid derivative into which steryl groups have been introduced is 0.0001 parts by mass or more and 15000 parts by mass or less.

34. A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a steryl group has been introduced, (B) a solubilizing agent, and (C) an active ingredient, The process includes the steps of: dispersing the active ingredient (C) in the solubilizing agent (B) to obtain a dispersion (I); and preparing an aqueous solution of a hyaluronic acid derivative or an aqueous solution of hyaluronic acid containing a solubilizer to obtain an aqueous solution (II). The step includes mixing the dispersion (I) and the aqueous solution (II), The aforementioned (B) solubilizing aid is polyethylene glycol with a molecular weight of 190 g / molL or more and 4000 g / molL or less. A method for producing the pharmaceutical composition, wherein the content of (B) solubilizing aid relative to 100 parts by mass of (A) hyaluronic acid derivative into which steryl groups have been introduced is 0.0001 parts by mass or more and 15000 parts by mass or less.

35. A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a steryl group has been introduced, (B) a solubilizing agent, and (C) an active ingredient, A method for producing a pharmaceutical composition according to claim 33 or 34, characterized in that it does not include a step of removing an organic solvent.

Images