Hyaluronic acid derivative pharmaceutical composition and method for producing pharmaceutical composition

Abstract

A hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative into which a hydrophobic group is introduced, (B) an association promoter, and (C) an active ingredient. Preferably, the association accelerator (B) contains at least four ether structures (R-O-R), and satisfies the condition that the number of carbon atoms is 4 or more.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid derivative pharmaceutical composition and a method for preparing 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, the contents of which are incorporated herein. Background Art

[0002] In recent years, biopharmaceuticals, which are drugs with 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 insufficient medical needs that were not met by conventional low-molecular-weight drugs.

[0003] However, there are the following problems in biopharmaceuticals: not only are they difficult to be absorbed from the digestive tract, mucous membranes, etc., but they are also unstable in vivo and have a short half-life in the blood. Therefore, biopharmaceuticals need to be frequently administered by injection, which brings a huge burden to both patients and medical staff. Therefore, a drug substrate (sustained-release drug delivery system substrate) that can encapsulate biopharmaceuticals without damaging pharmacological activity and slowly release active ingredients in vivo is being sought.

[0004] Against such a background, Patent Document 1 proposes a sustained-release drug delivery system substrate comprising a hyaluronic acid derivative having excellent safety. The hyaluronic acid derivative disclosed in patent documentation 1 associates spontaneously in aqueous solution, can be efficiently enclosed in medicine, particularly biopharmaceutical under the state of keeping its biological activity.Accordingly, coagulate under normal saline concentration (or disperse under normal saline concentration), and retention is good in blood.About this hyaluronic acid derivative, particularly when using biopharmaceutical as effective ingredient, it can be used as carrier that can efficiently enclose a large amount of medicines under the state of keeping pharmacological activity and blood in the blood in which retention is excellent slow-release carrier and targeting carrier are used, and can also become the local (such as subcutaneous etc.) slow-release carrier that can continue sustained-release medicine.

[0005] On the other hand, in the pharmaceutical industry, even active pharmaceutical ingredients with strong biological activity are often abandoned in development because they cannot exert their effects due to their low water solubility, or are marketed as preparations that only exhibit lower activity than their original activity.

[0006] As a method for solubilizing a water-insoluble or poorly water-soluble pharmaceutical active ingredient, there are methods described in the following (a) to (c).

[0007] (a) A method of changing a part of the structure of a drug to make a soluble derivative: a soluble derivative such as a hydrochloride, a hydrobromide, a sulfate, a methanesulfonate, a sodium salt, a potassium salt, or a sodium sulfonate is made.

[0008] (b) Method using an organic solvent monomer or a mixed solvent of an aqueous solvent and an organic solvent: a method of solubilizing using methanol, ethanol, or the like.

[0009] (c) Method of adding a dissolution aid: A method of solubilizing by adding a surfactant to form micelles or emulsify the mixture. A method of using serum albumin or plasma protein.

[0010] However, in the method of (a), a part of the structure of the pharmaceutical raw material drug itself as the active ingredient is changed, and the solubility of the raw material drug itself cannot be improved. In addition, by making a derivative, various problems such as the situation of reduced activity as a drug or the precipitation of the drug caused by the change of pH sometimes occur, which is not a desirable method.

[0011] Regarding the method using an organic solvent such as methanol described in (b) above, there are very few safe organic solvents that are inactive in biological activity and not associated with hemolysis, and they are almost not put into practical use in the field of medicine. For example, Patent Document 2 describes a production method that involves the steps of dissolving a poorly water-soluble cyclosporin composition 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 obtained solution was turbid and partial precipitation was confirmed, indicating that the solubilization was insufficient. This means that the poorly water-soluble active ingredient of the drug was precipitated, and sufficient activity could not be obtained, and the toxicity to the organism caused by the precipitation was not improved.

[0013] Regarding the method of using a surfactant in (c) above, the actual situation is that there are very few soluble surfactants that are safe and effective for living organisms. In the past, there were those who used polyoxyethylated castor oil (Cremophor EL) to dissolve the drug Taxol, but Non-Patent Document 1 reported that polyoxyethylated castor oil causes acute immunotoxicity shown by anaphylactic reaction (HSR) as a result of activation of the complement system, which is not mediated by IgE. Since there are very few safe and useful surfactants, there are preparations that use toxic Cremophor to dissolve paclitaxel.

[0014] In patent documentation 3, when the water-insoluble active ingredient is enclosed in the hyaluronic acid derivative, the water-insoluble active ingredient needs to be dissolved in an organic solvent such as methanol. Therefore, it is impossible to avoid using an organic solvent. In addition, in patent documentation 3, the solubilization amount of the water-insoluble active ingredient is insufficient. In addition, there is no specific research yet on the formulation method of the water-insoluble active ingredient being effectively solubilized from the state of the powder, and there is room for improvement.

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

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

[0017] Non-patent document 1: Toxicology 216 (2005) 106-121 Summary of the invention [Problems to be solved by the invention]

[0018] Sustained-release drug substrates are required to be able to maintain the concentration of active ingredients in vivo for a long period of time and to slowly release the active ingredients. In addition, they are required to be able to solubilize the active ingredients at high concentrations. The present invention is completed in view of the above situation, and its purpose is to provide a hyaluronic acid derivative pharmaceutical composition and a method for producing the same, wherein the hyaluronic acid derivative pharmaceutical composition can further maintain the concentration of the active ingredient in the body for a long time, slowly release the active ingredient, and can solubilize the active ingredient at a high concentration. Furthermore, the present invention is completed in view of the above situation, and its purpose is to provide a hyaluronic acid derivative pharmaceutical composition and a method for producing the same, wherein the hyaluronic acid derivative pharmaceutical composition does not use an organic solvent, can reduce the amount of highly toxic surfactants used, and can solubilize water-insoluble active ingredients at a high concentration. [Methods of solving the problem]

[0019] That is, the present invention includes the following aspects. [1] A hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient. [2] The hyaluronic acid derivative pharmaceutical composition according to [1], wherein the (B) association promoter contains at least 4 ether structures (ROR) and has 4 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 polysorbate 80, polysorbate 20, poloxamer, oxyethylene castor oil, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 4000, sorbitan fatty acid esters, tocopherol polyethylene glycol succinate and polyvinyl alcohol. [4] The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to [3], which produces a precipitate at a 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 and poorly water-soluble drugs. [6] The hyaluronic acid derivative pharmaceutical composition according to [5], wherein the solubility of the poorly water-soluble drug in water is 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 hyaluronic acid derivative pharmaceutical composition according to any one of [5] to [7], wherein the poorly water-soluble drug is a poorly water-soluble peptide. [9] The pharmaceutical composition of a hyaluronic acid derivative according to [8], wherein at least one of the nitrogen atoms constituting the amide bond of the poorly water-soluble peptide has a methyl group.

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

[11] The hyaluronic acid derivative pharmaceutical composition according to any one of [8] to

[10] , wherein the poorly water-soluble peptide is a cyclic peptide.

[12] The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[11] , wherein the content of the (C) active ingredient is 10 parts by mass or more and 100 parts by mass or less relative to 100 parts by mass of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced.

[13] The hyaluronic acid derivative pharmaceutical composition according to any one of [1] to

[12] , wherein the (A) hyaluronic acid derivative into which a hydrophobic group is introduced has one or more repeating units represented by the following general formula (I). (Where R1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 The group in the alkylcarbonyl group. Z represents a direct bond or a peptide linker composed of 2 or more and 30 or less arbitrary amino acid residues. X 1 For free-NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b The group among the groups represented by -SR and -SSR. R a , R b and R c are each independently selected from a hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. a , R b and R c The alkyl portion may or may not be inserted into -O- and -NR f -Groups in. R f is selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The group in the alkyl group. f The alkyl portion may or may not be inserted with a group selected from -O- and -NH-. R is a steryl group. Y is C 2-30 Alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 Here, the alkylene group of Y may be inserted or not inserted and is selected from -O-, -NR g - and -SS- groups. R g is selected from hydrogen atoms, C1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. g The alkyl portion may or may not be inserted with a group selected from -O- and -NH-. Y a C 1-5 Alkylene. Y b C 2-8 Alkylene or C 2-8 Alkenylene. m is an integer of 1 to 100.)

[14] The hyaluronic acid derivative pharmaceutical composition according to

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

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

[13] to

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

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

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

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

[16] , which is filter sterilizable.

[18] A method for producing a hyaluronic acid derivative pharmaceutical composition, comprising: (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient, wherein the method for producing the hyaluronic acid derivative pharmaceutical composition comprises: A step of mixing the (A) hyaluronic acid derivative having a hydrophobic group introduced therein with the (B) association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter; and A mixing step of mixing the (C) 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 having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient, wherein the method for producing the hyaluronic acid derivative pharmaceutical composition comprises: A step of dispersing the active ingredient (C) in the association promoter (B) to obtain a dispersion (I); A step of 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 A step of mixing the dispersion (I) and the aqueous solution (II).

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

[18] or

[19] , wherein the method for producing a hyaluronic acid derivative pharmaceutical composition does not include a step of removing the organic solvent.

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

[21] A hyaluronic acid derivative pharmaceutical composition comprising (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing agent, and (C1) an effective ingredient, wherein the solubilizing agent (B1) comprises at least 4 ether structures (ROR) and has a carbon number of 4 or more, and the content of the solubilizing agent (B1) is greater than 0.0001 parts by mass and less than 15,000 parts by mass relative to 100 parts by mass of the hyaluronic acid derivative (A1) having a hydrophobic group introduced therein.

[22] The hyaluronic acid derivative pharmaceutical composition according to

[21] , wherein the solubilizing agent (B1) is one or more selected from a nonionic surfactant, a polyethylene glycol having a molecular weight of 190 g / moL to 4000 g / moL, and a cyclodextrin derivative.

[23] The hyaluronic acid derivative pharmaceutical composition according to

[21] or

[22] , wherein the solubilizing agent (B1) is one or more selected from 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 tocopherol polyethylene glycol succinate.

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

[21] to

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

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

[21] to

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

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

[21] to

[25] , wherein the active ingredient (C1) is a poorly water-soluble drug having a solubility in water of 1 mg / mL or less.

[27] The hyaluronic acid derivative pharmaceutical composition according to

[26] , wherein the molecular weight of the poorly water-soluble drug is 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 hyaluronic acid derivative pharmaceutical composition according to

[28] , wherein at least one of the nitrogen atoms constituting the amide bond of the poorly water-soluble peptide has 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 hyaluronic acid derivative pharmaceutical composition according to any one of

[28] to

[30] , wherein the poorly water-soluble peptide is a cyclic peptide.

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

[26] to

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

[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). (Where R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 The group in the alkylcarbonyl group. Z represents a direct bond or a peptide linker composed of 2 or more and 30 or less arbitrary amino acid residues. X 1 For free-NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b The group among the groups represented by -SR and -SSR. Ra , R b and R c are each independently selected from a hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. a , R b and R c The alkyl portion may or may not be inserted into -O- and -NR f -Groups in. R f is selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The group in the alkyl group. f The alkyl portion may or may not be inserted with a group selected from -O- and -NH-. R is a steryl group. Y is C 2-30 Alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 Here, the alkylene group of Y may be inserted or not inserted and is selected from -O-, -NR g - and -SS- groups. R g is selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. g The alkyl portion may or may not be inserted with a group selected from -O- and -NH-. Y a C 1-5 Alkylene. Y b C 2-8 Alkylene or C 2-8 Alkenylene. m is an integer of 1 to 100.)

[34] The hyaluronic acid derivative pharmaceutical composition according to

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

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

[33] to

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

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

[21] to

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

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

[21] to

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

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

[39] A method for producing a pharmaceutical composition, which is a method for producing a pharmaceutical composition comprising (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing aid, and (C1) an active ingredient, wherein the method for producing the pharmaceutical composition comprises: A step of dispersing the active ingredient (C1) in the solubilizing agent (B1) to obtain a dispersion (I); A step of 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); and A step of mixing the dispersion (I) and the aqueous solution (II).

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

[38] or

[39] is characterized in that the method for producing the pharmaceutical composition is a method for producing a pharmaceutical composition containing (A1) a hyaluronic acid derivative introduced with a hydrophobic group, (B1) a solubilizing agent and (C1) an active ingredient, and the method for producing the pharmaceutical composition does not include a step of removing an organic solvent. [Effects of the Invention]

[0021] According to the above-mentioned hyaluronic acid derivative pharmaceutical composition, a hyaluronic acid derivative pharmaceutical composition and a production method thereof can be provided, wherein the hyaluronic acid derivative pharmaceutical composition can further maintain the concentration of the active ingredient in the body for a long time, slowly release the active ingredient, and can solubilize the active ingredient at a high concentration.

[0022] According to the above-mentioned hyaluronic acid derivative pharmaceutical composition, a hyaluronic acid derivative pharmaceutical composition and a production method thereof can be provided, wherein the hyaluronic acid derivative pharmaceutical composition does not use an organic solvent, can reduce the amount of highly toxic surfactant used, and can solubilize a poorly water-soluble active ingredient at a high concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [ Figure 1 ] is a chromatogram of hyaluronic acid derivatives. [ Figure 2 ] is a chromatogram of a hyaluronic acid derivative pharmaceutical composition. [ Figure 3 ] is a graph showing changes in the plasma concentration of cyclosporine. [ Figure 4 ] is a graph showing changes in the plasma concentration of cyclosporine. DETAILED DESCRIPTION

[0024] Hereinafter, an embodiment 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 invention.

[0025] The following is an explanation of the terms used in this specification.

[0026] The “C 1-20 The term "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, etc. 1-4 "Alkyl" further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, etc. 1-20 The alkyl group also includes a C 1-12 Alkyl, C having 1 to 6 carbon atoms 1-6 alkyl.

[0027] The “C 1-6 The term "alkylcarbonyl" means that the alkyl portion is already the C 1-6 The alkylcarbonyl of the alkyl group includes, for example, acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc. 1-4 "Alkylcarbonyl".

[0028] The term "amino C" used in this specification 2-20The term "alkyl" refers to a linear or branched alkyl group having 2 or more and 20 or less carbon atoms and having an amino group as a substituent. For example, the amino group may be located on a carbon atom at the terminal of the alkyl group. 2-20 The alkyl group also includes an amino group having 2 or more and 12 carbon atoms. 2-12 alkyl.

[0029] The term "hydroxyl C 2-20 The term "alkyl" refers to a linear or branched alkyl group having 2 or more and 20 or less carbon atoms and having a hydroxyl group as a substituent. For example, the hydroxyl group may be located on a carbon atom at the terminal of the alkyl group. 2-20 The alkyl group also includes a hydroxyl group having 2 or more and 12 carbon atoms. 2-12 alkyl.

[0030] The “C 2-30 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, for example, ethylene, propylene, etc., including C 2-20 Alkylene, C 2-8 Alkylene, group "-(CH 2 ) n -"(Here, n is 2 to 30, preferably 2 to 20, more preferably 2 to 15.).

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

[0032] The term "C 2-8 The term "alkenylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms and containing one or more double bonds, for example, -CH=CH-, -C(CH 3 )=CH-, 2-butene-1,4-diyl, hept-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0033] <Hyaluronic acid derivative pharmaceutical composition 1> This embodiment is a hyaluronic acid derivative pharmaceutical composition containing (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient. Hereinafter, the "hyaluronic acid derivative pharmaceutical composition containing (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient" is sometimes referred to as "pharmaceutical composition 1".

[0034] The hyaluronic acid derivatives can be formulated with active ingredients and used as pharmaceutical compositions. In the pharmaceutical composition 1 of the present embodiment, the hyaluronic acid derivative forms a complex with the active ingredient (hereinafter sometimes referred to as "active ingredient-hyaluronic acid derivative complex"). Specifically, the steryl group in the hyaluronic acid derivative forms a complex with the hydrophobic part of the active ingredient through hydrophobic interaction, and it is estimated that the following cylindrical structure or core-shell cylindrical structure is presented: the hydrophobic parts such as the active ingredient and the steryl group are present in the central part, and on the other hand, the hydrophilic parts such as the part of the hyaluronic acid from the hyaluronic acid derivative are present in the outer edge. That is, it is estimated that the structure in which the active ingredient is enclosed or enclosed in the hyaluronic acid derivative is presented.

[0035] In the pharmaceutical composition 1 of the present embodiment, the average particle diameter of the structure containing the active ingredient-hyaluronic acid derivative complex can be set to more than 20nm and less than 220nm, can be set to more than 20nm and less than 150nm, can be set to more than 30nm and less than 100nm. By the average particle diameter being the above-mentioned numerical range, sterilization filtration can be performed, and agglutination and precipitation can be stably performed in vivo, and the effect as a sustained-release substrate can be played. The average particle diameter can be measured, for example, by DLS (dynamic light scattering: Dynamic Light Scattering), nanoparticle tracking measurement device, size exclusion chromatography, high performance liquid chromatography and electron microscopy, etc. More specifically, for example, in a DLS device, the ratio of 1mg / mL with a hyaluronic acid derivative concentration is measured by diluting a 10mM phosphate buffer containing 10w / v% sucrose.

[0036] Preferably, the (B) association promoter contained in the pharmaceutical composition 1 of this embodiment contains at least 4 ether structures (ROR) and has 4 or more carbon atoms.

[0037] The pharmaceutical composition 1 of this embodiment comprises (B) an association promoter. (B) association promoter in this specification refers to a substance that interacts with a hyaluronic acid derivative introduced with a hydrophobic group to promote association between hyaluronic acid derivative molecules. The association promoter of the present embodiment forms a complex with a hyaluronic acid derivative to improve the drug compounding ability. Improve the precipitation performance under physiological salt concentration. In this regard, after subcutaneous precipitation, the drug in the gel is released by replacing the exchange reaction of hydrophobic components such as albumin or other hydrophobic proteins in the organism or the decomposition of the gel, but the sustained release time can be controlled by controlling the sustained release of the drug based on the exchange reaction, inhibiting and shielding the influx of the decomposing enzyme of the gel, and giving stealth.

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

[0039] 《Introduction of hyaluronic acid derivatives with hydrophobic groups》 The pharmaceutical composition 1 of this embodiment contains (A) a hyaluronic acid derivative into which a hydrophobic group is introduced. Hereinafter, "(A) a hyaluronic acid derivative into which a hydrophobic group is introduced" may be described 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 hyaluronic acid or bonded via a linker. The "linker" mentioned here may be any peptide linker or synthetic compound linker that can be introduced by genetic engineering. In the (A) hyaluronic acid derivative used in the present 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 according to the purpose, but preferably the length is more than 2 amino acids, and particularly preferably 15 amino acids. The upper limit of the length of the peptide linker is not particularly limited, and is usually less than 30 amino acids, and is preferably less than 20 amino acids. (A) The peptide linker contained in the hyaluronic acid derivative can use peptide linkers of all the same length, or can use peptide linkers of different lengths.

[0042] When (A) the hyaluronic acid derivative has a steryl group as a hydrophobic group, the steryl group in the hyaluronic acid derivative self-associates in water to form a nano-sized hydrogel through single-molecule or multi-molecule association.

[0043] [Steryl] The term "steryl" used in this specification is not particularly limited as long as it is a group with a steroid skeleton. Here, as steroid, specifically, cholesterol, cholestanol, campestanol, ergosterol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, sagosterol, bile acid, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycorticosterone, etc. can be mentioned. As steryl, cholesteryl, stigmasteryl, lanosteryl, ergosteryl, etc., wherein, cholesteryl (particularly cholest-5-ene-3β-yl) is preferred.

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

[0045] When the steryl introduction rate is within the above range, the (A) hyaluronic acid derivative can strongly interact with the hydrophobic part of the poorly water-soluble drug or protein and the hydrophobic part of the association promoter. In addition, when the steryl introduction rate is within the above range, the stability of the preparation of the hyaluronic acid derivative-drug complex in which the (A) hyaluronic acid derivative in the pharmaceutical composition 1 is complexed with the drug is improved, and the complex aggregates and precipitates at a physiological salt concentration, thereby enabling sustained release of the drug.

[0046] The rate of steryl incorporation can be determined by 1 That is, the pharmaceutical composition 1 can be used to measure 1 The integrated value of the peak derived from the steryl group of the (A) hyaluronic acid derivative and the peak derived from the acetyl group of N-acetyl-D-glucosamine contained in the (A) hyaluronic acid derivative (COCH 3 , 1.6ppm or more and 2.0ppm or less, the integral value of 3H) is calculated according to the following formula. It should be noted that, in the formula, n H The peak is represented by the number of hydrogen atoms. Specifically, for example, it can be measured by the method described in the Examples described below. [Steryl introduction rate (%)] = [(integrated value of the peak derived from the steryl group) / (integrated value of the peak derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0048] The molecular weight of the hyaluronic acid derivative (A) is not particularly limited, but from the perspective of improving the sustained-release function derived from diffusion delay in topical administration, the hyaluronic acid derivative (A) with relatively large molecular weight is preferred. On the other hand, when the final dosage form is a solution preparation, from the perspective of syringeability, the hyaluronic acid derivative (A) with relatively small molecular weight is preferred. The molecular weight of the hyaluronic acid derivative (A) can be appropriately adjusted according to the purpose or dosage form.

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

[0050] When the weight average molecular weight of the hyaluronic acid derivative is equal to or greater than the above lower limit, the viscosity increase 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 a corresponding molecular weight. More specifically, from the viewpoint of viscosity, the weight average molecular weight of the hyaluronic acid derivative is preferably 100 k or less, more preferably 50 k or less, and particularly preferably 40 k or less. From the viewpoint of production of a hyaluronic acid derivative, 4k to 100k is preferred, and 6k to 50k is particularly preferred. From the viewpoint that the hyaluronic acid derivative strongly exhibits the properties of hyaluronic acid, for example, in order to be strongly recognized by the CD44 receptor, the size is preferably 100 kDa or more, particularly preferably 200 kDa or more, and most preferably 300 kDa or more.

[0051] The "molecular weight of the hyaluronic acid derivative" described herein is a weight average molecular weight determined by size exclusion chromatography-multi-angle light scattering detector (SEC-MALS).

[0052] Specific examples of preferred hyaluronic acid derivatives include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter, sometimes referred to as “repeating unit (I)”).

[0053]

[0054] (Where R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C1-6 Alkyl, formyl and C 1-6 The group in the alkylcarbonyl group. Z represents a direct bond or a peptide linker composed of 2 to 30 arbitrary amino acid residues. X 1 For free-NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b The group among the groups represented by -SR and -SSR. R a , R b and R c are each independently selected from a hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. Here, R a , R b and R c The alkyl portion may be inserted into the f -Groups in. R f is selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The group in the alkyl group. f The alkyl portion of the alkyl group may be inserted with a group selected from -O- and -NH-. R is a steryl group. Y is C 2-30 Alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 Here, the alkylene group of Y may be inserted into -O-, -NR g - and -SS- groups. R g is selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group. g The alkyl portion of the alkyl group may be inserted with a group selected from -O- and -NH-. Y a C 1-5 Alkylene. Y b C 2-8 Alkylene or C 2-8 Alkenylene. m is an integer of 1 to 100.)

[0055] Preferably, the hyaluronic acid derivative includes a hyaluronic acid derivative having one or more repeating units represented by the following general formula (Ia) (hereinafter, sometimes referred to as “repeating unit (Ia)”).

[0056]

[0035] (Where R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 The group in the alkylcarbonyl group. X is -NR a -Y-NR b -COO-R represents a hydrophobic group. a and R b are each independently selected from a hydrogen atom and C 1-6 The group in the alkyl group. R is a steryl group. Y is C 2-30 Alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 -, m is an integer greater than or equal to 1 and less than or equal to 100. )

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

[0059] The hyaluronic acid derivative may be modified at a position other than the repeating unit (I) or the repeating unit (Ia), for example, the hydroxyl group may be converted into -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 The carboxyl group may be converted into an amide or an ester, or may form a salt.

[0060] [Repeating unit (I)] The group "-ZN(R a )YX 1 " includes a group selected from the group represented by the following formula: -NH-(CH 2) mz -NH-R; -NH-(CH 2 ) mz -NH-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NH-COO-R; -NH-(CH 2 ) mz -COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -COO-R, -NH-(CH 2 ) mz -O-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -O-COO-R, -NH-(CH 2 ) mz -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -S-R; -NH-(CH 2 ) mz -O-CO-CH(R 8 )-CH 2 -S-R; -NH-(CH 2 ) mz -NHCO-CH(R 8 )-CH 2 -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NHCO-CH(R 8 )-CH 2 -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -O-CO-CH(R 8 )-CH 2 -S-R; -NH-(CH 2 ) mz -S-S-R; and -Z-NRa -Y-NR b -COO-R (wherein mz is an integer from 2 to 30, R 8 is a hydrogen atom or a methyl group, and R and m are as defined in the present specification).

[0061] The group is preferably selected from -NH-(CH 2 ) mz -NH-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NH-COO-R; and -NH-(CH 2 ) mz -SSR (herein, mz, R and m are as defined in the present specification).

[0062] (Z) In the general formula (I), Z is preferably directly bonded. In another embodiment, when Z is a peptide linker, X 1 Preferably -NR b In another embodiment, Z may be -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a )-CO-, wherein n is an integer of 2 to 30, and Z a Each independently represents H 2 N-CH(-Z a )-COOH in an α-amino acid. The peptide linker is bound to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R a )-YX 1 As examples of amino acids that can be used as amino acid residues of the peptide linker, α-amino acids can be used, for example, natural amino acids (L-type) 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, their D-types, and all α-amino acids including synthetic amino acids. That is, as Z a For example, -CH 3 , H 2 NC(NH)NH(CH 2 ) 3 -、H 2 NCOCH2 -, etc. In addition, n Zs may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, more preferably 2 to 4. Preferred examples of peptide linkers include: -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, etc.

[0063] (Y) In the general formula (I), Y is preferably selected from -(CH 2 ) n1 - and - (CH 2 CH 2 O) m1 -CH 2 CH 2 -(herein, n1 is an integer of 2 to 20, preferably an integer of 2 to 15, more preferably an integer of 2 to 12, and further preferably an integer of 2 to 6. m1 is an integer of 1 to 4). Specifically, -(CH 2 ) 2 -、-(CH 2 ) 6 -、-(CH 2 ) 8 -、-(CH 2 ) 12 -or-(CH 2 CH 2 O) 2 -CH 2 CH 2 In addition, from the perspective of achieving high solubility in pure water to low salt concentrations and exhibiting high precipitation forming ability at physiological saline concentrations, Y is preferably selected from -(CH 2 ) 2 -、-(CH 2 ) 6 -、-(CH 2 ) 8 - and - (CH 2 ) 12 -, more preferably -(CH 2 ) 6 -.

[0064] Y can be, for example, -CH 2 CH 2 O-CH 2 CH 2 -SS-CH 2 CH 2 O-CH 2 CH 2 -、-(CH2 CH 2 O) 2 -CH 2 CH 2 -SS-CH 2 CH 2 O-CH 2 CH 2 -、-CH 2 CH 2 O-CH 2 CH 2 -SS-(CH 2 CH 2 O) 2 -CH 2 CH 2 -、-(CH 2 CH 2 O) 2 -CH 2 CH 2 -SS-(CH 2 CH 2 O) 2 -CH 2 CH 2 -wait.

[0065] (Y a ) As Y a , preferably -CH 2 -or-CH 2 -CH 2 -.

[0066] (Y b ) As Y b , preferably -CH 2 -CH 2 -、-CH(CH 3 )CH 2 -, 2-butene-1,4-diyl, hept-2,4-diene-1,6-diyl or octa-2,4,6-triene-1,8-diyl, more preferably -CH 2 -CH 2 - or -CH(CH 3 )CH 2 -.

[0067] As the group "-ZN(R a )YX 1 Specific examples of " can include: -NH-(CH 2 ) 2 -NH-CO-cholesteryl, -NH-(CH 2 ) 4-NH-(CH 2 ) 3 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH 2 ) 4 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-CO-NH-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-CO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2) 3 -NH 2 )-cholesteryl, etc. As a preferred group "-ZN(R a )YX 1 ”, R a , R b and R c is a hydrogen atom, and Y is a straight-chain C 2-30 Alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 -, Y a A straight chain C 1-5 Alkylene, or Y b A straight chain C 2-8 Alkylene or straight chain C 2-8 Alkenylene.

[0068] [Repeating unit (Ia)] In the general formula (Ia), X is preferably -NH-(CH 2 ) 2 -NH-COO-cholesteryl, -NH-(CH 2 ) 6 -NH-COO-cholesteryl, -NH-(CH 2 ) 12 -NH-COO-cholesteryl or -NH-(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-COO-cholesteryl, more preferably -NH-(CH 2 ) 2 -NH-COO-cholesteryl, -NH-(CH 2 ) 6 -NH-COO-cholesteryl or -NH-(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-COO-cholesteryl.

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

[0070]

[0071] (Where R 1a , R2a , R 3a and R 4a are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 The group in the alkylcarbonyl group. a is selected from hydroxyl and -OQ + The group in Q + is a counter cation.)

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

[0073] [Repeating unit (II)] In the general formula (II), Q + There are no particular limitations on the counter cations as long as they form salts with carboxyl groups in water. In the case of a valency of 2 or more, salts are formed with multiple carboxyl groups according to the valency. Examples of counter cations include metal ions such as lithium ions, sodium ions, rubidium ions, cesium ions, magnesium ions, and calcium ions; + R j R k R l R m (Where R j , R k , R l and R m are independently selected from hydrogen atoms and C 1-6 alkyl) represented by ammonium ions, etc. Among them, Q + It is preferably a sodium ion, a potassium ion or a tetraalkylammonium ion (for example, a tetra-n-butylammonium ion). j , R k , R l and R m Preferably selected from C 1-6 The same group of alkyl groups is preferably n-butyl.

[0074] R 1 , R 2 , R 3 and R 4 and R 1a , R 2a , R 3a and R 4a All of them are preferably hydrogen atoms. a and R b Both are preferably hydrogen atoms.

[0075] Among them, the hyaluronic acid derivative (A) is preferably a hyaluronic acid derivative substantially composed of repeating units (I) and repeating units (II). Regarding the hyaluronic acid derivative (A), among the repeating units of the disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative, for example, more than 80%, preferably more than 90%, and more preferably more than 95% are repeating units (I) and repeating units (II). The hyaluronic acid derivative (A) may be composed only of repeating units (I) and repeating units (II).

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

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

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

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

[0080] Figure 2 The gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment is recorded in. Here, the refractive index intensity at the beginning of the measurement is set to zero, and the line drawn from this level is used as a baseline. For example, before the measurement starts, the increase and decrease of the refractive index intensity is adjusted to within the scope of ± 0.5mV, and the refractive index intensity is adjusted to the increase and decrease below 0.5mV in 5 minutes. For example, the initial point of the amount of 3 times the amount equivalent to 5 times of the noise value is exceeded as the "starting point" of the chromatogram, and the elution time is set to 0 minute.

[0081] For example, the point where the refractive index intensity reaches one thousandth of the maximum refractive index intensity is set as the "end point" of the chromatogram, and when the refractive index intensity does not reach one thousandth of the maximum refractive index intensity, "Tlim" is set as the "end point." "Tlim" is set as the elution time at which the maximum refractive index intensity is exhibited 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 of the GPC workstation EcoSECElite-WS.

[0083] When calculating each area value, peaks due to the developing solvent used in gel permeation chromatography and the like and pseudo peaks due to baseline fluctuations caused by the chromatography column or apparatus used were removed.

[0084] If the ratio A2 / A1 of area A2 to area A1 is 1.2 or more, it means that the association between hyaluronic acid derivative molecules or between hyaluronic acid derivative and association promoter is promoted. It should be noted that when the association between hyaluronic acid derivative molecules or between hyaluronic acid derivative and association promoter is not promoted, the ratio A2 / A1 is in the range of 0.95 to 1.19.

[0085] The ratio A2 / A1 is preferably 1.20 or more, more preferably 1.30 or more, further preferably 1.40 or more, and particularly preferably 1.50 or more. When the ratio A2 / A1 of the area A2 to A1 is above the above lower limit, a relatively large amount of hyaluronic acid derivatives whose association is promoted can be contained, and when the (C) active ingredient is formulated, a large amount of the (C) active ingredient can be retained, and the (C) active ingredient can be solubilized at a high concentration. In addition, the (B) association promoter can control the release of the (C) active ingredient, and can maintain the sustained release time of the drug in the organism for a long time.

[0086] On the other hand, as for the upper limit of A2 / A1, as long as the ratio of area A2 is higher than area A1, the higher the better. Therefore, the upper limit of A2 / A1 is not particularly limited, but can 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] The (B) association promoter is not particularly limited as long as it is a surfactant that can be generally used in pharmaceutical applications. The (B) association promoter is preferably a component containing at least 4 ether structures (ROR) and having 4 or more carbon atoms.

[0088] (B) The association promoter includes, for example, polysorbate (having 5 or more ether structures and 10 or more carbon atoms), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene castor oil, and the like. Examples of the association promoter (B) 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 glycol monolaurate (having 7 or more ether structures and 28 or more carbon atoms), stearic acid polyoxyl 40 (having 39 or more ether structures and 98 or more carbon atoms), stearic acid polyoxyl 45 (having 44 or more ether structures and 108 or more carbon atoms), stearic acid polyoxyl 55 (having 54 or more ether structures and 128 or more carbon atoms), Cremophor EL (having 35 or more ether structures and 127 or more carbon atoms), etc. Among them, polysorbate 20, polysorbate 80 and polyoxyethylene castor oil are particularly preferred.

[0089] (B) The association promoter is not limited to the above-mentioned surfactants, and examples thereof 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).

[0090] The amount of the association promoter (B) added in the 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, relative to 100 parts by mass of the hyaluronic acid derivative.

[0091] When (B) the association promoter is at least one selected from the above-mentioned surfactants, the amount of the (B) association promoter added in 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, relative to 100 parts by mass of the hyaluronic acid derivative.

[0092] When the association promoter (B) is at least one selected from the group other than the above-mentioned surfactants, the amount of the association promoter (B) added in the pharmaceutical composition 1 is preferably 10 parts by mass or more and 15,000 parts by mass or less relative to 100 parts by mass of the hyaluronic acid derivative. It is more preferably 100 parts by mass or more and 10,000 parts by mass or less, further 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.

[0093] The less the association promoter (B), the better. However, if it is below the above lower limit, the active ingredient cannot be fully solubilized. In addition, if it is greater than the upper limit, the toxicity tends to increase due to the excessive amount of solubilizing agent (B), and the viscosity of the preparation also increases, so it is preferably within the above range.

[0094] The means for dissolving the (B) association promoter is not particularly limited, and examples thereof include stirring, shaking, and ultrasonic treatment.

[0095] Regarding the pharmaceutical composition 1 of the present embodiment, by containing (B) an association promoter, a large amount of active ingredients can be retained, and further, by preventing hydrophobic biological components from flowing into the subcutaneous gelled hyaluronic acid derivative composition, the release rate of the active ingredient can be controlled, and the active ingredient can be slowly released for a long time. It should be noted that the desired effect can also be obtained by a mechanism different from the above mechanism.

[0096] 《(C) Active ingredients》 The active ingredient is not particularly limited, and examples thereof include pharmaceutically active peptides or proteins, nucleic acids, low molecular weight compounds, medium molecular weight compounds, antigens (cancer antigens, antigens from infectious diseases, autoantigens in immune diseases, etc.), etc. Among them, low molecular weight compounds, medium molecular weight compounds, and peptides are more preferred.

[0097] The applicable diseases of the pharmaceutical composition 1 of this embodiment are not particularly limited, and can be widely used for the prevention or treatment of currently known diseases or diseases to be discovered in the future. It can be a chronic disease or an acute disease. Examples of currently known diseases include cancer, infectious diseases, immune diseases, inflammatory relaxation, allergic diseases, skin diseases, hypertension, diabetes, nervous system diseases, hereditary diseases, cardiovascular diseases, cerebrovascular diseases, respiratory diseases, eye diseases, ear diseases, bone and joint diseases, pain diseases, etc.

[0098] [Pharmacologically active peptides or proteins] As a pharmaceutically active peptide or protein, it refers to a peptide or protein that has a positive or favorable effect on the state or condition of the subject when a therapeutically effective amount is administered to the subject. As a preferred pharmaceutically active peptide or protein, it has a radical or symptomatic effect, and can be administered to improve, slow down, alleviate, reverse, delay one or more symptoms of a disease or condition, or reduce the severity of the symptoms. The pharmaceutically active peptide or protein has a preventive property and can be used to delay the development of the disease or reduce the severity of such a disease or condition. The term "pharmaceutically active peptide or protein" refers to a full-length protein or polypeptide, and may also refer to a pharmaceutically active fragment thereof. The term also includes pharmaceutically active analogs of peptides or proteins.

[0099] Examples of pharmaceutically active proteins include, but are not limited to, immunologically active compounds such as cytokines and immune system proteins (e.g., interleukins, colony stimulating factors, factor, CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferon, integrin, addressin, selectin, homing receptor, T cell receptor, immunoglobulin, antibody, hormone (insulin, thyroid hormone, catecholamine, gonadotropin, stimulating hormone, prolactin, oxytocin, dopamine, bovine growth hormone, leptin, etc.), growth hormone (e.g., human growth hormone), growth factor (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptor. Enzyme (tissue plasminogen activator, streptokinase, cholesterol biosynthetic enzyme or degrading enzyme, steroidogenic enzyme, kinase, phosphodiesterase, methylase, demethylase The invention also includes but is not limited to enzymes (such as phospholipases, cytochromes, adenylate cyclases or guanylate cyclases (guanylastes), ceramidase, 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 (for example, proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin, etc.), 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), etc.

[0100] [Nucleic Acids] Examples of the nucleic acid include DNA and RNA, and include short-chain interfering RNA (siRNA), double-chain RNA (dsRNA), micro RNA (miRNA), short-chain hairpin RNA (shRNA), and nucleic acid aptamers.

[0101] [Low molecular weight compounds] Low molecular weight compounds are compounds with a molecular weight of less than about 500, for example, anticancer agents (for example, alkylating agents, anti-metabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroids, non-steroidal anti-inflammatory agents, etc.), anti-rheumatic agents, antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfonamides, etc.), etc.

[0102] The active ingredient may be a Rho kinase inhibitor, an endothelin A receptor inhibitor, a transmembrane conductance regulator (CFTR) regulator, a TRPV1 inhibitor, an NK1 receptor inhibitor, a purine receptor inhibitor and an angiotensin receptor inhibitor, a peroxisome proliferator-responsive receptor, a P2Y receptor inhibitor, a VEGF inhibitor, or the like, or may be an active ingredient having two of these inhibitory effects at the same time.

[0103] In addition, as an active ingredient, from the perspective of being able to fully exert the interaction with the steryl group of the above-mentioned hyaluronic acid derivative, a substance with high hydrophobicity, i.e., poor water solubility, may also be preferably used. It should be noted that "poor water solubility" means that the amount of water required to dissolve 1g of solute in the 17th revised "Japanese Pharmacopoeia" needs to be more than 30mL.

[0104] Examples of the poorly water-soluble solid active ingredient include active ingredients of pharmaceuticals described in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia Extra-Pharmacopoeia Standards, USP, NF, and EP, such as acetaminophen, ibuprofen, benzoic acid, ethenzamide, caffeine, camphor, quinine, calcium gluconate, dimercaprol, sulfonamide, theophylline, theobromine, riboflavin, toluenepropanol, phenobarbital, aminophylline, thiosemicarbazide, quercetin, rutin, salicylic acid, theophylline sodium salt, pyrabital, quinine hydrochloride, irgapyrin, digoxin, Antipyretic analgesics such as griseofulvin and phenacetin, nervous system drugs, sedatives and hypnotics, muscle relaxants, hypotensive agents, antihistamines, etc.; antibiotics such as acetylspiramycin, ampicillin, erythromycin, kitasamycin, chloramphenicol, triacetyloleandomycin, nystatin, colistin sulfate, etc.; steroid hormones such as methyltestosterone, methylandrostenediol, progesterone, estradiol benzoate, ethinylestradiol, deoxycorticosterone acetate, cortisone acetate, hydrocortisone, hydrocortisone acetate, prednisolone, etc.; non-steroidal yolk hormones such as dienystrol, hexestrol, diethylstilbestrol, diethylstilbestrol dipropionate, chloroethenol, etc.; other fat-soluble vitamins, etc. One selected from these active ingredients may be used, or two or more may be used in combination.

[0105] The active ingredient can be an oily or liquid active ingredient that is poorly soluble in water. As an oily or liquid active ingredient that is poorly soluble in water, for example, it can be enumerated: "Japanese Pharmacopoeia", "Japanese Pharmacopoeia External Drug Standards", "USP", "NF", "EP" in the drug active ingredients recorded in the active ingredients, such as teprenone, indomethacin farnesate, menatetrenone, phytonadione, vitamin A oil, phenylpentyl alcohol, vitamin D, vitamin E and other vitamins, DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), liver oil and other higher unsaturated fatty acids, coenzyme Q, orange oil, lemon oil, peppermint oil and other oil-soluble spices, etc. Vitamin E has various homologues and derivatives, but as long as it is liquid at room temperature, it is not particularly limited, for example, dl-α-tocopherol, dl-α-tocopherol acetate, d-α-tocopherol and d-α-tocopherol acetate can be enumerated. One type selected from these active ingredients may be used, or two or more types may be used in combination.

[0106] The active ingredient may be a semi-solid active ingredient that is poorly soluble in water. As a semi-solid active ingredient that is poorly soluble in water, for example, it can be enumerated: earthworms, licorice, cinnamon, peony, peony bark, valerian, pepper, ginger, tangerine peel, ephedra, bamboo, cherry peel, Polygala, Platycodon, Plantago, Plantago, Lycoris, Polygala, Fritillaria, fennel, Phellodendron, Coptis, Curcuma, chamomile, gentian, bezoar, animal gall, Adenophora, ginger, Atractylodes, cloves, tangerine peel, Atractylodes, bamboo ginseng, carrots, Pueraria Decoction, Guizhi Decoction, Xiangsusan, Zihu Guizhi Decoction, Xiaozihu Decoction, Xiaoqinglong Decoction, Maimendong Decoction, Banxia Houpo Decoction, Mahuang Decoction and other Chinese medicines or crude drug extracts; oyster meat extract, propolis and propolis extracts, coenzyme Q, etc. One selected from these active ingredients can be used, or two or more can be used in combination.

[0107] [Medium molecular weight compounds] In this specification, "medium molecule" refers to peptides, macrolide compounds, nucleic acids and natural products or their derivatives with a molecular weight of about 500 to 5000, which are neither low molecular weight (organic compounds with a molecular weight of about 500 or less) nor high molecular weight (proteins with a molecular weight of more than 10,000, etc.). Peptides with a molecular weight of about 500 to 2000 are preferred, that is, linear or cyclic peptides with about 5 to 20 amino acid residues. As peptides, cyclic peptides are preferred, and the details will be described later. Macrolide compounds refer to macrocyclic lactones, which are a general term for compounds with 12 or more ring members. Examples of the middle molecular compound include FK506 and rapamycin.

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

[0109] The antigenic proteins that can be used in the pharmaceutical composition 1 of this embodiment are not limited, and examples thereof include: ERK1, ERK2, WT1, MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal related antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate specific membrane antigen (PSMA), T cells Cellular receptor / CD3-ζ chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE- A11, 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 and 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, adenomatous polyposis coli protein (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, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, and c-erbB-2.

[0110] As the above-mentioned antigen protein, the entire sequence thereof may be used, or a partially deleted sequence may be used.

[0111] The antigenic peptide that can be used in the pharmaceutical composition 1 of this embodiment is an antigenic peptide that contains one or more epitopes selected from CD8 positive cytotoxic T cell recognition epitopes and CD4 positive helper T cell recognition epitopes in the sequence of the antigenic protein. In one embodiment, from the perspective of being carried on class I MHC molecules or class II MHC molecules after being decomposed in antigen presenting cells, the above-mentioned antigenic peptide is preferably an antigenic peptide containing two or more epitopes. Specifically, as the above-mentioned antigenic peptide, an antigenic peptide containing an epitope of an antigenic protein of a tumor cell can be cited.

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

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

[0114] In one embodiment, when two or more epitopes are included, an amino acid linker can be configured 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. As the amino acids used in the linker, glycine (G), tyrosine (Y), leucine (L), tryptophan (W), etc. can be cited. Preferably, tyrosine (Y), leucine (L), tryptophan (W). Specific examples of amino acid linkers include: a linker (4Y) composed of four consecutive tyrosines (Y), a linker (4L) composed of four consecutive leucines (L), a linker (4W) composed of four consecutive tryptophans (W), a linker (6G) composed of six consecutive glycines (G), a linker (6Y) composed of six consecutive tyrosines (Y), a linker (6L) composed of six consecutive leucines (L), a linker (6W) composed of six consecutive tryptophans (W), a linker (8Y) composed of eight consecutive tyrosines (Y), a linker (8L) composed of six consecutive leucines (L), and a linker (8W) composed of eight consecutive tryptophans (W), preferably 6Y, 6L or 6W.

[0115] (Antigens from infectious diseases) As the antigen from infectious diseases, there is no particular limitation as long as it is an infectious pathogen and an antigen from an infectious pathogen. As infectious pathogens, viruses, bacteria, fungi, nematodes, etc. can be cited. The antigen from an infectious pathogen can be an antigen protein or an antigen peptide.

[0116] The diseases caused by the infectious pathogens are not particularly limited, and examples thereof include adenoviruses, herpes viruses (e.g., HSV-I, HSV-II, CMV, VZV), poxviruses (e.g., orthopoxviruses of smallpox, vaccinia, molluscum contagiosum, etc.), picornaviruses (e.g., rhinoviruses, enteroviruses), orthomyxoviruses (e.g., influenza viruses), paramyxoviruses (e.g., parainfluenza viruses, mumps viruses, measles viruses, respiratory syncytial virus (RSV)), coronaviruses (e.g., SARS coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2), papovaviruses (e.g., papillomaviruses that cause genital warts, common warts, plantar warts, etc.), hepadnaviruses (e.g., hepatitis B virus), flaviviruses (e.g., hepatitis C virus, Viral diseases such as diseases caused by viral infection with Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, Bordetella, etc.; fungal diseases represented by but not limited to chlamydiosis, candidiasis, aspergillosis, histoplasmosis, and cryptococcal meningitis; malaria, Pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, trypanosome infection, etc.

[0117] The structure of the 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 the various components constituting the pathogen, and examples thereof include live bacteria vaccines, inactivated whole particles, parts thereof, protein subunits, proteins, peptides, etc. Among them, protein subunits, proteins, or peptides are preferred from the perspective of being complexed with a hyaluronic acid derivative.

[0118] In addition, herein, the above-mentioned influenza virus refers to an RNA enveloped virus with a particle size of about 100nm in diameter belonging to the Orthomyxoviridae family, and is divided into A, B and C types based on the antigenicity of the internal protein. The above-mentioned influenza virus is composed of the core and external glycoprotein of ribonucleic acid (RNA), and ribonucleic acid (RNA) is associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope with a lipid bilayer structure. The inner layer of the above-mentioned viral envelope is mainly composed of matrix protein, and the outer layer is mostly composed of lipid substances from the host. In addition, the RNA of the above-mentioned influenza virus has a segmental structure. It should be noted that the influenza that is in the global pandemic is caused by type A influenza virus, which has two kinds of envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and according to the difference in antigenicity, HA is divided into 16 subtypes, and NA is divided into 9 subtypes. As the antigen derived from the infectious disease, it is preferable to use antigens derived from influenza virus type A and type B. In addition, the subtypes of the influenza virus type A and type B are not particularly limited, and may be subtypes isolated so far or may be subtypes isolated in the future.

[0119] In addition, as an antigen from influenza virus, as long as it is at least a part of the various components constituting the above-mentioned influenza virus, there is no particular limitation, for example, it can be cited: a complete virus particle obtained by inactivating the purified virus particle with an organic solvent / surfactant or other reagent, or a virus subunit prepared by removing impurities from the complete virus particle and purifying HA and / or NA, etc. From the perspective of immunogenicity, HA subunits or complete virus particles are preferred. The above-mentioned complete virus particles are preferably inactivated by formalin or the like. In addition, HA subunits (splits) having few impurities and requiring adjuvants such as immunostimulants are particularly effective.

[0120] The method for preparing the influenza virus antigen is not particularly limited, and known methods can be used without limitation. For example, eggs or the like are infected with a virus strain isolated from an influenza-infected animal or influenza patient, and the virus is cultured by a conventional method to prepare the antigen from the purified virus stock solution. In addition, antigens from viruses prepared in cultured cells by genetic engineering can also be used.

[0121] (Antigens in immune diseases) As an antigen in an immune disease, there is no particular limitation as long as the epitope of the target protein of the immune disease is included. As an immune disease, there is no particular limitation, for example, it can include: psoriasis vulgaris, ankylosing spondylitis, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, bronchial asthma, chronic urticaria, hay fever, atopic dermatitis, etc. As a target protein, there is no particular limitation, for example, it can 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, etc. Reference 1 (International Publication WO No. 2017 / 164409) records a peptide from IL-17A.

[0122] When the (C) active ingredient 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 classified as slightly soluble, slightly poorly soluble, poorly soluble, very poorly soluble, or almost insoluble among the terms indicating solubility in the 17th revision of the Japanese Pharmacopoeia, namely, very soluble, easily soluble, slightly poorly soluble, poorly soluble, very poorly soluble, and almost insoluble.

[0123] Specifically, (C) the active ingredient may be a poorly water-soluble drug having a solubility in water of 1 mg / mL or less. The pharmaceutical composition 1 of the present invention can solubilize even such a poorly water-soluble drug at a high concentration without using an organic solvent while reducing the amount of a highly toxic surfactant used.

[0124] (C) The molecular weight of the active ingredient is preferably 200 or more, more preferably 300 or more, more preferably 400 or more, more preferably 500 or more, further preferably 600 or more, further preferably 700 or more, further preferably 800 or more, further 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] The poorly water-soluble drug as (C) active ingredient is preferably a poorly water-soluble peptide. The poorly water-soluble peptide may have an acidic amino acid, a basic amino acid, or a neutral amino acid, preferably a basic amino acid or a neutral amino acid. When compounding, the isoelectric point may be considered and the pH of the solution may be appropriately selected. In addition, the amino acid may include a natural amino acid or a non-natural amino acid.

[0127] Preferably, at least one of the nitrogen atoms constituting the amide bond of the poorly water-soluble peptide has a methyl group. The hydrophobicity imparted by methylation improves the interaction with the hydrophobic part of the hyaluronic acid derivative, and more solubilization is expected. In addition, it can be inferred that the interaction improves, and the stability as a preparation also improves.

[0128] Preferably, the poorly water-soluble peptide contains at least one selected from cyclic peptides and long-chain peptides, hydrophobic group-modified peptides, membrane-destructive peptides, and peptide-drug complexes.

[0129] Preferably, the poorly water-soluble peptide is a cyclic peptide. The cyclic and rigid skeleton maximizes the interaction with the hydrophobic part of hyaluronic acid and forms a stable structure with the hyaluronic acid derivative.

[0130] The cyclic peptide is preferably composed of 4 to 49 amino acids, more preferably 6 to 30 amino acids, and most preferably 8 to 25 amino acids. The number of rings in the molecule is not particularly limited, but is preferably 1 or more and 8 or less.

[0131] As an example of hydrophobic peptides, alkylated polypeptides are included. " Insulin detemir " sold by Novo Nordisk Pharmaceuticals is an insulin analog, which is designed to bind C14 fatty acid side chains to the lysine at position 29 of human insulin B chain, and shows affinity with albumin. The fatty acid side chain promotes the self-association between insulin detemir hexamers, and is bound to albumin at the subcutaneous injection site, so it is subject to stabilization effect, and the absorption rate from the administration site is reduced. By making the hyaluronic acid derivative pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later, the hydrophobic part of the complex of hyaluronic acid derivative and association promoter or solubilizing aid in vivo interacts firmly with the hydrophobic part possessed by peptide, and the sustained-release time can be extended accordingly.

[0132] Furthermore, the hyaluronic acid derivative pharmaceutical composition 1 or the pharmaceutical composition 2 described later comprising the above peptide can be expected to provide a longer-term sustained release by effectively suppressing the degradation of the hyaluronic acid derivative by the peptide degrading enzyme in the body.

[0133] In the present embodiment, the amount of the active ingredient (C) 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, relative to 100 parts by mass of the hyaluronic acid derivative (A).

[0134] (C) The active ingredient is not particularly limited, and the poorly water-soluble drug may be blended alone or in combination of two or more.

[0135] About the pharmaceutical composition 1 of the present embodiment, when (C) active ingredient as a poorly water-soluble drug is prepared, it is possible to solubilize without using an organic solvent, and it is possible to reduce the high solubilizing agent of toxicity in the past. In addition, by (B) solubilizing aid, it is possible to promote the hydrophobic interaction between the steryl of (A) hyaluronic acid derivative. It can be inferred that powdered (C) active ingredient can be solubilized with high concentration. It should be noted that it can be the situation of obtaining the desired effect by a mechanism different from the above-mentioned mechanism.

[0136] That is, the pharmaceutical composition 1 of the present embodiment can also be referred to as a composition that does not use an organic solvent, or a composition for solubilizing a powdered drug.

[0137] 《Other additives》 The pharmaceutical composition 1 of the present embodiment can also be administered alone, or administered according to a conventional method together with a pharmacologically acceptable carrier. In the case of being used in combination with a pharmacologically acceptable carrier, for example, the above-mentioned hyaluronic acid derivative and the above-mentioned active ingredient and an adjuvant as required, and water or a physiologically acceptable liquid (such as saline, phosphate buffered saline (PBS)) etc. in addition can be mixed, and physiologically acceptable buffer, excipient, carrier (vehicle), preservative, stabilizer, binding agent, lyophilizing aid etc. can also be included.

[0138] Examples of the buffer include Tris, sodium phosphate, potassium phosphate, histidine, and citric acid.

[0139] The hyaluronic acid derivative is dissolved in a pharmaceutically acceptable medium at any concentration for use. For example, water for injection, phosphate buffer and other buffers are preferred. As a buffer, there is no particular restriction as long as it is a compound with a buffering capacity in the pH range of 4 to 10 of the composition. As examples of buffers, there can be cited: acetates such as sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphates such as dipotassium hydrogen phosphate, ε-aminocaproic acid, amino acid salts such as sodium glutamate, boric acid and its salts, and mixtures thereof. Among them, water for injection, phosphate buffer, water for injection containing sucrose, phosphate buffer containing sucrose, 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 be blended. These pH adjusters may be used alone or in any combination of two or more.

[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, and is, for example, in the range of 4.0 to 9.0, preferably in the range of 4.0 to 8.8, and more preferably in the range of 6.5 to 8.8.

[0143] Examples of the preservative in the pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described below include benzalkonium chloride, methylparaben, propylparaben, chlorobutanol, sorbic acid, and alkylpolyaminoethylglycine. If necessary, one or more preservatives may be further compounded, and there are no particular limitations as long as they are pharmaceutically acceptable preservatives. Examples of the preservative in the present invention include benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, parabens such as ethyl paraben, benzyl alcohol, m-cresol, phenol, phenethyl alcohol, sorbic acid or its salts, and thimerosal.

[0144] The pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later may further contain one or more thickeners as necessary, and there are no particular limitations as long as the thickeners are pharmaceutically acceptable. Examples of thickeners for the pharmaceutical composition 1 of the present invention or the pharmaceutical composition 2 described below include cellulose polymers (methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, etc.), vinyl polymers (polyvinyl pyrrolidone, polyvinyl alcohol, etc.), sugars (hyaluronic acid, its salts and other mucopolysaccharides, gellan gum, sodium alginate, dextran, cyclodextrin and other polysaccharides), oxyalkylene polymers (polyoxyethylene polyoxypropylene block copolymers), etc. The molecular weight of the thickener in the present invention can be, for example, from a number average molecular weight of 0.5×10 4 ~100×10 4 Select from the left and right ranges.

[0145] Examples of the stabilizer include edetate sodium hydrate and polyvinyl pyrrolidone (povidone).

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

[0147] As isotonic agents, sucrose, glucose, dextrose, lactose, mannitol, calcium or magnesium compounds, such as CaCl 2 wait. According to JP Patent No. 4758893, it is described that the antibacterial and antiseptic effects can be improved by mixing glycerol at a concentration (2 to 2.5% (v / v)) that substantially makes the osmotic pressure. Therefore, not only the function as an isotonic agent but also the antibacterial and antiseptic effects can be expected. The content of glycerol is, for example, 0.01 to 10% (w / v), preferably 0.05 to 5% (w / v), more preferably 0.1 to 3.0% (w / v), further preferably 0.3 to 3.0% (w / v), and particularly preferably 0.3 to 2.5% (w / v).

[0148] The pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later may be blended with a base as needed. There are no particular limitations on the base as long as it is pharmaceutically acceptable, and examples thereof include sodium hydroxide, potassium hydroxide, monoethanolamine, diethanolamine, triethanolamine, tromethamine, and meglumine.

[0149] The pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later may contain an inorganic salt as needed. There are no particular limitations on the base as long as it is a pharmaceutically acceptable base, and examples thereof include zinc chloride and zinc acetate.

[0150] As the pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later, a pharmaceutically formulated substance can be used. The form of the pharmaceutical composition may be solid, semisolid, or liquid. In the case of a solid, the form may be powder, granules, pills, pellets, tablets, capsules, etc. Among them, freeze-dried powder is preferred as a solid. In the case of a semisolid, the form of a gel or the like is exemplified. In the case of a liquid, the form may be a suspension in which the powder is diluted or suspended in water or a buffer such as phosphate buffer (PB) or phosphate buffered saline (PBS).

[0151] [Physical properties of pharmaceutical composition] Preferably, the pharmaceutical composition of this embodiment produces a precipitate at a physiological salt concentration. Specifically, it is preferred that the pharmaceutical composition has an in vitro precipitation rate of 20%, preferably 50% or more, more preferably 70% or more, further 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 precipitation samples 200 μL of concentrated buffer (40 mM PB, 600 mM NaCl aqueous solution) was added to a microtube (1.5 mL), and 600 μL of a preparation containing a hyaluronic acid derivative pharmaceutical composition was added. Then, the mixture was mixed with voltex for 30 seconds, incubated at 37°C for 20 minutes, and then the precipitate was precipitated with a centrifuge (2000 G, 5 min). Next, the hyaluronic acid derivative in the supernatant was subjected to GPC measurement.

[0153] Preparation of blank samples Add 200 μL of water for injection to a microtube (1.5 mL), add 600 μL of a preparation containing a hyaluronic acid derivative pharmaceutical composition (the theoretical concentration of the hyaluronic acid derivative is X mg / mL. X>1.33). Then, mix with voltex for 30 seconds, incubate at 37°C for 20 minutes, and centrifuge (2000G, 5 minutes). Next, dilute with water for injection so that the hyaluronic acid derivative in the supernatant becomes 1 mg / mL, and provide for GPC determination.

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

[0155] Confirmation of precipitation In the present specification, a hyaluronic acid derivative pharmaceutical composition having a precipitation rate represented by the following formula of 20% or more is evaluated as producing precipitation at a physiological salt concentration. Precipitation rate (%) = {1-(area value of hyaluronic acid derivative of precipitation sample) ÷ ((area value of hyaluronic acid derivative of blank sample) × (0.75X))} × 100

[0156] In the above test, the centrifugal separation conditions and incubation time can be set arbitrarily when preparing the precipitate sample. For example, when the precipitation rate satisfies 20% or more when left to stand at 37°C for 2 weeks under the above conditions, it is also judged that a precipitate is produced at a physiological salt concentration.

[0157] Preferably, in the pharmaceutical composition 1 of the present embodiment or the pharmaceutical composition 2 described later, no precipitate is visually observed in an environment at 20°C. If the presence of precipitates is confirmed, the insoluble particle test method for injections specified in the Japanese Pharmacopoeia, the light-shielding automatic particle measuring device (liquid particle counter "KL-05") used in the first method "light-shielding particle counting method" can be used for detection. From the supersaturated solution in which the amorphous drug is completely dissolved, drug crystals of micron size may sometimes precipitate. In the absence of precipitates, no particles can be observed in the above-mentioned light-shielding automatic particle measuring device. Alternatively, a DLS device may be used to verify the presence or absence of particles larger than 1 μm.

[0158] Preferably, the pharmaceutical composition 1 of this embodiment or the pharmaceutical composition 2 described later can be sterilized by filtration. Filter sterilizability can be determined by flow-through evaluation using a syringe filter as follows. Specifically, it was evaluated whether the sterile filtration filter with a pore size of 0.45 μm or 0.22 μm could be passed. More specifically, the filter membrane used When a pore size of 0.45 μm and a 3 mL sterile syringe filter is used, more than 50% of the hyaluronic acid derivative passing through the filter membrane of 2 mL of the hyaluronic acid derivative composition is liquid-permeable, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, and most preferably 90% or more.

[0159] <Hyaluronic acid derivative pharmaceutical composition 2> This embodiment is a hyaluronic acid derivative pharmaceutical composition containing (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing agent, and (C1) an effective ingredient. Hereinafter, the "hyaluronic acid derivative pharmaceutical composition containing (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing agent, and (C1) an effective ingredient" is sometimes referred to as "pharmaceutical composition 2".

[0160] In the pharmaceutical composition 2 of the present embodiment, the average particle diameter of the spherical structure containing the active ingredient-hyaluronic acid derivative complex can be more than 20nm and less than 220nm, can be more than 20nm and less than 150nm, can be more than 30nm and less than 100nm. By the average particle diameter being the above-mentioned numerical range, it can be present in the organism with a stable structure, and can pass through the lymph nodes more easily. The average particle diameter can be measured, for example, by DLS (dynamic light scattering: Dynamic LightScattering), nanoparticle tracking measurement device, size exclusion chromatography, high performance liquid chromatography and electron microscopy. More specifically, for example, in a DLS device, the hyaluronic acid derivative concentration is 1mg / mL, and it is measured by diluting with 10mM phosphate buffer or 10mM phosphate buffer containing 10w / v% sucrose.

[0161] The solubilizing agent (B1) contained in the pharmaceutical composition 2 of the present embodiment contains at least 4 or more ether structures (ROR) and has 4 or more carbon atoms. In the pharmaceutical composition 2 of the present embodiment, the content of the solubilizing agent (B1) is 0.0001 parts by mass or more and 15000 parts by mass or less per 100 parts by mass of the hyaluronic acid derivative (A1) into which a hydrophobic group is introduced.

[0162] The pharmaceutical composition 2 of the present embodiment contains a specific solubilizing agent in a specific amount. The pharmaceutical composition of the present embodiment containing the above-mentioned (B1) solubilizing aid can interact with the hyaluronic acid derivative introduced with a hydrophobic group by the moderate polarity brought by the ether structure and the moderate hydrophobicity brought by the alkyl skeleton, and can promote the interaction between the hydrophobic groups in the hyaluronic acid derivative introduced with a hydrophobic group by the above-mentioned (A1). Thus, a pharmaceutical composition that can solubilize a poorly water-soluble active ingredient at a high concentration can be provided.

[0163] When the content of the solubilizing aid (B1) is at least the above lower limit, the active ingredient can be sufficiently solubilized in the pharmaceutical composition. When the content of the solubilizing aid (B1) is below the upper limit, the active ingredient can be sufficiently solubilized in the pharmaceutical composition within a range that does not excessively increase the viscosity of the pharmaceutical composition and does not affect the living body.

[0164] 《(A1) Hyaluronic acid derivatives having hydrophobic groups introduced》 The description of the (A1) hyaluronic acid derivative into which a hydrophobic group is introduced contained in the pharmaceutical composition 2 is substantially the same as the description of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced contained in the above-mentioned pharmaceutical composition 1.

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

[0166] When the steryl introduction rate is within the above range, the (A1) hyaluronic acid derivative can interact firmly with the water-poorly soluble drug and the hydrophobic part of the surfactant. In addition, when the steryl introduction rate is within the above range, the stability of the preparation of the hyaluronic acid derivative-drug complex in which the (A1) hyaluronic acid derivative in the pharmaceutical composition 2 is complexed with the drug can be improved.

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

[0168] By the weight average molecular weight of hyaluronic acid derivative being more than the above-mentioned lower limit, the complexation of molecule can be further improved, and the retention in blood can be further improved. On the other hand, by the weight average molecular weight of hyaluronic acid derivative below the above-mentioned upper limit, the rise of viscosity can be suppressed, and the hyaluronic acid derivative of higher concentration can be dissolved in the pharmaceutical composition. The weight average molecular weight of hyaluronic acid derivative can be regulated by using the raw material with corresponding molecular weight usually.

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

[0170] From the viewpoint that the hyaluronic acid derivative strongly exhibits the properties of hyaluronic acid, for example, in order to be strongly recognized by the CD44 receptor, it is preferably 100 k or more, particularly preferably 200 k or more, and most preferably 300 k or more.

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

[0172] 《(B1) Solubilizing agent》 In the present invention, "solubilization" means that the active ingredient (C1) described later can be dissolved in water until it becomes visually transparent. The "solubilizing agent" refers to an agent that has the effect of increasing the solubility of the (C) active ingredient when added to water together with the (C1) active ingredient and the (A1) hyaluronic acid derivative.

[0173] As the solubilizing agent (B1), there is no particular limitation as long as it has the function of dissolving (solubilizing) the active ingredient (C1) in the aqueous phase, and it can be appropriately selected according to the purpose, for example, nonionic surfactants can be mentioned. The solubilizing agent (B1) used in the present invention is a reagent that can be generally used for medical purposes, and is a reagent that contains at least 4 or more ether structures (ROR) and satisfies the requirement of 4 or more carbon atoms.

[0174] Preferably, the solubilizing aid (B1) is at least one selected from a nonionic surfactant, polyethylene glycol having a molecular weight of 190 g / moL or more and 4000 g / moL or less, and a cyclodextrin derivative.

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

[0176] As the solubilizing aid (B1), preferably at least one selected from the group consisting of 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 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), The present invention relates to 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 tocopherol polyethylene glycol succinate (having 21 or more ether structures and 35 or more carbon atoms).

[0177] In addition to the above, for example, polyethylene glycol 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), Cremophor EL (having 35 or more ether structures and 127 or more carbon atoms), etc. can also be mentioned.

[0178] Examples of the solubilizing aid (B1) 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 aid (B1) is 0.0001 parts by mass or more and 15,000 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, further preferably 1 parts 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, relative to 100 parts by mass of the hyaluronic acid derivative (A1).

[0180] When the solubilizing aid (B1) is a nonionic surfactant, the content of the nonionic surfactant 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, relative to 100 parts by mass of the hyaluronic acid derivative (A1).

[0181] When the solubilizing aid (B1) is polyethylene glycol having a molecular weight of 190 g / moL or more and 4000 g / moL or less, the content of polyethylene glycol 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 further preferably 500 parts by mass or more and 5000 parts by mass or less, relative to 100 parts by mass of the hyaluronic acid derivative (A1).

[0182] The less the solubilizing agent (B1), the better. However, if it is below the above lower limit, the active ingredient cannot be fully solubilized. In addition, if it is greater than the upper limit, the toxicity is likely to increase due to the excessive amount of solubilizing agent (B1), and the viscosity of the preparation is also increased, so it is preferably within the above range.

[0183] The means for dissolving the solubilizing aid (B1) is not particularly limited, and examples thereof include stirring, vibration, and ultrasonic treatment.

[0184] 《(C1) Active ingredient》 The description of the (C1) active ingredient contained in the pharmaceutical composition 2 is substantially the same as the description of the (C) active ingredient contained in the above-mentioned pharmaceutical composition 1.

[0185] In the pharmaceutical composition 2, the amount of the poorly water-soluble drug as the active ingredient (C1) 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 further preferably 23 parts by mass or more and 50 parts by mass or less, relative to 100 parts by mass of the hyaluronic acid derivative (A1).

[0186] The (C1) active ingredient is not particularly limited, and the poorly water-soluble drug may be blended alone or in combination of two or more.

[0187] Regarding the pharmaceutical composition 2 of this embodiment, when formulating the (C1) active ingredient which is a poorly water-soluble drug, it is possible to solubilize it without using an organic solvent, and the amount of the conventional highly toxic solubilizing agent used can be reduced. In addition, by containing (B1) solubilizing aid, the hydrophobic interaction between the steryl of (A1) hyaluronic acid derivative can be promoted.Thus, the hydrophobic region possessed by the hyaluronic acid derivative pharmaceutical composition expands, and the carrying capacity of hydrophobic drugs increases.In addition, except being bound to the hydrophobic portion of the hyaluronic acid polymer by chemical bond, the solubilizing aid can also play a role in the hydrophobic region of the high mobility in the inside of the hyaluronic acid derivative pharmaceutical composition, therefore can be solubilized powdered (C1) active ingredient with high concentration.It should be noted that the situation that the desired effect can be obtained by a mechanism different from the above-mentioned mechanism.

[0188] That is, the pharmaceutical composition 2 of the present embodiment may also be referred to as a composition that does not use an organic solvent, or a composition for solubilizing a powdered drug.

[0189] The content of the organic solvent relative to the pharmaceutical composition 2 is preferably less than 0.8%. Here, the organic solvent contained in the pharmaceutical composition 2 is a solvent corresponding to categories 1 to 3 as defined in the Guidelines for Residual Solvents in Pharmaceuticals.

[0190] Specifically, examples of the first type organic solvent include benzene, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethane, and 1,1,1-trichloroethane.

[0191] Examples of the second type of organic solvent include acetonitrile, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethylene, 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-trichloroethylene, and xylene.

[0192] Examples of the third class of organic solvents include acetic acid, acetone, anisole, 1-butanol, 2-butanol, n-butyl acetate, tert-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] The organic solvents of categories 1 to 3 are preferably not contained in the pharmaceutical composition 2, but are components that inevitably remain in the production process of the hyaluronic acid derivative. Even if they are organic solvents of categories 1 to 3, if the content of the organic solvent relative to the pharmaceutical composition 2 is less than 0.8%, it is within the range that can be used safely.

[0194] 《Other additives》 The pharmaceutical composition 2 of the present embodiment can be administered alone, or can be administered according to a conventional method together with a pharmacologically acceptable carrier. In the case of being used in combination with a pharmacologically acceptable carrier, for example, the above-mentioned hyaluronic acid derivative and the above-mentioned active ingredient and adjuvant as required and water or physiologically acceptable liquids (such as normal saline, phosphate buffered saline (PBS)) etc. in addition can be mixed, and physiologically acceptable buffer, excipient, carrier, preservative, stabilizer, bonding agent, lyophilizing aid etc. can also be included. The explanation about additive is the same as the explanation about << other additives >> in the above-mentioned pharmaceutical composition 1.

[0195] 《Method for producing hyaluronic acid derivatives》 Hyaluronic acid derivatives can be obtained, for example, by converting the carboxyl group of glucuronic acid into an amide and introducing a steryl group. In addition, the introduction rate of the steryl group can be controlled by adjusting the amount of the compound having a steryl group to be reacted relative to the raw material hyaluronic acid or its derivative.

[0196] As a method for converting the carboxyl group of glucuronic acid into an amide and introducing a steryl group, specifically, for example, there can be mentioned the following steps: ion-exchanging a raw material hyaluronic acid or a derivative thereof (preferably a hyaluronic acid or a derivative thereof consisting only of repeating unit (II)) with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), reacting the hyaluronic acid salt with an introduced steryl group represented by the formula: "HNR a -Y-NR b -R, NHR a -Y-NR b -COO-R, HNR a -Y-NR b -COO-R, HNR a -Y-NR b -CO-R、HNR a -Y-NR b -CO-NR c -R, HNR a -Y-COO-R, HNR a -YO-COO-R, HNR a -YSR, HNR a -Y-CO-Y a-SR, HNR a -YO-CO-Y b -SR, HNR a -Y-NR b -CO-Y b -SR, HNR a -YSSR or -Z-NR a -Y-NR b -COO-R (where R a , R b , R c , Y, Y a , Y b A method for reacting an amine of a steryl group (particularly a cholesteryl group) represented by "(Z, Z and R are as defined in the present specification)".

[0197] The condensing agent that can be used in the above reaction is not particularly limited, and examples thereof include: 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholine (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroethylene, borate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tripyrrolidine-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), etc.

[0198] Although not particularly limited, DMT-MM is preferred based on the angle of reacting efficiently in a mixed solvent of water and an organic solvent. In addition, by using DMT-MM as a condensing agent, it is possible to utilize amino and carboxyl to form amide bonds in a system where a large amount of hydroxyls coexist while suppressing ester bond formation. By using this condensing agent, for example, it is possible to prevent the alcohol as a solvent from reacting with the carboxyl of the hyaluronic acid part or the carboxyl and hydroxyl that exist simultaneously in the hyaluronic acid part from forming undesirable crosslinking in the molecule or intermolecular bonding.

[0199] Examples of the solvent used in the steryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixed solvents thereof. The polyol may be a diol or a triol. Examples of the diol include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of the triol include glycerol and trimethylolpropane.

[0200] Alternatively, the raw material hyaluronic acid or its derivatives can be ion-exchanged into tetraalkylammonium salts (e.g., tetrabutylammonium (TBA) salts), and the hyaluronate is reacted with the spacer group part in a solvent in the presence of an appropriate condensing agent (at this time, a protection reaction and a deprotection reaction can be performed as needed), and the carboxyl group (-COOH) of the raw material hyaluronic acid or its derivatives is converted, and then reacted with an appropriate reagent. Examples of the combination of the group derived from the carboxyl group and the reaction reagent 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 -YO-CO-CH=CH 2 +HS-R; -CONR a -Y-NR b -CO-CH(CH 3 )=CH 2 +HS-R; -CONR a -Y-SH+HS-R; -COZ-OH+HNR a -Y-NR b -COO-R; -COZ-NR a -Y-NR b H+Hal-COO-R (Where R a , R b , R c , Y, Y a , Y b and Z are as defined in the present specification, and Hal represents a halogen atom selected from a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0201] As the reaction mode, there can be mentioned: dehydrohalogenation reaction, condensation reaction, dehydration reaction, nucleophilic addition reaction such as Michael addition, oxidative disulfide formation reaction, etc. These are well-known reactions, and those skilled in the art can appropriately select and find the preferred reaction conditions to carry out. When the transformant or reactant has a carboxyl group, it can be made into N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester and reacted.

[0202] In addition, the following method can be mentioned: 2-aminoethyl 2-pyridyl disulfide is reacted with the carboxyl group of the raw material hyaluronic acid or its derivative to prepare a hyaluronic acid derivative introduced with a spacer group having a thiol group modified with a leaving group at the terminal, and thiol cholesterol is subjected to a nucleophilic substitution reaction to form a disulfide bond.

[0203] In addition, the following method can also be cited: prepare the obtained product with a part of the spacer group introduced into the carboxyl of hyaluronic acid or its derivative and the obtained product with a part of the spacer group introduced into the steryl, and react them. A part of the specific example is as described above, and further, in the case of -SS- being inserted into Y, the following method can also be cited: prepare the hyaluronic acid derivative with a spacer group having a thiol group at the end and the steryl with a spacer group having a thiol group at the end respectively on the carboxyl of hyaluronic acid, and make them undergo oxidation reaction to form a disulfide bond. At this time, one of the thiol groups can also be reacted with 2-thiopyridine to form a disulfide, and then substituted with another thiol group.

[0204] In addition, after the preparation of the hyaluronic acid derivative of the present invention, other substituents may be further introduced. For example, 0.1% or more and 99.5% or less, preferably 40% or more and 65% or less of the carboxyl groups in the hyaluronic acid derivative substantially composed of the repeating units (I) and (II) may be converted into -CO-X z , [Here, X z Selected from the following groups: -NH-(CH 2 ) p1 -O-CO-C(R 17 )=CH 2 ; -NH-(CH 2 ) p1 -O-CO-CH(R 17 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH; -NH-(CH 2 ) p1 -SH; -NH-(CH 2 ) p1 -NH-CO-C(R 17 )=CH 2 ; -NH-(CH 2 ) p1 -NH-C(=NH)-(CH 2 ) 3 -SH; -NH-(CH 2 ) p1 -NH-CO-(CH 2 ) r-SH;-NH-(CH 2 ) p1 -NH-CO-CH(R 17 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH;-NH-(CH 2 ) p1 -NH-CO-CH(NH 2 )-CH 2 -SH;-NH-(CH 2 ) p1 -NH-CO-CH(NH 2 )-(CH 2 ) 2 -SH;-NH-NH-CO-(CH 2 ) 4 -CO-NH-NH-C(=NH)-(CH 2 ) 3 -SH;-NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -O-CO-C(R 17 )=CH 2 ;-NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -O-CO-CH(R1 7 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH;-NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -SH;-NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-C(R 17 )=CH 2 ;-NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2-NH-C(=NH)-(CH 2 ) 3 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-(CH 2 ) r -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-CH(R 17 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-CH(NH 2 )-CH 2 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-CH(NH 2 )-(CH 2 ) 2 -SH; -NH-CH(CO 2 H)-(CH 2 )-SH;-NH-CH(CO 2 H)-(CH 2 ) 2 -SH; and, -NH-CH(CO 2 H)-(CH 2 ) 2 -CONH-CH(CONH-CH 2 -CO 2 H)-CH 2 -SH (here, R 17 Represents a hydrogen atom or C 1-6 The alkyl group (p1 represents an integer of 2 to 10, q represents an integer of 1 to 200, and r represents an integer of 1 to 3)] chemically crosslinks within a molecule or between molecules including other molecules to form a gel.

[0205] The obtained hyaluronic acid derivative can be dried. As the drying method, for example, it can be enumerated: ventilation drying, drying in a thermostatic bath, reduced pressure drying, hot air circulation drying, freeze drying, etc. Among them, freeze drying is preferred. When freeze drying is carried out, from the perspective of more effectively suppressing the increase in the particle size of the microparticles formed by the hyaluronic acid derivative, the hyaluronic acid derivative preferably further contains a cryoprotectant.

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

[0207] As disaccharides, there is no particular limitation, for example, can be enumerated: sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiodiulose, mannobiose, melibiose, plantagobiose, neolactose, galactosucrose, scallionbiose, neohesperidose, rutinose, rutinose, fava sugar, xylobiose, primeverose etc. Among them, from the perspective of being widely used as a cryoprotectant, preferably sucrose, trehalose, maltose or lactose. In addition, from the perspective of the use performance as a pharmaceutical additive, more effectively suppressing the increase of the particle size of the microparticles formed by the hyaluronic acid derivative during freeze drying, more preferably sucrose.

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

[0209] The addition amount of the cryoprotectant is not particularly limited, and is preferably 20 parts by mass or more relative to 100 parts by mass of the hyaluronic acid derivative. By adding the cryoprotectant above the above lower limit, a more sufficient particle size increase inhibitory effect can be obtained. On the other hand, the upper limit of the addition amount of the cryoprotectant is not particularly limited, and for example, can be 100,000 parts by mass.

[0210] The device used in freeze drying is not particularly limited either, for example, a commercially available freeze drying machine can be used. Wherein, from the perspective of controlling the vacuum degree, a freeze drying machine that can monitor the vacuum degree in the device during freeze drying is preferred, and from the perspective of controlling the product temperature, a shelf-type freeze drying machine is preferred.

[0211] <Method for producing pharmaceutical composition 1> The pharmaceutical composition 1 of the present embodiment can be produced by the following method 1 or method 2 for producing the pharmaceutical composition 1 . Hereinafter, each manufacturing method will be described.

[0212] 《Method 1 for producing pharmaceutical composition 1》 Production method 1 is a method for producing a pharmaceutical composition 1 comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient. Production method 1 includes: a step of combining (A) a hyaluronic acid derivative having a hydrophobic group introduced therein and (B) an association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter; and a mixing step of mixing (C) an active ingredient with the aqueous solution of the hyaluronic acid derivative containing the association promoter.

[0213] 《Method 2 for producing pharmaceutical composition 1》 Production method 2 is a method for producing a pharmaceutical composition 1 comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) an active ingredient. Manufacturing method 2 includes: a step of dispersing (C) an active ingredient in (B) an association promoter to obtain a dispersion (I); and a step of 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), including a step of mixing the dispersion (I) with the aqueous solution (II). In the production method 1 and the production method 2, an appropriate pH or a buffer material can be used appropriately according to the structure of the drug.

[0214] In the production method 1 and the production method 2, it is preferred that the step of removing the organic solvent is not included.

[0215] <Method for producing pharmaceutical composition 2> The pharmaceutical composition 2 of the present embodiment can be produced by the following method 1 or method 2 for producing the pharmaceutical composition 2. Hereinafter, each manufacturing method will be described.

[0216] 《Method 1 for producing pharmaceutical composition 2》 The method 1 for producing the pharmaceutical composition 2 is a method for producing the pharmaceutical composition 2 comprising (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing aid, and (C1) an active ingredient. The manufacturing method 1 of the pharmaceutical composition 2 comprises: a step of mixing (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein with (B1) a solubilizing agent to obtain an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent; and a mixing step of mixing (C1) an active ingredient with the aqueous solution of the hyaluronic acid derivative containing the solubilizing agent.

[0217] 《Method 2 for producing pharmaceutical composition 2》 Method 2 for producing pharmaceutical composition 2 is a method for producing pharmaceutical composition 2 comprising (A1) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B1) a solubilizing aid, and (C1) an active ingredient. Method 2 for preparing pharmaceutical composition 2 comprises: a step of dispersing the active ingredient (C1) in the solubilizing agent (B1) to obtain a dispersion (I); and a step of 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), comprising: a step of mixing the dispersion (I) with the aqueous solution (II).

[0218] In the production method 1 and the production method 2 of the pharmaceutical composition 2, it is preferred that the step of removing the organic solvent is not included.

[0219] <Administration Method> The subjects to whom the pharmaceutical composition 1 or 2 of the present embodiment can be administered include animals classified as mammals (monkeys, marmosets, mice, rats, cows, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.), including humans.

[0220] The administration route of the pharmaceutical composition 1 or the pharmaceutical composition 2 of the present embodiment is not particularly limited, and a conventionally known administration route can be appropriately used depending on the intended use, the location of the treatment tissue, and the like. For example, the administration route may include subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intracerebral administration, intraarticular administration, intraperitoneal administration, intravaginal administration, intracapsular administration, intrarectal administration, intravitreal administration, periocular administration, intradermal administration, intraperitoneal administration, intranasal administration, transbronchial administration, transpulmonary administration, transdermal administration, sublingual administration, oral administration, oral administration, and eye drop administration. Among them, subcutaneous administration, intramuscular administration, intravitreal administration, intrathecal administration, and intraarticular administration are preferred. In addition, for topical administration, it may be in the form of powder, cream, ointment, or eye drops. Examples of injection administration include subcutaneous injection, intramuscular injection, intravenous injection, intraarterial injection, intrathecal injection, intraarticular injection, intraperitoneal injection, intravitreal injection, periocular injection, intradermal injection, and intratumor injection.

[0221] In eye drops, by using hyaluronic acid derivatives, it is possible to reduce the initial outbreak of drugs, suppress the aggregation that may occur between drugs during storage and after administration of the preparation, reduce drug efficacy changes, and precipitate and adhere to and remain on the corneal epithelium under physiological conditions. The drug can be sustained-released for a longer time and the number of eye drops can be reduced. Further, it is believed that the absorption of drugs by corneal epithelial cells and conjunctival epithelial cells can also be promoted. In addition, it has high moisture retention, viscosity, and biocompatibility from hyaluronic acid, and can alleviate inflammation, pain, stress, damage, etc. when the preparation contacts, and a preparation with high safety can be provided. Through the above, a hyaluronic acid derivative pharmaceutical composition 1 or a pharmaceutical composition 2 having excellent tear layer stabilization effect, corneal epithelial disorder treatment effect, meibomian gland insufficiency treatment effect, and pain suppression effect is provided by effectively exerting the effect of the drug.

[0222] In the pharmaceutical composition 1 or the pharmaceutical composition 2 of the present embodiment, in the case of parenteral administration, the dosage can be appropriately selected in consideration of the type of the administration subject (including age, gender, etc.), but generally, for example, in humans (weighing 60 kg), the amount of the active ingredient (preferably protein, peptide, low molecular weight compound) per time can be more than 0.01 μg and less than 20 mg, can be more than 0.1 μg and less than 15 mg, or can be more than 1 μg and less than 10 mg.

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

[0224] [Other embodiments] In one embodiment, the present invention provides a method for preventing or treating one or more diseases selected from cancer, infectious diseases, 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. In addition, examples of infectious diseases include the infectious diseases exemplified in the “antigen derived from an infectious disease” in the above-mentioned “antigen”. In addition, the "effective amount" mentioned here includes an amount effective for prevention or treatment, that is, an amount suitable for the prevention or treatment of the onset of the above-mentioned diseases.

[0225] In one embodiment, the present invention provides a composition comprising the above-mentioned active ingredient-hyaluronic acid derivative complex, which is a composition for preventing or treating one or more diseases selected from cancer, infectious diseases, immune diseases, inflammatory relaxation, allergic diseases, skin diseases, hypertension, diabetes, nervous system diseases, hereditary diseases, cardiovascular diseases, cerebrovascular diseases, respiratory diseases, eye diseases, ear diseases, and bone and joint diseases.

[0226] In one embodiment, the present invention provides use of the above-mentioned active ingredient-hyaluronic acid derivative complex in the manufacture of Pharmaceutical Composition 1 or Pharmaceutical Composition 2. Example

[0227] Hereinafter, the present invention will be described in detail by way of Examples, but these are not intended to limit the scope of the present invention to the Examples.

[0228] <Synthesis of Hyaluronic Acid Derivatives> [Synthesis example 1] A hyaluronic acid derivative was prepared according to the following steps 1-A, 2-A and 3-A.

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

[0230] [Process 1-1-A] Under an argon atmosphere, triethylamine (TEA, 1.05 mL) was added to a solution of cholesterol chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) and stirred. Under ice cooling, 6-(tert-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred directly under ice cooling for 30 minutes, then warmed to room temperature (about 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 then distilled off the solvent under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:4), the target fractions were combined, and the solvent was distilled off under reduced pressure.

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

[0232] [Process 2-A] (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) The TBA salt of hyaluronic acid (HA-TBA) was prepared according to the following step 2-1-A and the subsequent step 2-2-A.

[0233] [Process 2-1-A] DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water about three times by decantation. About 1.5 times the molar equivalent of 40 mass % tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added to the cation exchange capacity of the resin, and stirred for 30 minutes. After the remaining TBA-OH solution was removed by decantation, it was further washed with excess ultrapure water to obtain a cation exchange resin after TBA chlorination.

[0234] [Process 2-2-A] The raw material hyaluronic acid sodium salt (HA-Na) with a molecular weight of 35,000 (35 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. The suspension of the cation exchange resin after TBA chlorination in [Step 2-1-A] was added in a 5-fold molar equivalent relative to the molar number of HA units (unit molecular weight 401.3) in accordance with the ion exchange capacity of the resin. After stirring for 15 minutes, the suspension was filtered through a 0.45 μm filter, and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid.

[0235] [Process 3-A] Prepare an anhydrous DMSO solution (10 mg / mL) of the HA-TBA prepared in the above [Step 2-2-A]. Then, add Chol hydrochloride at a molar ratio of 19 / 100 relative to the disaccharide repeating unit (HA unit) present in the HA-TBA synthesized in the above [Step 1-A]. Next, add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) at a molar ratio of 24 / 100 relative to the HA unit, and stir overnight at room temperature (about 25°C). The reaction solution is dialyzed in the order of 0.3M ammonium acetate / DMSO solution, 0.15M NaCl aqueous solution, and ultrapure water (SpectraPor 7, molecular weight cutoff (MWCO): 3,500). The obtained dialyzate is freeze-dried to obtain the target product (HA-C 6 -Chol) as a white solid.

[0236] The product 1 In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH 3 , 1.6 ppm or more and 2.0 ppm or less, 3H), the peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7ppm, 3H), the cholesteryl introduction rate was 19%.

[0237] [Synthesis example 2] A hyaluronic acid derivative was prepared according to the following step 1-B, step 2-B and step 3-B.

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

[0239] [Process 1-1-B] Under an argon atmosphere, triethylamine (TEA, 1.05 mL) was added to a solution of cholesterol chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) and stirred. Under ice cooling, 6-(tert-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred directly under ice cooling for 30 minutes, then warmed to room temperature (about 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 then distilled off the solvent under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:4), the target fractions were combined, and the solvent was distilled off under reduced pressure.

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

[0241] [Process 2-B] (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) The TBA salt of hyaluronic acid (HA-TBA) was prepared according to the following step 2-1-B and the subsequent step 2-2-B.

[0242] [Process 2-1-B] DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water about three times by decantation. About 1.5 times the molar equivalent of 40 mass % tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added to the cation exchange capacity of the resin, and stirred for 30 minutes. After the remaining TBA-OH solution was removed by decantation, it was further washed with excess ultrapure water to obtain a cation exchange resin after TBA chlorination.

[0243] [Process 2-2-B] The raw material sodium hyaluronate (HA-Na) with a molecular weight of 35,000 (35 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. The suspension of the cation exchange resin after TBA chlorination in "(1) Step 2-1-B" was added in a 5-fold molar equivalent relative to the molar number of HA units (unit molecular weight 401.3) in accordance with the ion exchange capacity of the resin. After stirring for 15 minutes, the suspension was filtered through a 0.45 μm filter and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid.

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

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

[0246] The dialysate obtained was freeze-dried to obtain the target product (HA-C 6 -Chol), as a white solid. 1 In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH 3 , 1.6 ppm or more and 2.0 ppm or less, 3H), the peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7ppm, 3H), the cholesteryl introduction rate was 30%.

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

[0248] [Example 1-1] A CyA preparation using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing an association promoter (polysorbate 80) was prepared. The following operations were carried out at room temperature at 20°C. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL under stirring at 20° C. for 24 hours. In another vial, polysorbate 80 (Fujifilm Wako, product number: 164-21591) was diluted with water for injection at a ratio of 2 mg / mL.

[0249] In addition, further, in another vial, 200.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed, and then 1.0 mL of ethanol was added to uniformly dissolve CyA. Then, 7.2 mL of a 12 mg / mL hyaluronic acid derivative aqueous solution and 1.2 mL of a 2 mg / mL polysorbate 80 were added, mixed, and stirred for 1 hour, and 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 drug.

[0250] Further, after all CyA is compounded, the concentration is adjusted with water for injection, and sucrose (Fuji Film and Light, special for manufacturing) is added in powder form in a ratio of 10 mass % sucrose solution, stirred for 3 hours, homogenized, and prepared by a hyaluronic acid derivative pharmaceutical composition containing an association promoter. Finally, the solution was visually clear. In addition, no precipitate was found during 2 weeks of refrigerated storage. The above operations were carried out at 20°C. The final formulation composition is shown in Table 1.

[0251] [Example 1-2] A CyA formulation using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing an association enhancer (polyethylene glycol 400) was prepared. The following operations were carried out at room temperature of 20°C. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL at 20° C. by stirring for 24 hours. In another vial, 88.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed, and then 10.0 mL of polyethylene glycol 400 (Fuji Film Wako, product number: 161-09065) was added to uniformly dissolve CyA.

[0252] Then, while stirring, add 2.0mL of 8.8mg / mL of the polyethylene glycol 400 solution containing CyA in the vial of the hyaluronic acid derivative aqueous solution of 12mg / mL of 6mL, carry out the compounding of medicine. Further, after all the compounding of CyA, in the ratio of becoming 10 mass % sucrose solution, sucrose (Fuji Film and Light, special for manufacturing) is added in powder form, stirred for 3 hours, homogenized, and the preparation of the hyaluronic acid derivative pharmaceutical composition containing association promoter is carried out. Finally, the solution is visually clarified. In addition, no precipitate is found in the refrigerated storage of 2 weeks. The final preparation composition is shown in Table 1.

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

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

[0255] In addition, further, after CyA is fully compounded, 0.10mL of 100mg / mL polysorbate 80 is added. In order to become a ratio of 10 mass % sucrose solution, sucrose (Fuji Film and Light, manufacturing special) is added in powder form, stirred for 3 hours, homogenized, and the preparation of the hyaluronic acid derivative pharmaceutical composition containing an association promoter is carried out. Finally, the solution is visually clear. In addition, no precipitate is found in the refrigerated storage of 2 weeks. The final preparation composition is shown in Table 1.

[0256] [Comparative Example 1-1] Preparation of CyA formulations using alpha-cyclodextrin. The following operations were carried out at room temperature of 20°C. The powder of CyA was dissolved in a 10% by mass sucrose solution containing 10% by mass α-cyclodextrin (manufactured by Aldrich) at a ratio of 0.5 mg / mL and adjusted. Finally, the solution was visually clear. In addition, no precipitate was found during the 2-week refrigerated storage. The final formulation composition is shown in Table 1.

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

[0258] In addition, further, in another vial, weigh 200.0mg of powdered CyA (Tokyo Chemical Industry, product number: C2408), then, add 1.0mL of ethanol to evenly dissolve CyA. Then, drip 0.046mL of 200mg / mL of ethanol solution containing CyA in 12mg / mL of hyaluronic acid derivative aqueous solution to compound the medicine. Further, after all the compounding of CyA, after adjusting the concentration with water for injection, add sucrose (Fuji Film and Light, special for manufacturing) in the ratio of becoming 10% by mass sucrose solution in powder form, stir for 3 hours to homogenize it. Finally, the solution is visually clear. The above operation was carried out at 20° C. The final formulation composition is shown in Table 1.

[0259] [Table 1]

[0260] <Test Example 2> Pharmacokinetic studies of CyA in rats were performed. The solution preparations of the hyaluronic acid derivative pharmaceutical compositions prepared in Examples 1-1, 1-2, 1-3 and Comparative Example 1-2 and the solution preparation of Comparative Example 1-1 were subcutaneously administered to normal rats (SD, 6 weeks old, male) at the dosages (mg / kg) shown in Table 2 using 25G needles, respectively.

[0261] After administration, blood was collected from the jugular vein with a heparin-treated syringe over time, and aprotinin was added as a protease inhibitor. The obtained blood was separated into plasma and measured by LC-MS / MS. The changes in the plasma concentration of CyA when various CyA preparations were administered and the changes in the plasma concentration of CyA in Comparative Example 1 are shown together. Figure 3 . Figure 3 In the embodiment 2-1, a solution preparation containing the hyaluronic acid derivative pharmaceutical composition of embodiment 1-1 is administered. Embodiments 2-2 to 2-3 and Comparative Examples 2-1 to 2-2 are respectively administered solutions containing the hyaluronic acid derivative pharmaceutical composition of embodiments 1-2 to 1-3 and Comparative Example 1-2, and a solution preparation of Comparative Example 1-1.

[0262] In addition, pharmacokinetic parameters (drug half-life (T 1 / 2 ) and mean residence time (MRT), area under the serum drug concentration-(0 to infinity) time curve (AUC inf )), the values ​​of which are shown in Table 2.

[0263] [Table 2]

[0264] In the present specification, sustained release is evaluated by mean residence time (MRT). When the MRT of a hyaluronic acid derivative pharmaceutical composition containing an association promoter is greater than the MRT of a hyaluronic acid derivative pharmaceutical composition, the composition is evaluated to have sustained release.

[0265] More specifically, when the following formula (A) is satisfied, it is evaluated that "the concentration of the active ingredient can be maintained in the living body for 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] Relative to the comparative example 2-2 not containing the association promoter, in the embodiments 2-1, 2-2, and 2-3 of the preparations containing the association promoter of the hyaluronic acid derivative, the half-life of the concentration in the blood is long, and the MRT is a large value. The sustained release is evaluated by formula (A), and relative to the comparative example 2-2, the embodiment 2-1 is 1.35, the embodiment 2-2 is 1.42, and the embodiment 2-3 is 1.49. That is, it can be known that the drug is retained in the blood for a long time. According to the above, it can be confirmed that the longer-term sustained release of the hyaluronic acid derivative composition of the present invention is excellent.

[0267] <Test Example 3> The degree of association promotion was evaluated by GPC.

[0268] [Example 3-1] For Example 1-1 prepared in Test Example 1, the degree of association promotion of the hyaluronic acid derivative was evaluated by GPC.

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

[0270] (GPC measurement conditions) Apparatus: HLC8320-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 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] As in Example 1-1, Examples 1-2, 1-3 and Comparative Example 1-2 were also diluted with water for injection and measured at a concentration ratio of 1 mg / mL in the concentration of the hyaluronic acid derivative. Comparative Example 2-1 was not diluted and directly subjected to GPC measurement. At this time, the area value A2 surrounded by each chromatogram and the baseline was calculated respectively, and the value of the area ratio A2 / A1 with A1 is shown in Table 3.

[0272] [Comparative Examples 3-3 to 3-6] Aqueous solutions were prepared in such a ratio that the concentration of the additive single component became the same as that of Examples 3-1 to 3-3 and Comparative Example 3-2, and the same was applied to GPC measurement.

[0273] [Table 3]

[0274] This result shows that the association of the hyaluronic acid derivatives of Examples 3-1 to 3-3 is promoted by polysorbate 80 and polyethylene glycol 400. On the other hand, it was confirmed that the area value of the hyaluronic acid derivative peak in GPC with respect to ethanol hardly changed regardless of the presence or absence of the addition of ethanol, indicating that the association of the hyaluronic acid derivative was not promoted.

[0275] <Test Example 4> Evaluation of the association promoter by GPC was carried out. [Examples 4-1, 4-2, 4-3 and Comparative Examples 4-1, 4-2] In the same manner as in Test Example 1, except that the active ingredient CyA and sucrose as an isotonic agent were not contained, hyaluronic acid derivative aqueous solutions were prepared at the same composition ratio according to the following Table 4. Each adjusted sample was diluted with water for injection at a ratio to give a hyaluronic acid derivative concentration of 1 mg / mL and subjected to GPC measurement.

[0276] [Table 4]

[0277] <Test Example 5> Precipitation behavior at physiological salt concentration in vitro was confirmed.

[0278] [Examples 5-1 to 5-3, Comparative Examples 5-1 and 5-2] In a 1.5mL microtube, 150μL of each preparation obtained in Example 1 was sampled. Then, a concentrated buffer (40mM PB (pH7.4), 600mM NaCl) was added to a ratio of 10mM PB (pH7.4) and 150mM NaCl in the final buffer composition to precipitate the hyaluronic acid derivative. After incubation at 37°C for 20 minutes, centrifugation was performed at 2000G for 5 minutes, 50μL of the supernatant was sampled, diluted 2 times with an HP-β-CD aqueous solution (300mM), and after incubation for 1 hour, 350μL of an HP-β-CD aqueous solution (10mM) was added, and after dilution, it was provided for GPC determination. According to the peak area of ​​the hyaluronic acid derivative detected, the residual rate of the hyaluronic acid derivative in the solution relative to the initial amount used was calculated, and the ratio (precipitation rate) of the precipitated hyaluronic acid derivative was calculated. The precipitation evaluation method is consistent with the results of the precipitation evaluation method described in the preparation of the above-mentioned [physical properties of the pharmaceutical composition] (test conditions) and the precipitation sample.

[0279] (GPC measurement conditions) Apparatus: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Eluent: 10 mM HP-β-CD / phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50μL Detector: RI (differential refractive index) Temperature: 30°C

[0280] [Table 5]

[0281] This result shows that the hyaluronic acid derivatives of Examples 5-1 to 5-3 not only promote association by polysorbate 80 and polyethylene glycol 400, but also improve precipitation performance at a physiological salt concentration in vitro.

[0282] <Test Example 6> The types of association promoters were studied. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL by stirring at 20°C for 24 hours and used in Examples 6-1 to 6-11 and Comparative Examples 6-1 to 6-6.

[0283] [Example 6-1] The dissolved hyaluronic acid derivative aqueous solution was diluted with water for injection at a ratio of 2 mg / mL. In addition, in another vial, polyethylene glycol 300 was diluted with water for injection at a concentration of 10 mg / mL. The hyaluronic acid derivative aqueous solution and the polyethylene glycol 300 aqueous solution were mixed at a volume ratio of 1:1 and adjusted to a ratio of the composition of Table 6 below. Then, after incubation at 20°C for 24 hours, it was subjected to GPC measurement.

[0284] (GPC measurement conditions) Apparatus: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 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] In the same manner as in Example 6-1, a solution was prepared in a ratio to give the composition shown in Table 6 below, and subjected to GPC measurement.

[0286] [Table 6]

[0287] This result shows that the association of the hyaluronic acid derivatives of Examples 6-1 to 6-11 is promoted by polyethylene glycol 300, poloxamer 188, and Cremophor EL other than polysorbate 80 and polyethylene glycol 400. On the other hand, it can be confirmed that in Comparative Examples 6-1 to 6-4, with respect to tetrahydrofuran (having one ether structure and 4 carbon atoms), ethanol (having no ether structure and 2 carbon atoms), and ethylene glycol (having no ether structure and 2 carbon atoms), there is almost no change in the area value of the hyaluronic acid derivative peak in GPC, regardless of whether ethanol is added, and the association of the hyaluronic acid derivative is not promoted. Here, for poloxamer 188 and Cremophor EL, similarly to polysorbate 80 and polyethylene glycol 400, no GPC peak of the association promoter alone is detected at the peak position of the hyaluronic acid derivative.

[0288] <Test Example 7> Preparation of hyaluronic acid derivative pharmaceutical compositions. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL by stirring at 20°C for 24 hours and used in Examples 7-1 to 7-10 and Comparative Examples 7-1 to 7-6.

[0289] [Example 7-1] A CyA formulation using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing an association enhancer (polysorbate 80) was prepared. The following operations were carried out at room temperature of 20°C. In a vial, weigh 1.8 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408), then, in another vial, add water for injection to dilute polysorbate 80 to 0.5 mg / mL. The weighed CyA is suspended in 18 μL of a polysorbate 80 aqueous solution with a concentration of 0.5 mg / mL. Next, while stirring, 450 μL of a 12 mg / mL hyaluronic acid derivative aqueous solution is added to the polysorbate 80 aqueous solution containing CyA, and 432 μL of water for injection is added to compound the drug, so that the composition during preparation becomes: CyA concentration is 2 mg / mL, hyaluronic acid derivative is 6 mg / mL, and polysorbate 80 is 0.01 mg / mL. After stirring at 20°C for 24 hours, the precipitate is filtered through a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the preparation is quantified by the following HPLC determination. The final composition is listed in Table 7.

[0290] [Examples 7-2 to 7-10] A hyaluronic acid derivative pharmaceutical composition containing an association enhancer was prepared by the same method as in Example 7-1, so that the association enhancer concentration in the final composition reached the concentration described in the following Table 7. In addition, the CyA concentration in the preparation after 0.45 μm sterilization filtration was quantified by HPLC in the same manner as in Example 7-1, and the results are summarized in Table 7.

[0291] [HPLC quantitative conditions: reverse phase chromatography analysis conditions] Chromatographic column: InertSustain C18 particle size 5μm × inner diameter 4.6mm × length 150mm Column temperature: 40°C 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 various pharmaceutical compositions prepared in Test Example 7 at a physiological salt concentration in vitro was evaluated by the following method.

[0293] [Preparation of precipitation samples] In a microtube (1.5 mL), 200 μL of concentrated buffer (40 mM PB, 150 mM NaCl aqueous solution) was added, and 600 μL of a preparation containing a hyaluronic acid derivative pharmaceutical composition was added. Then, the mixture was mixed with voltex for 30 seconds, incubated at 37° C. for 20 minutes, and then the precipitate was precipitated with a centrifuge (2000 G, 5 min). Next, the hyaluronic acid derivative in the supernatant was subjected to GPC measurement.

[0294] [Preparation of blank samples] In a microtube (1.5 mL), add 200 μL of water for injection and 600 μL of a preparation containing a hyaluronic acid derivative pharmaceutical composition (the theoretical concentration of the hyaluronic acid derivative is X mg / mL. X>1.33). Then, mix with voltex for 30 seconds, incubate at 37°C for 20 minutes, and centrifuge (2000G, 5 minutes). Next, dilute the hyaluronic acid derivative in the supernatant with water for injection to 1 mg / mL and provide for GPC determination.

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

[0296] [Confirmation of precipitation] In the present specification, a hyaluronic acid derivative pharmaceutical composition having a precipitation rate of 20% or more represented by the following formula is defined as precipitating at a physiological salt concentration. Precipitation rate (%) = {1-(area value of hyaluronic acid derivative of precipitation sample) ÷ ((area value of hyaluronic acid derivative of blank sample) × (0.75X))} × 100

[0297] [Table 7]

[0298] The results show that only a small amount of various association promoters such as polysorbate 80, polysorbate 20, poloxamer 188, Cremophor EL can be added to solubilize the water-insoluble active ingredient from powder at a high concentration even without using an organic solvent. Further, it is shown that the precipitation performance is also improved by the association promoter. In summary, it can be expected that the association of hyaluronic acid derivatives is promoted, the content of active ingredients is high, and the pharmaceutical composition can be released over a long period of time by the association promoter.

[0299] <Test Example 8> The types of hyaluronic acid derivatives were studied. The freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in water for injection at 20°C with stirring for 24 hours at 2.0 mg / mL, and used in Examples 8-1 to 8-11 and Comparative Examples 8-1 to 8-6.

[0300] [Example 8-1] In a glass bottle, polysorbate 80 was diluted with water for injection at a concentration of 10 mg / mL. A 6 mL clean vial was prepared, 400 μL of the above hyaluronic acid derivative aqueous solution and 10 μL of polysorbate 80 aqueous solution were added, mixed with voltex for 30 seconds, and then 390 μL of water for injection were added to adjust the total to 800 μL. The composition was adjusted in the proportion of the composition in Table 8 below. Then, after incubation at 20°C for 24 hours, it was subjected to GPC measurement.

[0301] (GPC measurement conditions) Apparatus: HLC8420-GPC (manufactured by Tosoh Corporation) Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 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] By the same method as in Example 8-1, a solution was prepared in a ratio to give the composition shown in Table 8 below, and subjected to GPC measurement.

[0303] [Table 8]

[0304] The results show that the association of the hyaluronic acid derivatives of Examples 8-1 to 8-4 is promoted by polysorbate 80, polysorbate 20, and Cremophor EL. On the other hand, it can be confirmed that in Comparative Examples 8-1 to 8-6, whether or not tetrahydrofuran is added to the area value of the hyaluronic acid derivative peak in GPC, there is almost no change, and the association of the hyaluronic acid derivative is not promoted. Therefore, in the hyaluronic acid derivative (10k HA-C6-Chol-30%) with a small molecular weight and a high introduction rate of hydrophobic groups, it can also be expected that the association of the hyaluronic acid derivative is promoted by the association promoter, and a pharmaceutical composition with a high content of active ingredients and long-term sustained release can be obtained.

[0305] <Test Example 9> The preparation of a hyaluronic acid derivative pharmaceutical composition using human growth hormone (protein) was carried out.

[0306] [Example 9-1] The freeze-dried hyaluronic acid derivative (35kHA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 4.0 mg / mL by stirring at 20°C for 24 hours, and used in Examples 9-1 to 9-2 and Comparative Example 9-1.

[0307] [Example 9-1 to Example 9-2] An association promoter and water for injection are added to the above aqueous solution of the hyaluronic acid derivative. In addition, in another vial, human growth hormone (hGH: Genotropin (registered trademark) for injection) powder was dissolved in water for injection at a concentration of 2 mg / mL. After preparation, 250 μL was added to the above-mentioned hyaluronic acid derivative aqueous solution containing the association promoter to adjust the total amount to 1 mL. Then, the complexation with hGH was promoted by incubation at 37° C. for 24 hours. The composition of the final preparation was adjusted to the concentrations described in Table 9.

[0308] [Comparative Example 9-1] Tetrahydrofuran (THF) and water for injection were added to a hyaluronic acid derivative aqueous solution having a concentration of 4.0 mg / mL. In addition, 250 μL of a 2.0 mg / mL hGH aqueous solution was added to the above-mentioned hyaluronic acid derivative aqueous solution containing THF to adjust the total amount to 1 mL. Then, by incubating at 37° C. for 24 hours, the complexation with hGH was promoted. As in Example 9-1, the composition of the final preparation was adjusted to the concentrations described in Table 9.

[0309] Examples 9-1 to 9-2 and Comparative Example 9-1 were all adjusted to a concentration of 1.0 mg / mL of hyaluronic acid derivative and 0.5 mg / mL of hGH. After 24 hours, Examples 9-1 to 9-2 obtained clear solutions. Here, only Comparative Example 9-1 had a turbid solution after incubation at 37°C for 24 hours, and it was difficult to obtain a composite solution of hyaluronic acid derivative and hGH.

[0310] [Table 9]

[0311] Next, regarding Examples 9-1 and 9-2, the complexation ratio of hGH was evaluated by GPC as shown below.

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

[0313] Prepare a single hGH sample with a concentration of 0.50 mg / mL and 0.25 mg / mL and provide it for GPC measurement. At this time, the peak of the hGH single component was confirmed at 11.5 minutes. When compounded and mixed with the hyaluronic acid derivative composition, the peak area value of the hGH single component at 11.5 minutes decreased. According to the reduction rate of the peak area, the concentration of hGH contained in the hyaluronic acid derivative composition was calculated as the hGH composite rate. It was also confirmed that the peak area value from the hyaluronic acid derivative of the hyaluronic acid derivative composition containing hGH increased. According to the hGH composite rate calculated above, the actual mass parts of (C) active ingredient (hGH) relative to 100 mass parts of hyaluronic acid derivatives are shown in Table 9.

[0314] According to the results, the hyaluronic acid derivative composition containing polyethylene glycol 400 as an association promoter is effective in compounding hGH as a protein drug. Long-term sustained release function is expected not only for poorly soluble drugs but also for water-soluble drugs. In addition, it is speculated that long-term sustained release can be achieved for various modalities by applying it 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 was a turbid sample, probably because THF inhibited the complexation. It can be inferred that hGH was not well complexed with the hyaluronic acid derivative. In conclusion, complexation with hGH also proceeds efficiently in the presence of an association promoter, and it can be expected that a protein-hyaluronic acid derivative pharmaceutical composition capable of long-term sustained release in vivo can be obtained.

[0316] <Test Example 10> [Synthesis example 3] Using Test Example 6 as a reference, the freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-40%) obtained by the same method as Synthesis Example 2 was evaluated for the degree of association promotion of the hyaluronic acid derivative by GPC.

[0317] (GPC measurement conditions) Apparatus: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (differential refractive index) Temperature: 30℃

[0318] By the same method as in Test Example 3, the area ratio A2 / A1 for evaluating the degree of association promotion of the hyaluronic acid derivative was calculated.

[0319] The hyaluronic acid derivative (10kHA-C6-Chol-40%) obtained in Synthesis Example 3 was added to 10 mM PB (phosphate buffer, pH 7.4) at a concentration of 1.0 mg / mL and stirred overnight to completely dissolve. The area value calculated in the GPC evaluation of this sample was taken as A1.

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

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

[0322] Then, using the solution prepared in Example 10-1, the following solution adjustment is performed. In a 6ml sterilized vial, 600μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, 50μL of 10mg / mL polysorbate 80 aqueous solution is added while stirring, mixed, 100μL of 100mM PB phosphate buffer is added, and finally 250μL of water for injection is added, and the hyaluronic acid derivative aqueous solution is prepared in a ratio of 10mM PB with a final concentration. Then, after cultivating at 20°C for 24 hours, the concentration of the hyaluronic acid derivative becomes a ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0323] [Example 10-3] Using the solution prepared in Example 10-1, the following solution adjustment is performed. In a 6ml sterile vial, 600μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, while stirring, 50μL of 10mg / mL polysorbate 80 aqueous solution is added, mixed, and finally 350μL of water for injection is added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating at 20°C for 24 hours, the concentration of the hyaluronic acid derivative becomes a ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0324] [Example 10-4] Then, using the solution prepared in embodiment 10-1 and embodiment 10-2, the following solution adjustment is performed. In a 6ml sterilized vial, 600 μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, 100 μL of polyethylene glycol 400 (manufactured by Nacalai Tesque) is added while stirring, mixed, 100 μL of 100mM PB phosphate buffer is added, and finally 200 μL of water for injection are added, and the hyaluronic acid derivative aqueous solution is prepared in the ratio of 10mM PB with a final concentration. Then, after cultivating for 24 hours at 20°C, the ratio of 1mg / mL is made with the concentration of hyaluronic acid derivative, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0325] [Example 10-5] Then, using the solution prepared in Example 10-1, the following solution adjustment is performed. In a 6ml sterilized vial, 600μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, 100μL of polyethylene glycol 400 (manufactured by Nacalai Tesque) is added while stirring, mixed, and finally 300μL of water for injection is added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating for 24 hours at 20°C, the ratio of 1mg / mL with the concentration of hyaluronic acid derivative is made, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0326] [Example 10-6] Then, using the solution prepared in Example 10-1, the following solution adjustment is performed. In a 6ml sterilized vial, 600μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, while stirring, 300μL of polyethylene glycol 400 (manufactured by Nacalai Tesque) is added, mixed, and finally 100μL of water for injection is added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating for 24 hours at 20°C, the concentration of the hyaluronic acid derivative becomes a ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0327] [Example 10-7] First, Poloxamer 338 (Aldrich company system) is added to water for injection in the ratio of 150mg / mL with concentration, and it is dissolved.Then, using the solution prepared in embodiment 10-1, the following solution adjustment is carried out.In the sterilizing bottle of 6ml, the above-mentioned hyaluronic acid derivative aqueous solution of 600μL is added, then, the Poloxamer 338 aqueous solution of 150mg / mL of 100μL is added while stirring, mix, finally add the water for injection of 300μL, prepare the hyaluronic acid derivative aqueous solution.Then, after cultivating 24 hours at 20 ℃, become the ratio of 1mg / mL with the concentration of hyaluronic acid derivative, after diluting with 10mM PB (phosphate buffer, pH7.4), supply to GPC and measure.

[0328] [Example 10-8] Using the solution prepared in Example 10-1, Example 10-2, and Example 10-7, the following solution adjustment is performed. In a 6ml sterilized vial, 600 μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, 300 μL of Poloxamer 338 aqueous solution is added while stirring, mixed, 100 μL of 100mM PB phosphate buffer is added, and the hyaluronic acid derivative aqueous solution is prepared in a ratio of 10mM PB with a final concentration. Then, after cultivating for 24 hours at 20°C, the concentration of the hyaluronic acid derivative becomes a ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH 7.4), it is provided for GPC determination.

[0329] [Example 10-9] Use the solution prepared in embodiment 10-1 and embodiment 10-7, carry out the following solution adjustment.In a 6ml sterilized vial, add 600μL of the above-mentioned hyaluronic acid derivative aqueous solution, then, add 300μL of 150mg / mL of Poloxamer 338 aqueous solution while stirring, mix, finally add 100μL of water for injection, prepare the hyaluronic acid derivative aqueous solution.Then, after cultivating 24 hours at 20 ℃, become the ratio of 1mg / mL with the concentration of hyaluronic acid derivative, after diluting with 10mM PB (phosphate buffer, pH7.4), supply to GPC measurement.

[0330] [Example 10-10] First, Chol-PEG600 (manufactured by Avanti) was added to water for injection at a concentration of 50 mg / mL to dissolve it. Then, the hyaluronic acid derivative (10kHA-C6-Chol-40%) obtained in Synthesis Example 3 was stirred at 10.0 mg / mL in water for injection for 24 hours at 20°C to dissolve it, and it was used in Examples 10-10 to 10-12. In a 6ml sterilized vial, 750 μL of the above-mentioned hyaluronic acid derivative aqueous solution was added, and then, while stirring, 2.8 μL of 50 mg / mL Chol-PEG600 aqueous solution was added, mixed, and finally 747.2 μL of water for injection was added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating at 20°C for 24 hours, the concentration of the hyaluronic acid derivative was made into a ratio of 1 mg / mL, and after diluting with 10mM PB (phosphate buffer, pH 7.4), it was provided for GPC determination.

[0331] [Examples 10-11] Using the solution prepared in Example 10-10, the following solution adjustment is performed. In a 6ml sterile vial, 750μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, while stirring, 11.2μL of 50mg / mL Chol-PEG600 aqueous solution is added, mixed, and finally 738.8μL of water for injection is added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating for 24 hours at 20°C, the concentration of the hyaluronic acid derivative becomes a ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0332] [Examples 10-12] Using the solution prepared in Example 10-10, the following solution adjustment is performed. In a 6ml sterilized vial, 750 μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, while stirring, 28 μL of the 50mg / mL Chol-PEG600 aqueous solution is added, mixed, and finally 722 μL of water for injection is added to prepare the hyaluronic acid derivative aqueous solution. Then, after cultivating for 24 hours at 20°C, the concentration of the hyaluronic acid derivative becomes the ratio of 1mg / mL, after diluting with 10mM PB (phosphate buffer, pH7.4), it is provided for GPC determination.

[0333] The area value A2 surrounded by each chromatogram and the baseline of Examples 10-1 to 10-12 was calculated by the same method as in Test Example 3, and the values ​​of the area ratio A2 / A1 to A1 are shown in Table 10.

[0334] [Table 10]

[0335] This result shows that the association of the hyaluronic acid derivatives of Examples 10-1 to 10-12 is promoted by polysorbate 80, polyethylene glycol 400, Poloxamer 338, and Chol-PEG 600. Here, for Poloxamer 338 and Chol-PEG 600, similarly to polysorbate 80 and polyethylene glycol 400, no GPC peak of the association promoter alone was detected at the peak position of the hyaluronic acid derivative.

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

[0337] [Example 11-1] A CyA formulation using a hyaluronic acid derivative (35k HA-C6-Chol-19%) containing an association enhancer (polysorbate 80) was prepared. The following operations were carried out at room temperature of 20°C. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was added to a 10% sucrose aqueous solution at a concentration of 12.0 mg / mL and dissolved.

[0338] Then, stir at 20°C for 24 hours to dissolve. For the 10% sucrose solution, a solution was used in which sucrose powder (manufactured by Fujifilm Wako Co., Ltd., for exclusive use) was fully dissolved in water for injection and then sterilized and filtered using a 0.22 μm filter (membrane material: PTFE). In another vial, 10.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408) was weighed, and then 0.50 mL of an aqueous solution of polysorbate 80 dissolved in water for injection at a concentration of 100 mg / mL was added, and the mixture was stirred with a stirrer chip to disperse the CyA.

[0339] Next, in the vial containing the above-mentioned CyA suspension, 3.75mL of 12mg / mL hyaluronic acid derivative aqueous solution was added while stirring, and after further adding 0.75mL of 10% sucrose aqueous solution, the drug was compounded. The solution of the hyaluronic acid derivative pharmaceutical composition containing the association promoter of CyA was visually clear. In addition, no precipitate was found during the refrigerated storage of 2 weeks. The final formulation composition is shown in Table 11.

[0340] [Comparative Example 11-1] Preparation of CyA formulations using alpha-cyclodextrin. The following operations are carried out under an environment of 20°C and room temperature. First, weigh 2.0g of sucrose and 2.0g of α-Cyclodextrin (α-cyclodextrin) (manufactured by Tokyo Chemical Industry Co., Ltd.) in a beaker, add about 15mL of water for injection, and dissolve. Transfer to a measuring cylinder and make up to 20mL. Then, filter with a 0.45μm filter to make a 10% sucrose / 10% α-Cyclodextrin aqueous solution. Weigh 2.0mg of Cyclosporin A (manufactured by Tokyo Chemical Industry Co., Ltd.) in a conical tube, add 10mL of 10% sucrose / 10% α-Cyclodextrin aqueous solution, and stir to dissolve. Finally, filter with a 0.2μm filter to make a dosing solution. The final formulation composition is shown in Table 11.

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

[0342] In addition, further, in another vial, weigh 200.0 mg of powdered CyA (Tokyo Chemical Industry, product number: C2408), then add 1.0 mL of ethanol to dissolve CyA evenly. Then, relative to 3.75 mL of 12 mg / mL hyaluronic acid derivative aqueous solution, 0.0225 mL of 200 mg / mL ethanol solution containing CyA was added dropwise, and then 1.2275 mL of 10% sucrose aqueous solution was added to compound the drug. Then, slowly stir at room temperature for more than 12 hours to make it homogenous. The solution is visually clear and is used as a dosing solution after confirming that the powder is completely dissolved. The final formulation composition is shown in Table 11.

[0343] [Table 11]

[0344] <Test Example 12> Pharmacokinetic studies of CyA in rats were performed. The solution preparations containing the hyaluronic acid derivative pharmaceutical compositions prepared in Example 11-1 and Comparative Example 11-2 and the solution preparation of Comparative Example 11-1 were subcutaneously administered to normal rats (SD, 6 weeks old, male) using 25G needles in the dosages (mg / kg) shown in Table 12.

[0345] After administration, blood was collected from the subclavian vein over time. An equal amount of 0.1% ZnSO 4 After the aqueous solution was hemolyzed, it was pre-treated using MicroVolumeQuEChERSKit. The supernatant after centrifugation was filtered through a filter and measured by LC-MS / MS. The changes in the blood concentration of CyA when various CyA preparations were administered and the changes in the blood concentration of CyA in Comparative Example 1 are shown together. Figure 4 . Figure 4 In the embodiment 12-1, a solution preparation of the hyaluronic acid derivative pharmaceutical composition of the embodiment 11-1 is administered. Separately, the comparative example 12-2 is a case where a solution preparation of the hyaluronic acid derivative pharmaceutical composition of the comparative example 11-2 is administered, and the comparative example 12-1 is a case where the solution preparation of the comparative example 11-1 is administered.

[0346] In addition, the maximum plasma concentration (C max ) using the measured value. 0→∞ ) and blood concentration-area under the first moment curve (AUMC 0→∞ ), the area up to the detectable measurement time was calculated using the linear trapezoidal method, and the area thereafter until time infinity was calculated using the disappearance rate constant (k e Finally, the mean residence time (MRT) was calculated using the following formula and its value is shown in Table 12.

[0347] MRT=AUMC 0→∞ / AUC 0→∞

[0348] [Table 12]

[0349] In Example 12-1, which is a preparation containing an association promoter of a hyaluronic acid derivative, the value of Cmax / Dose, which is an indicator of abnormal initial release, is small, and MRT is a large value relative to Comparative Example 12-2, which does not contain an association promoter. The sustained release was evaluated by formula (A), and the value of Example 12-1 was 2.49 relative to Comparative Example 12-2. That is, it can be seen that the drug is retained in the blood for a long time. In summary, it can be confirmed that the hyaluronic acid derivative composition of the present invention has excellent long-term sustained release. <Test Example 13> Manufacturing hyaluronic acid derivative pharmaceutical compositions. The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL by stirring at 20°C for 24 hours, and used in Examples 13-1 to 13-2 and Comparative Example 13-1. The following operations were carried out at room temperature of 20°C.

[0350] [Example 13-1] First, polysorbate 80 (manufactured by Thermo Scientific) was added to water for injection at a concentration of 10 mg / mL to dissolve it. Then, glycerol (manufactured by Nacalai Tesque, product number: 17045-94) was dissolved in water for injection at a concentration of 170 mg / mL for adjustment. Further, 1 mL of 0.5 mol / L-EDTA solution (pH 8.0) (manufactured by Nacalai Tesque, product number: 06894-14) was added to 28.240 mL of water for injection to adjust 5.0 mg / mL EDTA buffer.

[0351] Here, in a 6ml sterilized vial, 416.7 μL of the above-mentioned hyaluronic acid derivative aqueous solution is added, then, 100 μL of 10mg / mL polysorbate 80 aqueous solution is added while stirring, mixed, then, 318.3 μL of water for injection is added, and finally, 15 μL of 5.0mg / mL EDTA buffer is added to prepare the hyaluronic acid derivative aqueous solution. At this time, EDTA concentration is 0.075mg / mL, glycerol concentration is 25.5mg / mL, polysorbate 80 concentration is 1mg / mL, and the concentration of hyaluronic acid derivative is 5mg / mL. Then, at 20°C, stir for 24 hours, become the ratio of 1mg / mL with the concentration of hyaluronic acid derivative, dilute with water for injection, and provide for GPC determination. Except not containing association promoter, hyaluronic acid derivative solution is adjusted with the same composition, the area value obtained is set to A1, the association promotion degree A2 / A1 of association promoter is calculated, and it is shown in Table 13.

[0352] Next, a CyA preparation using a hyaluronic acid derivative (35kHA-C6-Chol-19%) containing an association enhancer (polysorbate 80) was prepared. First, 2.5 mg of CyA was weighed in 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. Dissolution of the CyA powder was observed and the mixture was transferred to the next operation in a saturated state. The 0.22 μm PES filter was sterilized and filtered to remove the drug that could not be enclosed in the hyaluronic acid derivative and precipitated, and the CyA concentration in the preparation of the obtained filtrate was quantified by reversed-phase HPLC. This experiment was implemented with n=2, which was 1.50 mg / mL and 1.41 mg / mL. For the CyA concentration in the preparation, the obtained average values ​​are summarized in Table 13. In addition, the precipitation was verified by 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 at all the same concentrations except that the polysorbate concentration was 3 mg / mL. At this time, the EDTA concentration is 0.075 mg / mL, the glycerol concentration is 25.5 mg / mL, the polysorbate 80 concentration is 3 mg / mL, and the concentration of the hyaluronic acid derivative is 5 mg / mL. Then, the mixture is stirred at 20°C for 24 hours, diluted with water for injection to a concentration of 1 mg / mL of the hyaluronic acid derivative, and subjected to GPC measurement. The hyaluronic acid derivative solution is adjusted with the same composition except that the association promoter is not contained, and the obtained area value is taken as A1, and the association promotion degree based on the association promoter is calculated in the same manner as in Example 13-1, and is recorded in Table 13.

[0354] Next, a CyA preparation using a hyaluronic acid derivative (35kHA-C6-Chol-19%) containing an association enhancer (polysorbate 80) was prepared. First, 2.5 mg of CyA was weighed in 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. The dissolved residue of CyA powder was observed and the mixture was transferred to the next operation in a saturated state. The 0.22 μm PES filter was sterilized and filtered to remove the drug that could not be enclosed in the hyaluronic acid derivative and precipitated, and the CyA concentration in the preparation of the obtained filtrate was quantified by reversed-phase HPLC. This experiment was carried out with n=2, and the results were 1.89 mg / mL and 1.87 mg / mL. For the CyA concentration in the preparation, the obtained average values ​​are summarized in Table 13. In addition, whether precipitation was verified in the same manner as in Test Example 7. The results are also summarized in Table 13.

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

[0356] Next, a CyA preparation using a hyaluronic acid derivative (35kHA-C6-Chol-19%) was prepared. First, 2.5 mg of CyA was weighed in 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. Dissolution of the CyA powder was observed and the mixture was transferred to the next operation in a saturated state. Sterile filtration was performed through a 0.22 μm PES filter, but filtration was difficult.

[0357] Here, the GPC peak of the association promoter alone was also confirmed under the condition of not containing the hyaluronic acid derivative, but no peak derived from the association promoter was detected at the peak position of the hyaluronic acid derivative.

[0358] [Table 13]

[0359] The results showed that polysorbate 80 promoted the association of hyaluronic acid derivatives even in an EDTA solution containing glycerol as an isotonic agent (A2 / A1=1.90-2.10). In addition, the results showed that the sterilization filterability of the hyaluronic acid derivative pharmaceutical composition was improved by promoting the association.

[0360] Furthermore, the amount of CyA as an active ingredient was 29.1 or 37.6 parts by mass per 100 parts by mass of the hyaluronic acid derivative, and it was confirmed that it can be solubilized at a high concentration, and a longer-term sustained release can be expected by eye drops, intramuscular administration, subcutaneous administration, etc.

[0361] <Test Example 14> In the same manner as in Test Example 3, the other association promoters were evaluated for their association promotion degree by GPC.

[0362] [Example 14-1] The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was stirred at 20°C for 24 hours at 12.0 mg / mL in water for injection to dissolve it. In a 1.5 mL microtube (EppendorfTube), 416.7 μL of 12.0 mg / mL of the above-mentioned hyaluronic acid derivative aqueous solution was added, followed by 50 μL of polyvinyl alcohol (polymerization degree 500, manufactured by Nacalai Tesque, product number: 11738-62) dissolved in water for injection at a concentration of 100 mg / mL. Then, water for injection was added in a total of 1000 μL for adjustment (Table 14). Then, after incubation at 20°C for 2 hours, the concentration of the hyaluronic acid derivative was 1 mg / mL, and diluted 5 times with water for injection, and then provided for GPC determination.

[0363] [Example 14-2] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was stirred at 20°C for 24 hours at 62.6 mg / mL in water for injection to dissolve it. In a 1.5 mL microtube (EppendorfTube), 79.9 μL of 62.6 mg / mL of the above-mentioned hyaluronic acid derivative aqueous solution was added, followed by 50 μL of polyvinyl alcohol (polymerization degree 500, manufactured by Nacalai Tesque, product number: 11738-62) dissolved in water for injection at a concentration of 100 mg / mL. Then, water for injection was added in a total ratio of 1000 μL for adjustment (Table 14). Then, after incubation at 20°C for 2 hours, the concentration of the hyaluronic acid derivative was made into 1 mg / mL, diluted 5 times with water for injection, and then provided for GPC determination.

[0364] [Example 14-3] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was stirred at 20°C for 24 hours at 40.0 mg / mL in water for injection to dissolve it. In a 1.5 mL microtube (EppendorfTube), 125.0 μL of 40.0 mg / mL of the above-mentioned hyaluronic acid derivative aqueous solution was added, followed by 50 μL of polyvinyl alcohol (polymerization degree 500, manufactured by Nacalai Tesque, product number: 11738-62) dissolved in water for injection at a concentration of 100 mg / mL. Then, water for injection was added in a total ratio of 1000 μL for adjustment (Table 14). Then, after incubation at 20°C for 2 hours, the concentration of the hyaluronic acid derivative was made into 1 mg / mL, diluted 5 times with water for injection, and then provided for GPC determination.

[0365] Here, even under the condition that no hyaluronic acid derivative was contained, the GPC peak of the association promoter alone was confirmed, but no peak derived from PVA as the association promoter was detected at the peak position of the hyaluronic acid derivative.

[0366] [Example 14-4 to Example 14-6] The freeze-dried hyaluronic acid derivative (35k HA-C6-Chol-19%) obtained in Synthesis Example 1 was dissolved in water for injection at 12.0 mg / mL by stirring at 20° C. for 24 hours. Furthermore, the hyaluronic acid derivative aqueous solution containing the association promoter was adjusted to a ratio of concentrations shown in Table 14 using the 10 mg / mL polysorbate 80 aqueous solution prepared in Example 13-1, a 170 mg / mL glycerol aqueous solution, water for injection, and a 5.0 mg / mL EDTA solution. Then, after incubation at 20°C for 24 hours, the mixture was subjected to GPC measurement.

[0367] [Example 14-7 to Example 14-8] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-40%) obtained in Synthesis Example 3 was dissolved in water for injection at 62.6 mg / mL by stirring at 20°C for 24 hours. Furthermore, the hyaluronic acid derivative aqueous solution containing the association promoter was adjusted to a ratio of concentrations shown in Table 14 using the 10 mg / mL polysorbate 80 aqueous solution prepared in Example 13-1, a 170 mg / mL glycerol aqueous solution, water for injection, and a 5.0 mg / mL EDTA solution. Then, after incubation at 20°C for 24 hours, the mixture was subjected to GPC measurement.

[0368] [Example 14-9 to Example 14-11] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-30%) obtained in Synthesis Example 2 was dissolved in water for injection at 40.0 mg / mL by stirring at 20°C for 24 hours. Furthermore, the hyaluronic acid derivative aqueous solution containing the association promoter was adjusted to a ratio of concentrations shown in Table 14 using the 10 mg / mL polysorbate 80 aqueous solution prepared in Example 13-1, a 170 mg / mL glycerol aqueous solution, water for injection, and a 5.0 mg / mL EDTA solution. Then, after incubation at 20°C for 24 hours, the mixture was subjected to GPC measurement.

[0369] [Example 14-4 to Example 14-11] Each of the hyaluronic acid derivatives was diluted 5-fold with water for injection so that the concentration became 1 mg / mL, and then subjected to GPC measurement.

[0370] Here, the GPC peak of the association promoter alone was confirmed even when the hyaluronic acid derivative was not contained, but the peak derived from polysorbate 80 as the association promoter was not detected at all at the peak position of the hyaluronic acid derivative. The final composition and the degree of association promotion are shown in Table 14.

[0371] [Table 14]

[0372] According to the results, in Examples 14-1 to 14-3, even though the molecular weight of the hyaluronic acid derivatives was different and the cholesteryl introduction rate was different, the association promotion based on PVA was confirmed. Therefore, by expanding the hydrophobic part in the nanoparticles, it can be expected that the solubilization amount of poorly soluble drugs will be increased and the sustained release period after in vivo administration will be extended.

[0373] In addition, as can be seen from the results of Examples 14-4 to 14-11, the hyaluronic acid derivatives are hyaluronic acid derivatives with different molecular weights and different cholesteryl introduction rates, and the results obtained do not hinder the promotion of association even if other additives such as glycerol and EDTA are contained. In JP6271672B2, it is mentioned that EDTA plays the role of a protease inhibitor and can be added as a stabilizer for API. In the JP Patent 4758893 Gazette, it is recorded that by mixing glycerol, the effectiveness of antibacterial preservation is improved. It shows that: even based on such antibacterial preservatives or API stabilizers, the association promoter also has the function of fully interacting and promoting the association of hyaluronic acid derivatives. In other various buffers, antibacterial preservatives or stabilizers, the association promotion effect is also expected. By expanding the hydrophobic part in the nanoparticles, it can be expected that the solubilization amount of poorly soluble drugs increases and the sustained release period after administration in the organism is extended.

[0374] <Test Example 15> Preparation of a hyaluronic acid derivative pharmaceutical composition using Semaglutide (peptide) was carried out.

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

[0376] Specifically, 416.7 μL of 12.0 mg / mL of hyaluronic acid derivative (35kHA-C6-Chol-19%) aqueous solution was added, followed by adding 100 μL of 10 mg / mL of polysorbate 80 aqueous solution while stirring with a stirrer, and adding 150 μL of 170 mg / mL of glycerol solution. Then, 318.3 μL of water for injection and 15 μL of 5 mg / mL of EDTA solution were added and stirred overnight. In addition, semaglutide (manufactured by Funakoshi, product number: AG-CP3-0032) powder was dissolved in 10 mM phosphate buffer at a concentration of 1 mg / mL using a microtube (Eppendorf Tube), and after preparation, 150 μL of the above-mentioned hyaluronic acid derivative aqueous solution containing an association promoter and 150 μL of 1 mg / mL of semaglutide solution were mixed, and then, by cultivating for 1 hour at 20°C, the complexation with semaglutide was promoted. The composition of the final preparation was adjusted to the concentrations listed in Table 15.

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

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

[0379] All of Examples 15-1 to 15-2 were adjusted to a concentration of 2.5 mg / mL of the hyaluronic acid derivative and 0.5 mg / mL of semaglutide. The semaglutide complexation ratio was evaluated by GPC as shown below for Examples 15-1 to 15-2.

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

[0381] Prepare a sample of semaglutide alone at a concentration of 0.50 mg / mL and 0.25 mg / mL for GPC determination. At this time, the peak of semaglutide single component was confirmed at 9.0 minutes. When compounded and incorporated with the hyaluronic acid derivative composition, the peak area value of the semaglutide single component at 9.0 minutes was reduced. According to the reduction rate of the peak area, the concentration of semaglutide contained in the hyaluronic acid derivative composition was calculated as the semaglutide compounding rate. According to the semaglutide compounding rate calculated above, the actual mass parts of the (C) active ingredient (semaglutide) relative to 100 mass parts of hyaluronic acid derivatives are shown in Table 15.

[0382] According to the result, the hyaluronic acid derivative composition containing polysorbate 80 as an association promoter is effectively used for the semaglutide compounding of peptide drugs. In JP6271672B2, it is mentioned that EDTA plays the role of protease inhibitors and can be added as a stabilizer of API, but it is pointed out that even based on such a stabilizer, the association promoter can also be enclosed in the hyaluronic acid derivative while fully interacting. According to the result, it is inferred that: except the cyclic peptide drug such as CyA, by being applied to long-chain peptide drugs, for a variety of modalities, long-term sustained release can be achieved. In addition, it is believed that other stabilizers, metacresol, phenol and other preservatives, as other isotonic agents D-mannitol, propylene glycol, etc. can also be used. In summary, the complexation with semaglutide can also be effectively carried out in the presence of an association promoter, and 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] According to the following steps 1B to 3B, a hyaluronic acid derivative was prepared.

[0385] [Process 1B] (Synthesis of cholesteryl 6-aminohexyl carbamate hydrochloride) According to the following step 1B-1 and the subsequent step 1B-2, cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized.

[0386] (Process 1B-1) Under an argon atmosphere, triethylamine (TEA, 1.05 mL) was added to a solution of cholesterol chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) and stirred. Under ice cooling, 6-(tert-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred directly under ice cooling for 30 minutes, then warmed to room temperature (about 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 then distilled off the solvent under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:4), the target fractions were combined, and the solvent was distilled off under reduced pressure.

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

[0388] [Process 2B] (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) According to the following step 2B-1 and the subsequent step 2B-2, a TBA salt of hyaluronic acid (HA-TBA) was prepared.

[0389] (Process 2B-1) DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water about three times by decantation. About 1.5 times the molar equivalent of 40 mass % tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) was added to the cation exchange capacity of the resin, and stirred for 30 minutes. After the remaining TBA-OH solution was removed by decantation, it was further washed with excess ultrapure water to obtain a cation exchange resin after TBA chlorination.

[0390] [Process 2B-2] The raw material hyaluronic acid sodium salt (HA-Na) with a molecular weight of 10,000 (10 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. The suspension of the cation exchange resin after TBA chlorination in "Step 2B-1" was added in a 5-fold molar equivalent relative to the molar number of HA units (unit molecular weight 401.3) in accordance with the ion exchange capacity of the resin. After stirring for 15 minutes, the suspension was filtered through a 0.45 μm filter, and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid.

[0391] [Process 3B] Prepare an anhydrous DMSO solution (10 mg / mL) of the HA-TBA prepared in the above "Step 2B-2". Then, add Chol hydrochloride in an amount of 44 / 100 relative to the disaccharide repeating unit (HA unit) present in the HA-TBA synthesized in "Step 1B". Next, add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) in an amount of 48 / 100 relative to the HA unit, and stir overnight at room temperature (about 25°C). The reaction solution is dialyzed in the order of 0.3M ammonium acetate / DMSO solution, 0.15M NaCl aqueous solution, and ultrapure water (SpectraPor 7, molecular weight cutoff (MWCO): 3,500). The resulting dialyzate is freeze-dried to obtain the target product (HA-C 6 -Chol), as a white solid. 1 In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH 3 , 1.6 ppm or more and 2.0 ppm or less, 3H), the peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7ppm, 3H), the cholesteryl introduction rate was 44%.

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

[0393] [Step of Obtaining Aqueous Solution (II)] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL to obtain an aqueous solution of a hyaluronic acid derivative as an aqueous solution (II).

[0394] [Step of Obtaining Dispersion Liquid (I)] In another vial, polysorbate 80 was diluted with water for injection at a ratio of 10 mg / mL. Furthermore, 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. Thus, a CyA dispersion in which CyA was dispersed in polysorbate 80 was obtained as dispersion (I).

[0395] [Mixing process] A stirring bar was placed in a vial, and after adding 0.30 mL of a hyaluronic acid derivative aqueous solution as an aqueous solution (II) while stirring, a CyA dispersion was added in a ratio of 0.650 mL of liquid volume. Then, the mixture was stirred for 24 hours to solubilize the drug. The final formulation composition is shown in Table 16.

[0396] [Table 16]

[0397] The solution was then filtered through a 0.45 μm sterilizing filter to remove precipitated CyA, and the drug concentration in the preparation was quantitatively measured by HPLC as described below. The CyA concentration in the preparation after filtration is shown in Table 17.

[0398] [HPLC quantitative conditions: reverse phase chromatography analysis conditions] Chromatographic column: InertSustain C18 particle size 5μm × inner diameter 4.6mm × length 150mm Column temperature: 40°C 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] [Example 1B-2 to Example 1B-11] Except changing the type and amount of solubilizing agent, the same procedure as in Example 1B-1 was followed to prepare CyA using hyaluronic acid derivatives and solubilizing agents. For each preparation, the theoretical concentration in the preparation calculated from the amount added and the actual drug concentration in the preparation quantitatively measured by HPLC are shown in Tables 17 to 18.

[0400] [Comparative Example 1B-1] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, 4.0 mg of powdered CyA was weighed, and then 0.30 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution was added. A stirring bar was placed in the vial, and water for injection was added to make the liquid volume 0.650 mL. Then, the mixture was stirred for 24 hours to solubilize the drug. Next, the mixture was filtered with a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the quantitative preparation was determined by HPLC in the same manner as in Example 1B-1. Confirm that the filtrate is all transparent. Table 4 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantitatively determined by HPLC.

[0401] [Comparative Example 1B-2] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, weigh 4.0 mg of powdered CyA, then add 0.1783 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution. In the vial, put a stirrer and add water for injection to make the liquid volume 0.650 mL. Then, stir for 24 hours to solubilize the drug. Next, filter with a 0.45 μm sterilizing filter to remove the precipitated CyA, and determine the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Confirm that the filtrate is transparent. Table 19 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0402] [Comparative Example 1B-3] The raw material hyaluronic acid sodium salt (HA-Na) with a molecular weight of 10,000 (10 kDa) used in Synthesis Example 1B was dissolved in water for injection at 55 mg / mL. Then, polysorbate 20 and sodium hyaluronate were appropriately diluted with water for injection to give the concentrations listed in Table 4 to prepare aqueous solutions. In another vial, 4.0 mg of powdered CyA was weighed, and then 0.65 mL of the above-mentioned HA-Na aqueous solution containing polysorbate 20 was added. A stirring bar was placed in the vial and stirred for 24 hours to solubilize the drug. Next, the precipitated CyA was filtered through a 0.45 μm sterilizing filter, and the drug concentration in the quantitative preparation was determined by HPLC. The theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation determined by HPLC are shown in Table 19.

[0403] [Comparative Example 1B-4] Polysorbate 80 was diluted with water for injection at a ratio of 100 mg / mL. In another vial, 4.0 mg of powdered CyA was weighed, and then 0.65 mL of the above-mentioned 100.0 mg / mL polysorbate 80 was added. A stirring bar was placed in the vial and stirred for 24 hours to solubilize the drug. Then, the precipitated CyA was removed by filtration with a 0.45 μm sterilizing filter, and the drug concentration in the quantitative preparation was determined by HPLC in the same manner as in Example 1B-1. Table 19 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0404] [Comparative Examples 1B-5 to 1B-12] The same procedures as in Comparative Example 1B-4 or Example 1B-1 were followed to prepare the formulation and quantify the drug concentration, except that the concentration of the solubilizing agent was as described in the table below. The results are shown in Tables 19 and 20.

[0405] Span 83 used in Comparative Example 1B-9 was a reagent manufactured by Tokyo Chemical Industry Co., Ltd., and sucrose stearate used in Comparative Examples 1B-10 to 1B-12 was a reagent manufactured by Mitsubishi Chemical Corporation.

[0406] [Table 17]

[0407] [Table 18]

[0408] [Table 19]

[0409] [Table 20]

[0410] As shown in Tables 17 to 18 above, when a poorly water-soluble drug is formulated, it can be solubilized without using an organic solvent, and the amount of solubilizers such as polysorbate 80 and Cremophor EL, which are highly toxic in the past, can be reduced. Furthermore, the hydrophobic interaction between the sterol groups of the above-mentioned hyaluronic acid derivatives is promoted by the solubilizing agent, and as a result, the amount of hydrophobic drugs carried is increased by expanding the hydrophobic region of the hyaluronic acid derivatives. Further, it is speculated that the solubilizing agent in the hyaluronic acid derivative composition not only binds to the hydrophobic region of the hyaluronic acid polymer by chemical bonds, but also acts as a hydrophobic region with high mobility, thereby solubilizing the powdered drug at a high concentration. As a result, generally, the solubilization amount of a poorly water-soluble drug increases in proportion to the amount of solubilizer added, but in the present invention, although the total mass of the solubilizer is reduced, a high-concentration preparation of the active ingredient can be prepared.

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

[0412] In Comparative Example 1B-3, in the hyaluronic acid in which the hydrophobic group was not modified, no synergistic effect due to the solubilizing aid was observed.

[0413] Comparative Examples 1B-4 to 1B-8 show the solubilization ability of the solubilizing aid alone, but when compared with the drug concentration added with the solubilization ability of the hyaluronic acid derivative alone, the examples of the present invention can significantly improve the solubility of poorly water-soluble drugs.

[0414] Further, after in-depth research on the structure of the solubilizing agent, it was found that Comparative Examples 1B-9 to 1B-12 all satisfy the requirement of having more than 4 carbon atoms but only having 1 or 3 ether structures. In these examples, the drug and the solubilizing agent cannot be stably dissolved in the hyaluronic acid derivative and are almost difficult to filter.

[0415] According to the above results, the composition comprising a hyaluronic acid derivative and a solubilizing agent containing at least 4 ether structures (ROR) and having 4 or more carbon atoms does not use any organic solvent from the powder state. Even if the concentration of the solubilizing agent is reduced, the solubilizing ability of poorly water-soluble drugs can be greatly improved, and a simple formulation method and a low-toxic pharmaceutical composition can be provided.

[0416] <Test Example 2B> [Examples 2B-1 to 2B-12] The amount of solubilizing aid added was verified in detail. The freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection to a concentration of 5 mg / mL. In another vial, 1.0 mg of powdered CyA was weighed, and 0.2 mL was added so that the solubilizing agent concentration in the final composition of the preparation became the concentration described in Table 21, and a stirring bar was added and stirred.

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

[0418] [Comparative Example 2B-1] The freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection to a concentration of 5 mg / mL. In another vial, 1.0 mg of powdered CyA was weighed, and 0.1 mL of a 10 mg / mL sodium stearoyl lactylate aqueous solution was added so that the solubilizing agent concentration of the final component in the preparation became the concentration recorded in Table 21, and a stirring bar was added and stirred.

[0419] Next, 0.8 mL of the above-mentioned 5 mg / mL hyaluronic acid derivative aqueous solution was added while stirring, and then 0.1 mL of water for injection was added and stirred for 24 hours to solubilize the drug. Next, the mixture was filtered with a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the quantitative preparation was determined by HPLC in the same manner as in Example 1B-1. Table 21 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantitatively determined by HPLC measurement.

[0420] [Comparative Examples 2B-2 to 2B-6] In a vial, 1.0 mg of powdered CyA was weighed, 0.2 mL was added so that the concentration of the solubilizing agent in the final component of the preparation became the concentration recorded in Table 21, and a stirrer was placed and stirred. Then, 0.8 mL of water for injection was added while stirring, and the mixture was stirred for 24 hours to solubilize the drug only with the solubilizing agent. Then, the mixture was filtered with a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the preparation was quantitatively determined by HPLC in the same manner as in Example 1B-1. Table 21 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantitatively determined by HPLC measurement.

[0421] [Table 21]

[0422] From the above results, it can be seen that the solubilizing aids containing at least 4 or more ether structures (ROR) and satisfying the requirement of 4 or more carbon atoms increase the drug concentration in the preparation as shown in Examples 2B-1 to 2B-13. On the other hand, sodium stearoyl lactylate, which is one of the solubilizing aids that does not contain at least 4 or more ether structures (ROR), has almost no solubilizing effect.

[0423] In Comparative Examples 2B-2 to 2B-6, it was confirmed that the solubilizing agent itself had almost no solubilizing ability. These results show that the solubilizing ability can be greatly improved by adding a small amount of solubilizing agent to a hyaluronic acid derivative. As shown in Table 21, the solubilizing ability of the powdered product can be improved by adding only 0.003 parts by mass or more of the solubilizing agent to 100 parts by mass of the hyaluronic acid derivative without using an organic solvent.

[0424] <Test Example 3B> [Examples 3B-1 to 3B-14] Then, the solubilizing aids that can be used are verified in detail. Specifically, it was verified whether polyethylene glycol 300 and polyethylene glycol 400, which have at least 5 ether structures (ROR) and 12 or more carbon atoms, can be used as solubilizing aids.

[0425] The freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection to a concentration of 8 mg / mL. In another vial, 1.6 mg of CyA powder was weighed, and a solubilizing agent and water for injection were added to a total of 0.5 mL, and a stirring bar was added to stir. At this time, the concentration of the solubilizing agent in the final hyaluronic acid derivative pharmaceutical composition was adjusted to be as 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. Next, the mixture was filtered through a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the preparation was quantitatively determined by HPLC in the same manner as in Example 1B-1. Table 22 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0427] [Comparative Examples 3B-1 to 3B-6] In a vial, 1.0 mg of powdered CyA was weighed, and a solubilizing agent and water for injection were added to a total of 0.5 mL, and a stirring bar was added to stir. At this time, the concentration of the solubilizing agent in the pharmaceutical composition containing the finally obtained solubilizing agent was adjusted as shown in Table 22 below.

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

[0429] [Table 22]

[0430] According to the above results, for polyethylene glycol 300 and polyethylene glycol 400, the drug concentration in the preparation is increased in the solubilizing aid containing at least 4 ether structures (ROR) and satisfying the carbon number of 4 or more, as shown in Examples 3B-1 to 3B-14. Compared with the sum of the solubilization amount of the single component of the hyaluronic acid derivative and the single component of the solubilizing aid, the active ingredient can be solubilized at a higher concentration, showing a synergistic effect.

[0431] <Test Example 4B> [Example 4B-1] Next, the preparation method of the hyaluronic acid derivative pharmaceutical composition was verified in detail. The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, polysorbate 80 was diluted to 10 mg / mL with water for injection.

[0432] In addition, further, in another vial, 4.0 mg of powdered CyA was weighed, and then 160 μL of 10 mg / mL polysorbate 80 was added. In the vial, a stirrer was placed, and after adding 0.30 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution while stirring, water for injection was added so that the liquid volume became 0.650 mL. Then, stirring for 24 hours, the drug was solubilized. The final formulation composition is shown in Table 23. Then, filtered with a 0.45 μm sterilizing filter to remove the precipitated CyA, and the drug concentration in the quantitative formulation was determined by HPLC in the same manner as in Example 1B-1.

[0433] Table 23 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantitatively determined by HPLC.

[0434] [Example 4B-2] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, polysorbate 80 was diluted to 10 mg / mL with water for injection. Then, in another vial, add 320 μ L of 10 mg / mL of polysorbate 80, in a vial, put into a stirrer, add 0.60 mL of the above-mentioned 36.0 mg / mL of hyaluronic acid derivative aqueous solution while stirring, then, add water for injection so that the liquid volume becomes 1.30 mL. In addition, further, in another vial, weigh 4.0 mg of powdered CyA, add 0.65 mL of the above-mentioned hyaluronic acid derivative containing a solubilizing aid, stir for 24 hours, and carry out the solubilization of the drug. The final preparation composition is shown in Table 20. Then, filter with a 0.45 μ m sterilizing filter to remove the precipitated CyA, and measure the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Table 23 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantitatively determined by HPLC.

[0435] [Table 23]

[0436] It can be seen that any of the steps (I) of dispersing the above-mentioned active ingredient in a solubilizing agent and (II) of preparing the above-mentioned hyaluronic acid derivative aqueous solution or the above-mentioned hyaluronic acid aqueous solution containing a solubilizing agent, a formulation method prepared by the steps of mixing (I) and (II), and a formulation method prepared by the step of mixing the above-mentioned active ingredient with a hyaluronic acid derivative aqueous solution containing a solubilizing agent can obtain a hyaluronic acid derivative pharmaceutical composition that is excellent in improving the solubility of the water-slightly soluble active ingredient in a powder state. In addition, in addition to the solubilizing agent being polysorbate 80, this production method is also not greatly dependent on the mixing method, and a high solubilizing ability can be expected.

[0437] <Test Example 5B> [Example 5B-1] Next, the types of poorly water-soluble drugs were verified. The freeze-dried hyaluronic acid derivative (10kHA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at a concentration of 36.0 mg / mL.

[0438] In addition, further, in another vial, weigh 1.1mg of powdered paclitaxel (manufactured by Tokyo Chemical Industry Co., Ltd.), then, add 0.40mL of the above-mentioned 36.0mg / mL hyaluronic acid derivative aqueous solution. In the vial, put into a stirrer, stir, then, add 0.40mL of water for injection, then add 200μL of 10mg / mL polysorbate 80. Then, stir for 24 hours, solubilize the drug. The final formulation composition is shown in Table 24. Then, filter with a 0.22μm sterilizing filter, remove the precipitated paclitaxel, and measure the drug concentration in the quantitative formulation by HPLC in the same manner as in Example 1B-1.

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

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

[0441] [Comparative Example 5B-1] The preparation was prepared in the same manner as in Example 5B-1 except that no solubilizing aid was added.

[0442] [Comparative Example 5B-2] The preparation was prepared in the same manner as in Example 5B-2 except that no solubilizing agent was added.

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

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

[0445] Table 24 shows the theoretical concentrations in the preparations calculated from the added amounts and the actual drug concentrations in the preparations quantitatively determined by HPLC for Examples 5B-1 to 5B-2 and Comparative Examples 5B-1 to 5B-4.

[0446] [Table 24]

[0447] According to the above results, in the solubilizing aid containing at least 4 or more ether structures (ROR) and satisfying the carbon number of 4 or more, as shown in Examples 5B-1 to 5B-2, the drug concentration in the preparation is increased. On the other hand, in Comparative Examples 5B-3 to 5B-4, it was confirmed that the solubilizing aid alone had almost no solubilizing ability for water-poorly soluble drugs. According to these results, even in low-molecular water-poorly soluble drugs, by adding a small amount of solubilizing aid to hyaluronic acid derivatives, the solubilizing ability can be greatly improved.

[0448] [Example 6B-1] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, cholesterol-PEG600 (manufactured by Aldrich: C1145-250MG) was diluted to 10 mg / mL with water for injection. Then, in another vial, add 400 μ L of 10 mg / mL of cholesterol-PEG600, in the vial, put a stirrer, add 0.60 mL of the above-mentioned 36.0 mg / mL of hyaluronic acid derivative aqueous solution while stirring. In addition, further, in another vial, weigh 5.0 mg of powdered temsirolimus (temsirolimus) (Tokyo Chemical Industry Co., Ltd., product number: T3574), add 0.5 mL of the above-mentioned hyaluronic acid derivative containing a solubilizing agent, stir for 24 hours, and solubilize the drug. The final formulation composition is shown in Table X1. Then, filter with a 0.45 μ m sterilizing filter to remove the precipitated temsirolimus, and determine the drug concentration in the quantitative formulation by HPLC in the same manner as in Example 1B-1. Table 25 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0449] [Comparative Example 6B-1] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, weigh 5.0 mg of temsirolimus (manufactured by Tokyo Chemical Industry Co., Ltd., product number: T3574), and then add 0.30 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution. In the vial, put a stirring bar and add water for injection to make the liquid volume 0.50 mL. Then, stir for 24 hours to solubilize the drug. Next, filter with a 0.45 μm sterilizing filter to remove the precipitated temsirolimus, and determine the drug concentration in the quantitative preparation by HPLC in the same way as Example 1B-1. Confirm that the filtrate is transparent. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0450] [Comparative Example 6B-2] In a vial, weigh 5.0 mg of temsirolimus (manufactured by Tokyo Chemical Industry Co., Ltd., product number: T3574), then put a stirrer in the vial, add 200 μL of 10 mg / mL cholesterol-PEG600, and add water for injection to make the liquid volume 0.50 mL. Then, stir for 24 hours to solubilize the drug. Next, filter with a 0.45 μm sterilizing filter to remove the precipitated temsirolimus, and determine the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Confirm that the filtrate is all transparent. Table 25 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0451] [Table 25]

[0452] According to the above results, for cholesterol-PEG600, in a solubilizing agent containing at least 4 or more ether structures (ROR) and satisfying the requirement of more than 4 carbon atoms, the drug concentration in the preparation is increased as shown in Example 6B-1. Compared with the sum of the solubilization amounts of the single component of the hyaluronic acid derivative and the single component of the solubilizing agent, the active ingredient can be solubilized at a higher concentration, showing a synergistic effect.

[0453] [Example 7B-1] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, cholesterol-PEG600 (manufactured by Aldrich: C1145-250MG) was diluted to 10 mg / mL with water for injection. Then, in another vial, add 400 μ L of 10 mg / mL cholesterol-PEG600, in a vial, put into a stirrer, add 0.60 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution while stirring. In addition, further, in another vial, weigh 4.0 mg, weigh 5.0 mg of powdered CyA, add 0.5 mL of the above-mentioned hyaluronic acid derivative containing a solubilizing aid, stir for 24 hours, and carry out the solubilization of the drug. The final preparation composition is shown in Table Y1. Then, filter with a 0.45 μ m sterilizing filter to remove the precipitated CyA, and measure the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0454] [Example 7B-2] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, cholesterol-PEG600 (manufactured by Aldrich: C1145-250MG) was diluted to 10 mg / mL with water for injection. Then, weigh 10.0 mg of powdered CyA, then, add 0.60 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution, in a vial, put a stirrer, add 40 μL of 10 mg / mL cholesterol-PEG600 aqueous solution while stirring, and add 360 μL of water for injection. Then, stir for 24 hours to solubilize the drug. The final formulation composition is shown in Table 26. Then, filter with a 0.45 μm sterilizing filter to remove the precipitated CyA, and measure the drug concentration in the quantitative formulation by HPLC in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0455] [Example 7B-3] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, HP-β-CD (manufactured by Tokyo Chemical Industry Co., Ltd.: H0979) was diluted to 10 mg / mL with water for injection. Then, weigh 10.0 mg of powdered CyA, then, add 0.60 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution, put a stirrer in a vial, and add 272.2 μL of 10 mg / mL HP-β-CD aqueous solution while stirring. Then, add 127.8 μL of water for injection, stir for 24 hours, and solubilize the drug. The final formulation composition is shown in Table Y1. Then, filter with a 0.45 μm sterilizing filter to remove the precipitated CyA, and determine the drug concentration in the quantitative formulation by HPLC in the same manner as in Example 1B-1. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0456] [Comparative Example 7B-1] The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) obtained in Synthesis Example 1B was dissolved in water for injection at 36.0 mg / mL. In another vial, weigh 10.0 mg of powdered CyA, then add 0.60 mL of the above-mentioned 36.0 mg / mL hyaluronic acid derivative aqueous solution. In the vial, put a stirrer and add water for injection to make the liquid volume 1.00 mL. Then, stir for 24 hours to solubilize the drug. Next, filter with a 0.45 μm sterilizing filter to remove the precipitated CyA, and determine the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Confirm that the filtrate is transparent. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0457] [Comparative Example 7B-2] In a vial, weigh 10.0 mg of powdered CyA, then, in the vial, put a stirrer, add 400 μL of 10 mg / mL cholesterol-PEG600, and add water for injection to make the liquid volume 1.0 mL. Then, stir for 24 hours to solubilize the drug. Next, filter with a 0.45 μm sterilizing filter to remove the precipitated CyA, and determine the drug concentration in the quantitative preparation by HPLC in the same manner as in Example 1B-1. Confirm that the filtrate is transparent. Table 26 shows the theoretical concentration in the preparation calculated from the added amount and the actual drug concentration in the preparation quantified by HPLC measurement.

[0458] [Table 26]

[0459] According to the above results, for cholesterol-PEG600 or HP-β-CD, in the solubilizing agent containing at least 4 or more ether structures (ROR) and satisfying the carbon number of 4 or more, as shown in Examples 7B-1 to 7B-3, the drug concentration in the preparation is increased. Compared with the sum of the solubilization amount of the single component of the hyaluronic acid derivative and the single component of the solubilizing agent, the active ingredient can be solubilized at a higher concentration, showing a synergistic effect. Industrial Applicability

[0460] According to the hyaluronic acid derivative pharmaceutical composition of this embodiment, a pharmaceutical composition using a hyaluronic acid derivative can be obtained, which can solubilize a large amount of drugs from powder, maximize the solubilization amount, and control the release rate of the active ingredient from the subcutaneously gelled hyaluronic acid derivative composition, thereby achieving excellent long-term sustained release of the active ingredient.

Claims

1. A hyaluronic acid derivative pharmaceutical composition, characterized in that: Include: (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter, and (C) Active ingredient.

2. The hyaluronic acid derivative pharmaceutical composition according to claim 1, wherein The (B) association promoter contains at least 4 or more ether structures ROR and has 4 or more carbon atoms.

3. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein The (B) association promoter is one or more selected from polysorbate 80, polysorbate 20, poloxamer, oxyethylene castor oil, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 4000, sorbitan fatty acid ester, tocopherol polyethylene glycol succinate and polyvinyl alcohol.

4. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein The hyaluronic acid derivative pharmaceutical composition produces a precipitate at a physiological salt concentration.

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

6. The hyaluronic acid derivative pharmaceutical composition according to claim 5, wherein The solubility of the poorly water-soluble drug in water is below 1 mg / mL.

7. The hyaluronic acid derivative pharmaceutical composition according to claim 5, wherein The molecular weight of the poorly water-soluble drug is greater than 200.

8. The hyaluronic acid derivative pharmaceutical composition according to claim 5, wherein The poorly water-soluble drug is a poorly water-soluble peptide.

9. The hyaluronic acid derivative pharmaceutical composition according to claim 8, wherein At least one of the nitrogen atoms constituting the amide bond of the poorly water-soluble peptide has a methyl group.

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

11. The hyaluronic acid derivative pharmaceutical composition according to claim 8, wherein The poorly water-soluble peptide is a cyclic peptide.

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

13. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein: The hyaluronic acid derivative (A) having a hydrophobic group introduced therein has one or more repeating units represented by the following general formula (I): In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of 2 or more and 30 or less arbitrary amino acid residues, X 1 For free-NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b The group in the group represented by -SR and -SSR, R a , R b and R c are each independently selected from a hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group; R a , R b and R c The alkyl portion may or may not be inserted into -O- and -NR f - the group in R f is selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The group in the alkyl group; R f The alkyl part of the present invention may or may not be inserted into a group selected from -O- and -NH-, R is a steryl group, Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-; Here, the alkylene group of Y is inserted or not inserted and is selected from -O-, -NR g - and -SS-, R g is selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group; R g The alkyl part of the present invention may or may not be inserted into a group selected from -O- and -NH-, Y a C 1-5 Alkylene, Y b C 2-8 Alkylene or C 2-8 Alkenylene, m is an integer of 1 to 100 both inclusive.

14. The hyaluronic acid derivative pharmaceutical composition according to claim 13, wherein The steryl group is cholesteryl.

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

16. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein In the hyaluronic acid derivative pharmaceutical composition, no precipitate was visually observed.

17. The hyaluronic acid derivative pharmaceutical composition according to claim 1 or 2, wherein: The hyaluronic acid derivative pharmaceutical composition can be sterilized by filtration.

18. A method for preparing a hyaluronic acid derivative pharmaceutical composition, characterized in that: A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter and (C) an active ingredient. The method for preparing the hyaluronic acid derivative pharmaceutical composition comprises: A step of mixing the (A) hyaluronic acid derivative having a hydrophobic group introduced therein with the (B) association promoter to obtain an aqueous solution of the hyaluronic acid derivative containing the association promoter; and A mixing step of mixing the (C) active ingredient with the aqueous solution of the hyaluronic acid derivative containing the association promoter.

19. A method for preparing a hyaluronic acid derivative pharmaceutical composition, characterized in that: A method for producing a hyaluronic acid derivative pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) an association promoter and (C) an active ingredient. The method for preparing the hyaluronic acid derivative pharmaceutical composition comprises: A step of dispersing the active ingredient (C) in the association promoter (B) to obtain a dispersion (I); A step of 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 A step of mixing the dispersion (I) and the aqueous solution (II).

20. The method for producing a hyaluronic acid derivative pharmaceutical composition according to claim 18 or 19, wherein: The method for producing the hyaluronic acid derivative pharmaceutical composition does not include a step of removing the organic solvent.

21. A hyaluronic acid derivative pharmaceutical composition, characterized in that: Include: (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) a solubilizing aid, and (C) active ingredients, The solubilizing agent (B) contains at least 4 ether structures ROR and has at least 4 carbon atoms. The content of the solubilizing aid (B) is 0.0001 parts by mass or more and 15000 parts by mass or less relative to 100 parts by mass of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced.

22. The hyaluronic acid derivative pharmaceutical composition according to claim 21, wherein The (B) solubilizing agent is at least one selected from a nonionic surfactant, polyethylene glycol having a molecular weight of 190 g / moL or more and 4000 g / moL or less, and a cyclodextrin derivative.

23. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, wherein The (B) solubilizing agent is one or more selected from 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 tocopherol polyethylene glycol succinate.

24. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, wherein The solubilizing agent (B) is a nonionic surfactant. The content of the nonionic surfactant is 0.0001 parts by mass or more and 150 parts by mass or less relative to 100 parts by mass of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced.

25. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, wherein The solubilizing agent (B) is polyethylene glycol having a molecular weight of 190 g / moL or more and 4000 g / moL or less, The content of the polyethylene glycol is 25 parts by mass or more and 15000 parts by mass or less relative to 100 parts by mass of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced.

26. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, wherein The (C) active ingredient is a poorly water-soluble drug having a solubility in water of less than 1 mg / mL.

27. The hyaluronic acid derivative pharmaceutical composition according to claim 26, wherein The molecular weight of the poorly water-soluble drug is greater than 200.

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

29. The hyaluronic acid derivative pharmaceutical composition according to claim 28, wherein At least one of the nitrogen atoms constituting the amide bond of the poorly water-soluble peptide has a methyl group.

30. The hyaluronic acid derivative pharmaceutical composition according to claim 28, wherein The poorly water-soluble peptide includes at least one selected from cyclic peptides and long-chain peptides.

31. The hyaluronic acid derivative pharmaceutical composition according to claim 28, wherein The poorly water-soluble peptide is a cyclic peptide.

32. The hyaluronic acid derivative pharmaceutical composition according to claim 26, wherein The amount of the poorly water-soluble drug added is 21 parts by mass or more and less than 100 parts by mass based on 100 parts by mass of the (A) hyaluronic acid derivative into which a hydrophobic group is introduced.

33. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, wherein The hyaluronic acid derivative has one or more repeating units represented by the following general formula (I): In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of 2 or more and 30 or less arbitrary amino acid residues, X 1 For free-NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b The group in the group represented by -SR and -SSR, R a , R b and R c are each independently selected from a hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group; R a , R b and R c The alkyl portion may or may not be inserted into -O- and -NR f - the group in R f is selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The group in the alkyl group; R f The alkyl part of the present invention may or may not be inserted into a group selected from -O- and -NH-, R is a steryl group, Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, wherein the alkylene group of Y is inserted or not inserted and is selected from -O-, -NR g - and -SS-, R g is selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The group in the alkyl group; R g The alkyl part of the present invention may or may not be inserted into a group selected from -O- and -NH-, Y a C 1-5 Alkylene, Y b C 2-8 Alkylene or C 2-8 Alkenylene, m is an integer of 1 to 100 both inclusive.

34. The hyaluronic acid derivative pharmaceutical composition according to claim 33, wherein The steryl group is cholesteryl.

35. The hyaluronic acid derivative pharmaceutical composition according to claim 33, wherein The introduction rate of the steryl group relative to the hyaluronic acid derivative is 35% or more and less than 50%.

36. The hyaluronic acid derivative pharmaceutical composition according to claim 21 or 22, 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 claim 21 or 22, wherein Relative to the hyaluronic acid derivative pharmaceutical composition, the content of the organic solvent is less than 0.8%.

38. A method for producing a pharmaceutical composition, characterized in that: A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) a solubilizing agent and (C) an active ingredient. The method for preparing the pharmaceutical composition comprises: A step of mixing (A) a hyaluronic acid derivative having a hydrophobic group introduced therein and (B) a solubilizing agent to obtain an aqueous solution of the hyaluronic acid derivative containing the solubilizing agent; and A mixing step of mixing the (C) active ingredient with the aqueous solution of the hyaluronic acid derivative containing the solubilizing agent.

39. A method for producing a pharmaceutical composition, characterized in that: A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) a solubilizing agent and (C) an active ingredient. The method for preparing the pharmaceutical composition comprises: A step of dispersing the active ingredient (C) in the solubilizing agent (B) to obtain a dispersion (I); A step of 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); and A step of mixing the dispersion (I) and the aqueous solution (II).

40. The method for producing a pharmaceutical composition according to claim 38 or 39, characterized in that: A method for producing a pharmaceutical composition comprising (A) a hyaluronic acid derivative having a hydrophobic group introduced therein, (B) a solubilizing agent and (C) an active ingredient. The method for producing the pharmaceutical composition does not include a step of removing the organic solvent.

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