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

Abstract

A hyaluronic acid derivative into which a steryl group is introduced according to the present invention is such that a ratio A1 / A2 of areas A1 and A2, calculated by the following method from a chromatogram derived through gel permeation chromatography measurement, is 0.90 or greator.　A2 is an area value surrounded by: a curve from an intersection point (Ub), at which a perpendicular line drawn from a refractive index intensity maximum point (Kb) on a chromatogram of 50 kDa polyacrylic acid serving as a standard substance to a baseline (B) intersects a chromatogram of the hyaluronic acid derivative, to the end point of the chromatogram of the hyaluronic acid derivative; a perpendicular line drawn from the refractive index intensity maximum point (Kb) to the baseline (B); and the baseline (B). A1 is an area value surrounded by a curve from the starting point of the chromatogram of the hyaluronic acid derivative to the intersection point (Ub), a perpendicular line drawn from the refractive index intensity maximum point (Kb) to the baseline (B), and the baseline (B).

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid derivative, a pharmaceutical composition, and a method for producing the pharmaceutical composition. This application claims priority based on Japanese Patent Application No. 2022-088995 filed in Japan on May 31, 2022, and incorporates its content herein by reference.

Background Art

[0002] In recent years, biopharmaceuticals, which are pharmaceuticals having proteins, peptides, or nucleic acids as active ingredients, have been put into practical use, and the number of them has been increasing year by year. Biopharmaceuticals can meet unmet medical needs that could not be satisfied by conventional low-molecular-weight pharmaceuticals. However, there are problems that they are difficult to be absorbed from the digestive tract or mucosa, etc., and are unstable in the body and have a short blood half-life. Therefore, biopharmaceuticals require frequent administration by injection, which is a heavy burden on both patients and medical staff. Thus, there is a need for a drug substrate (sustained-release drug delivery system substrate) that can encapsulate biopharmaceuticals without impairing their pharmacological activity and gradually release the active ingredient in vivo.

[0003] Against such a background, Patent Document 1 proposes a sustained-release drug delivery system substrate composed of a hyaluronic acid derivative with excellent safety. This hyaluronic acid derivative spontaneously associates in an aqueous solution, can efficiently encapsulate drugs, particularly biopharmaceuticals, while maintaining their biological activity, aggregates under physiological saline concentration (or remains dispersed even under physiological saline concentration), and has good blood retention. This hyaluronic acid derivative can be used as a carrier that can efficiently encapsulate many drugs while maintaining their pharmacological activity, a blood sustained-release carrier with excellent blood retention, and a targeting carrier, particularly when using biopharmaceuticals as active ingredients, and can also be a local (e.g., subcutaneous, etc.) sustained-release carrier that can sustainably release drugs.

[0004] In addition, cancer vaccines using the above hyaluronic acid derivative as a carrier have also been reported (see, for example, Patent Document 2, etc.).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, the hyaluronic acid derivative used in Patent Document 2, etc. has a broad particle size distribution and is non-uniform, and the relationship between the particle size distribution of the hyaluronic acid derivative and the delivery ability to immune cells in lymph nodes and the activation ability of the immune cells has not been specifically studied, leaving room for improvement.

[0007] The present invention has been made in view of the above circumstances, and provides a hyaluronic acid derivative excellent in delivery ability to immune cells in lymph nodes and activation ability of the immune cells when formulated with a medicinal ingredient, and a pharmaceutical composition using the hyaluronic acid derivative and a method for producing the same.

Means for Solving the Problems

[0008] That is, the present invention includes the following aspects. (1) A hyaluronic acid derivative into which a steric group is introduced, A hyaluronic acid derivative in which the ratio A1 / A2 of the area A1 and A2 calculated by the following method is 0.90 or more from the chromatogram obtained by gel permeation chromatography measurement. (i) Let the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B and the chromatogram of the hyaluronic acid derivative be Ub; (ii) In the chromatogram of the hyaluronic acid derivative, the area value enclosed by the curve from the Ub to the end point, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A2. In the chromatogram of the hyaluronic acid derivative, the area value enclosed by the curve from the starting point to the Ub, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A1. (2) The hyaluronic acid derivative according to (1), wherein the ratio Da / Db of the distances Da and Db calculated by the following method from the chromatogram obtained by gel permeation chromatography measurement is more than 0.00 and 1.20 or less. (i) A perpendicular line is drawn from the refractive index intensity maximum point Ka of 150 kDa polyacrylic acid, which is a standard substance, to the baseline B, and the intersection point with the baseline is Ba. The length between the refractive index intensity maximum point Ka and Ba is defined as La. (ii) Among the two points on the chromatogram where the refractive index intensity is La / 20, the point with the earlier elution time is defined as point R1, and the point with the later elution time is defined as point S1. (iv) The intersection point of the straight line D1 connecting the point R1 and the point S1 and the perpendicular line drawn from the refractive index intensity maximum point Ka to the baseline B is defined as Ta, and the intersection point of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the straight line D1 is defined as Tb. (v) The distance between the point R1 and the Ta is defined as Da, and the distance between the Ta and the Tb is defined as Db. (3) The hyaluronic acid derivative according to (1) or (2), wherein the ratio A1 / A2 of the areas A1 and A2 is 1.70 or more. (4) A hyaluronic acid derivative into which a sterol group is introduced, The ratio Pt / Pr of the retention time Pt at the refractive index intensity maximum point of the hyaluronic acid derivative to the retention time Pr at the refractive index intensity maximum point of 50 kDa polyacrylic acid, which is a standard substance, calculated from the chromatogram obtained by gel permeation chromatography measurement is 0.5 or more and less than 1.0. (5) A hyaluronic acid derivative into which a sterol group is introduced, A hyaluronic acid derivative having a weight average molecular weight of 110,000 or more and less than 500,000, calculated based on a calibration curve prepared using polyacrylic acids having molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa, and 150 kDa as standard substances from a chromatogram obtained by gel permeation chromatography measurement. (6) The hyaluronic acid derivative according to any one of (1) to (5), wherein the hyaluronic acid derivative has one or more repeating units represented by the following general formula (I).

[0009] [Chemical formula]

[0010] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of 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 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -S-R, -CO-Y a -S-R, -O-CO-Y b -S-R, -NR b -CO-Y b -S-R, and -S-S-R, is a group selected from the group consisting of groups represented by; R a 、R b and R c are each independently selected from the group consisting of a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl, and hydroxy C 2-20 alkyl, and the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NR f - inserted therein; R f is a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl, or hydroxy C 2-12 alkyl, and the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NH- inserted therein; R is a sterol group; Y is C 2-30 alkylene, or -(CH 2 CH 2 O) m -CH 2 CH 2 -, where the alkylene may have a group selected from the group consisting of -O-, -NR g -, and -S-S- inserted therein; R g is a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl, or hydroxy C 2-20 alkyl, and the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NH- inserted therein; Y a is C 1-5 alkylene; Y b is C 2-8 alkylene or C 2-8 alkenylene; m is an integer of 1 or more and 100 or less.)

[0011] (7) The hyaluronic acid derivative according to any one of (1) to (6), wherein the sterol group is a cholesteryl group. (8) The hyaluronic acid derivative according to any one of (1) to (7), wherein the introduction rate of the sterol group with respect to the repeating unit of the disaccharide constituting the hyaluronic acid derivative is 30% or more and 60% or less. (9) The hyaluronic acid derivative according to any one of (1) to (8), wherein the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 4,000 or more and 1,000,000 or less. (10) The hyaluronic acid derivative according to (9), wherein the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 5,000 or more and 25,000 or less. (11) A pharmaceutical composition comprising the hyaluronic acid derivative according to any one of (1) to (10) and a medicinal ingredient. (12) The pharmaceutical composition according to (11), which is for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases. (13) The pharmaceutical composition according to (11) or (12), wherein the medicinal ingredient includes at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases, and further includes an adjuvant. (14) The pharmaceutical composition according to any one of (11) to (13), wherein the medicinal ingredient includes a cancer antigen or an antigen derived from an infectious disease, and further includes an adjuvant. (15) A method for producing a pharmaceutical composition comprising the hyaluronic acid derivative according to any one of (1) to (10) and a medicinal ingredient, comprising: a preparation step of dissolving the medicinal ingredient in an organic solvent or water containing an organic solvent to prepare an oil phase containing the medicinal ingredient; a mixing step of mixing the oil phase and an aqueous phase containing the hyaluronic acid derivative so that the mixing ratio of the oil phase to the aqueous phase is 20:100 to 0.01:100 by volume ratio; The method for producing a pharmaceutical composition comprising these steps. (16) The method for producing a pharmaceutical composition according to (15), wherein the pH of the aqueous phase containing the hyaluronic acid derivative is 6.00 or more and 11.00 or less.

Advantages of the Invention

[0012] According to the hyaluronic acid derivative of the above aspect, it is possible to provide a hyaluronic acid derivative that is excellent in the delivery ability to immune cells in lymph nodes and the ability to activate the immune cells when formulated with a drug efficacy component. The pharmaceutical composition of the above aspect contains the hyaluronic acid derivative and is excellent in the delivery ability to immune cells in lymph nodes and the ability to activate the immune cells. The method for producing the pharmaceutical composition of the above aspect uses the hyaluronic acid derivative, and a pharmaceutical composition excellent in the delivery ability to immune cells in lymph nodes and the ability to activate the immune cells can be obtained.

Brief Description of Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] Hereinafter, embodiments of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. However, the present invention is not limited thereto, and various modifications are possible without departing from the gist thereof.

[0015] Hereinafter, the terms used in this specification will be explained.

[0016] The term "C 1-20 alkyl" as used in this specification means a linear or branched alkyl group having 1 to 20 carbon atoms. For example, "C 1-4 alkyl" such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, etc. is included. Further, 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. are included. C 1-20 alkyl includes C 1-12 alkyl having 1 to 12 carbon atoms and C 1-6 alkyl groups having 1 to 6 carbon atoms.

[0017] The "C1-6 The term "alkylcarbonyl" means an alkylcarbonyl group in which the alkyl moiety is the alkyl already mentioned, C 1-6 alkyl, and includes, for example, "C 1-4 alkylcarbonyls" such as acetyl, propionyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, iso-butylcarbonyl, tert-butylcarbonyl, etc.

[0018] As used herein, the term "amino C 2-20 alkyl" means a linear or branched alkyl having 2 to 20 carbon atoms and having an amino group as a substituent. For example, the amino group may be located on the terminal carbon atom of the alkyl group. Amino C 2-20 alkyl also includes amino C 2-12 alkyl having 2 to 12 carbon atoms.

[0019] As used herein, the term "hydroxy C 2-20 alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxy group as a substituent. For example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. Hydroxy C 2-20 alkyl also includes hydroxy C 2-12 alkyl having 2 to 12 carbon atoms.

[0020] As used herein, the term "C 2-30 alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc., and includes C 2-20 alkylene having 2 to 20 carbon atoms, C2-8 alkylene having 2 to 8 carbon atoms, and the group "-(CH 2 ) n -", where n is from 2 to 30, preferably from 2 to 20, more preferably from 2 to 15.

[0021] As used herein, the term "C 1-5The term "alkylene" means a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, and includes, for example, methylene, ethylene, propylene, etc.

[0022] The term "C 2-8 "alkenylene" as referred to in this specification means a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms and containing one or more double bonds, and includes, for example, -CH=CH-, -C(CH 3 )=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomers exist, each isomer and their mixture are also included.

[0023] ≪Hyaluronic acid derivative≫ The hyaluronic acid derivative of the first embodiment of the present invention is a hyaluronic acid derivative into which a sterol group is introduced, From the chromatogram shown in FIG. 1 obtained by gel permeation chromatography measurement, the ratio A1 / A2 of the areas A1 and A2 calculated by the following method is 0.90 or more. (i) Let the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B and the chromatogram of the hyaluronic acid derivative be Ub, and the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the baseline be Bb; (ii) Let the area value surrounded by the curve from Ub to the end point in the chromatogram of the hyaluronic acid derivative, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline be A2, and the area value surrounded by the curve from the start point to Ub in the chromatogram of the hyaluronic acid derivative, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline be A1.

[0024] Furthermore, the hyaluronic acid derivative of the present embodiment preferably has a ratio Da / Db of the distances Da and Db calculated by the following method of 0.00 or more and 1.20 or less from the chromatogram shown in FIG. 2 obtained by gel permeation chromatography measurement. (i) A perpendicular line is drawn from the refractive index intensity maximum point Ka on the chromatogram of 150 kDa polyacrylic acid, which is a standard substance, to the baseline B, the intersection point with the baseline is Ba, and the length between the refractive index intensity maximum point Ka and Ba is La; (ii) Of the two points on the chromatogram where the refractive index intensity is La / 20, the point with the earlier elution time is designated as point R1, and the point with the later elution time is designated as point S1; (iii) The intersection point of the perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B and the baseline is designated as Bb; (iv) The intersection point of the straight line D1 connecting the points R1 and S1 and the perpendicular line drawn from the refractive index intensity maximum point Ka to the baseline B is designated as Ta, and the intersection point of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the straight line D1 is designated as Tb; (v) The distance between the point R1 and Ta is designated as Da, and the distance between Ta and Tb is designated as Db. In (ii), when there are three or more points on the chromatogram where the refractive index intensity is La / 20, the intersection point closest to the starting point is designated as R1, and the intersection point closest to the end point is designated as S1.

[0025] Unlike conventional hyaluronic acid derivatives, the hyaluronic acid derivative of the present embodiment contains a relatively large amount of hyaluronic acid derivatives with a specific particle size in the particle size distribution, and the particle size distribution is sharply controlled. The inventors have formulated the hyaluronic acid derivative with a controlled particle size distribution as a medicinal ingredient, and as shown in the examples described later, compared with hyaluronic acid derivatives with an uncontrolled particle size distribution and cholesterolated pullulan (CHP), which has conventionally been used as a carrier, they have found that the delivery ability to immune cells in lymph nodes and the ability to activate these immune cells are significantly superior, and thus have completed the present invention.

[0026] The immune cells referred to herein are preferably myeloid cells, more preferably macrophages or dendritic cells (DCs), still more preferably DCs, and particularly preferably conventional type 1 dendritic cells (cDC1).

[0027] Both macrophages and dendritic cells have the ability to present antigens. Dendritic cells are the most powerful antigen-presenting cells and are responsible for regulating the proliferation and function of T cells and natural killer cells in lymphoid and non-lymphoid tissues. Although there are several subtypes of dendritic cells, cDC1, which specifically expresses the chemokine receptor XCR1 and selectively expresses the C-type lectin endocytosis receptor CLEC9A, has a high cross-presentation ability and thus efficiently loads antigens onto MHC class I molecules (Reference 1: Noubade R et al., “Beyond cDC1: Emerging Roles of DC Crosstalk in Cancer Immunity”, Front Immunol., Vol. 10, Article 1014, pp. 1-13. 2019.). At the same time, by expressing co-stimulatory molecules, it is possible to activate T cells (cytotoxic T lymphocytes (CTLs)) that attack virus- or bacteria-infected cells and cancer cells. That is, enhancing the antigen delivery ability to macrophages or DCs (preferably cDC1) will improve the effects of cancer prevention or treatment and infectious disease prevention or treatment.

[0028] In addition, macrophages and DCs can load antigens onto MHC class II molecules. This activates helper T cells and promotes antibody production by B cells. That is, enhancing the antigen delivery ability to macrophages or DCs (preferably cDC1) will improve the effects of infectious disease prevention or treatment and immune disease treatment.

[0029] In addition, the ability to activate immune cells means the property of improving, promoting, or enhancing the activity of cells as described above. Among them, it is preferably the property of improving, promoting, or maintaining the expression of co-stimulatory molecules in DC (preferably cDC1). Examples of co-stimulatory molecules include CD80 and CD86. By improving, promoting, or maintaining the expression of these co-stimulatory molecules, T cells (cytotoxic T cells (CTL)) can be significantly induced. Therefore, it is preferable that the hyaluronic acid derivative of the present embodiment can improve, promote, or maintain the expression of both CD80 and CD86 in DC (preferably cDC1) when formulated with a medicinal ingredient.

[0030] The hyaluronic acid derivative of the present embodiment can form a stable association state with the medicinal ingredient when formulated with the medicinal ingredient, can stably and efficiently deliver the medicinal ingredient to immune cells (preferably DC, more preferably cDC1), and further can improve, promote, or enhance the uptake of the medicinal ingredient by immune cells. Thereby, it is presumed that the activity of immune cells is improved, promoted, or enhanced. Even when a desired effect is obtained by a mechanism different from the above mechanism, it is included in the technical scope.

[0031] That is, the hyaluronic acid derivative of the present embodiment can also be referred to as an agent for improving, promoting, or enhancing the delivery of a medicinal ingredient to immune cells. Alternatively, it can also be referred to as an agent for improving, promoting, or enhancing the uptake of a medicinal ingredient by immune cells. In addition, the pharmaceutical composition described later, which contains the hyaluronic acid derivative of the present embodiment and a medicinal ingredient, can also be referred to as a composition for activating immune cells, a composition for improving the expression of co-stimulatory molecules in DC (preferably cDC1), a composition for promoting expression, or a composition for maintaining expression.

[0032] The particle size distribution of the hyaluronic acid derivative of the present embodiment can be measured by gel permeation chromatography, and the fact that it has a controlled particle size distribution as described above can be indicated by the ratio A1 / A2 of the areas A1 and A2 calculated by the method shown below from the gel permeation chromatogram. The polyacrylic acid used as a standard substance in the gel permeation chromatography measurement is preferably sodium polyacrylate, and more preferably, it is the Polymer Standards Service-USA, ORDER No. PSS-Paa series (sodium polyacrylate).

[0033] The measurement of the particle size distribution of the hyaluronic acid derivative by gel permeation chromatography can be performed by the following method. Prepare a 1 mg / mL aqueous solution of the hyaluronic acid derivative and a 2 mg / mL aqueous solution of the polyacrylic acid standard substance, and perform measurement by gel permeation chromatography under the conditions shown below. (Measurement conditions) Apparatus: High-speed GPC (gel permeation chromatography) apparatus Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI (differential refractive index detector) Temperature: 30 °C

[0034] Figure 1 is an example of a gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment. With reference to Figure 1, the calculation methods of areas A1 and A2 will be described in detail below.

[0035] (i) Figure 1 shows the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment and the gel permeation chromatogram of 50 kDa polyacrylic acid as a standard substance. First, a perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid as the standard substance to the baseline B, and the intersection of the chromatogram of the hyaluronic acid derivative is Ub, and the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the baseline B is Bb. Here, the refractive index intensity at the start of measurement is set to zero, and a line drawn horizontally from here is used as the baseline. For example, before the start of measurement, adjust so that the increase and decrease of the refractive index intensity fall within the range of ±0.5 mV, and adjust so that the refractive index intensity falls within an increase and decrease of 0.5 mV or less in 5 minutes. For example, the first point where the increase amount of the refractive index intensity exceeds 5 times the noise value three times is taken as the "starting point" of the chromatogram, and the elution time is set to 0 minutes. For example, the point where the refractive index intensity becomes 1 / 1000 of the maximum maximum refractive index intensity is taken as the "end point" of the chromatogram. If the refractive index intensity does not reach 1 / 1000 of the maximum maximum refractive index intensity, "Tlim" is taken as the "end point". "Tlim" is the elution time at which the maximum refractive index intensity is exhibited when measuring 2 kDa polyacrylic acid. In this apparatus, the refractive index intensity is calculated every 0.00167 minutes.

[0036] (ii) Next, in the chromatogram of the hyaluronic acid derivative, the area value surrounded by the curve from the intersection Ub to the end point, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B (the straight line from the intersection Ub to the intersection Bb), and the baseline B is A2, and the area value surrounded by the curve from the starting point to the Ub in the chromatogram of the hyaluronic acid derivative, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B (the straight line from the intersection Ub to the intersection Bb), and the baseline B is A1. Here, each area value is calculated using the analysis application of the GPC workstation EcoSEC Elite-WS. When calculating each area value, pseudo-peaks due to peaks caused by the developing solvent used in gel permeation chromatography, etc., and fluctuations in the baseline caused by the columns and apparatuses used are excluded.

[0037] The ratio A1 / A2 of the areas A1 and A2 shown in FIG. 1 is 0.90 or more, more preferably 1.00 or more, still more preferably 1.10 or more, still more preferably 1.20 or more, still more preferably 1.30 or more, still more preferably 1.40 or more, still more preferably 1.50 or more, even more preferably 1.60 or more, and particularly preferably 1.70 or more. By the ratio A1 / A2 of the areas A1 and A2 being at least the above lower limit value, a relatively large amount of hyaluronic acid derivatives having a large particle size can be contained, and when formulated with a medicinal ingredient, a stable association state with the medicinal ingredient can be formed. Thereby, the medicinal ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, the uptake of the medicinal ingredient by immune cells can be improved, promoted, or enhanced.

[0038] On the other hand, the upper limit value of A1 / A2 is preferably as high as possible as long as the ratio of the area A1 is higher than the area A2. That is, in FIG. 1, the particle size is larger on the left side of the baseline (where the elution time is shorter) and smaller on the right side (where the elution time is longer). Therefore, the higher the ratio of the larger particle size, the more preferable. Thus, the upper limit value of A1 / A2 is not particularly limited, but may be, for example, 7.0 or less, 6.0 or less, or 5.0 or less.

[0039] The shape of the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment is not particularly limited, but there may be two or more maximum points (peak tops) of the refractive index intensity of the hyaluronic acid derivative. Although not particularly limited, on each side (left and right) of the perpendicular line drawn from the maximum point Kb of the refractive index intensity on the chromatogram of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B, there may be one or more maximum points (peak tops) of the refractive index intensity of the hyaluronic acid derivative. At this time, the refractive index intensity at the maximum point (peak top) of the refractive index intensity of the hyaluronic acid derivative on the left side of the perpendicular line drawn from the maximum point Kb of the refractive index intensity to the baseline B may be greater than the refractive index intensity at the maximum point (peak top) of the refractive index intensity of the hyaluronic acid derivative on the right side of the perpendicular line drawn from the maximum point Kb of the refractive index intensity to the baseline B. Also, the refractive index intensity at the left maximum point (peak top) relative to the refractive index intensity at the right maximum point (peak top) is not particularly limited, but may be 1.0 times or more, 2.0 times or more, 3.0 times or more, 5.0 times or more, or 10.0 times or more. The upper limit may be large, but for example, it may be 1000 times or less, 100 times or less, 50.0 times or less, 20.0 times or less, 10.0 times or less, 2.0 times or less, or 1.5 times or less.

[0040] Moreover, the fact that the hyaluronic acid derivative of this embodiment has a controlled particle size distribution as described above can also be defined by the ratio Da / Db of the distances Da and Db shown below.

[0041] Figure 2 is an example of the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment. The gel permeation chromatogram of the hyaluronic acid derivative shown in Figure 2 and the gel permeation chromatogram of 50 kDa polyacrylic acid, which is a standard substance, are the same as those in Figure 1. However, in Figure 2, the gel permeation chromatogram of 150 kDa polyacrylic acid, which is a standard substance, is additionally noted, and it is different from Figure 1 in terms of the points defined for various intersections and the like. With reference to Figure 2, the calculation methods of the distances Da and Db will be described in detail below.

[0042] (i) First, draw a perpendicular line from the refractive index intensity maximum point Ka on the chromatogram of 150 kDa polyacrylic acid, which is the standard substance, to the baseline B, and let the intersection point with the baseline be Ba, and the length between the refractive index intensity maximum point Ka and Ba be La.

[0043] (ii) Next, among the two points on the chromatogram where the refractive index intensity is La / 20, let the point with the earlier elution time be point R1 and the point with the later elution time be point S1.

[0044] (iii) Next, let the intersection point of the perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid, which is the standard substance, to the baseline B and the baseline be Bb.

[0045] (iv) Next, let the intersection point of the straight line D1 connecting the point R1 and the point S1 and the perpendicular line drawn from the refractive index intensity maximum point Ka to the baseline B be Ta, and the intersection point of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the straight line D1 be Tb.

[0046] (v) Finally, let the distance between the point R1 and the point Ta be Da, and the distance between the point Ta and the point Tb be Db.

[0047] Preferably, the ratio Da / Db of the distances Da and Db shown in Figure 2 is more than 0.00 and less than or equal to 1.20, more preferably 0.10 or more and 1.00 or less, still more preferably 0.20 or more and 0.8 or less, even more preferably 0.30 or more and 0.95 or less, yet even more preferably 0.40 or more and 0.94 or less, and particularly preferably 0.50 or more and 0.93 or less. By the ratio Da / Db of the distances Da and Db being within the above range, it is possible to contain a relatively large amount of hyaluronic acid derivatives having a particle size larger than that of 150 kDa polyacrylic acid, which is the standard substance, and when formulated with the medicinal ingredient, it is possible to form a stable association state with the medicinal ingredient. Thereby, the medicinal ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, the uptake of the medicinal ingredient by immune cells can be improved, promoted, or enhanced.

[0048] In the gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment, the horizontal axis represents the elution time, and the vertical axis represents the refractive index intensity obtained using a differential refractometer. There may be any number of refractive index maxima. The gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment preferably has a particle size distribution having 1 to 5 maxima, more preferably a particle size distribution having 1 to 3 maxima. Also, there may or may not be a minimum point. Furthermore, in the gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment, the chromatogram represented by the refractive index intensity obtained using a differential refractometer and the elution time may be asymmetric or symmetric about the left and right.

[0049] The hyaluronic acid derivative of the present embodiment is one in which the sterol group in the hyaluronic acid derivative self-associates in water, and a nano-sized hydrogel is formed by the association of single molecules or multiple molecules.

[0050] In the hyaluronic acid derivative, the sterol group may be directly bonded to the hyaluronic acid or may be bonded through a linker.

[0051] The "linker" referred to here can be any peptide linker or synthetic compound linker that can be introduced by genetic engineering. In the hyaluronic acid derivative, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 2 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. The peptide linkers contained in the hyaluronic acid derivative may all use peptide linkers of the same length or peptide linkers of different lengths.

[0052] [Sterol group] As used herein, the term "steryl group" is not particularly limited as long as it is a group having a steroid skeleton. Here, specific examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmasteranol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simiarenol, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycorticosterone, and the like. Examples of steryl groups include cholesteryl group, stigmasteryl group, lanosteryl group, ergosteryl group, etc. Among them, cholesteryl group (particularly, cholesta-5-en-3β-yl group) is preferred.

[0053] The weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is not particularly limited. However, from the viewpoints of increasing the number of steryl groups introduced per molecule of the hyaluronic acid derivative to form a complex with a medicinal ingredient and enhancing the entanglement of molecules to increase the residence time in blood, a hyaluronic acid derivative having a relatively large molecular weight is preferred. As such weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative, 4000 (4k) or more and 1,000,000 (1,000k) or less is preferred, 5k or more and 500k or less is more preferred, 6k or more and 500k or less is further preferred, 7k or more and 300k or less is even further preferred, 7k or more and 100k or less is even further preferred, 7k or more and 50k or less is even further preferred, 7k or more and 25k or less is even more further preferred, and 8k or more and 15k or less is particularly preferred. Alternatively, as the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative, 5k or more and 25k or less is more preferred. When the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is at least the above lower limit value, the entanglement of molecules can be further enhanced and the residence time in blood can be further increased. On the other hand, when the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is at most the above upper limit value, an increase in viscosity can be suppressed and a higher concentration of the hyaluronic acid derivative can be dissolved in the pharmaceutical composition. Generally, the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative can be adjusted by using a raw material having a corresponding molecular weight.

[0054] As used herein, the "molecular weight (absolute molecular weight) of the hyaluronic acid derivative" is the weight average molecular weight (absolute molecular weight) determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS).

[0055] In the chromatogram obtained by the gel permeation chromatography measurement, the ratio Pt / Pr of the retention time Pt of the refractive index intensity maximum point (peak top) of the hyaluronic acid derivative to the retention time Pr (elution time Bb) of the refractive index intensity maximum point (peak top) of the 50 kDa polyacrylic acid as the standard substance is not particularly limited, but Pt / Pr is preferably 0.5 or more and less than 1.0, more preferably 0.7 or more and less than 0.95, and even more preferably 0.75 or more and less than 0.92. When the ratio Pt / Pr is within the above range, a relatively large amount of hyaluronic acid derivatives with large particle sizes can be included, and when formulated with the active ingredient, a stable association state with the active ingredient can be formed. Thereby, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, the uptake of the active ingredient by immune cells can be improved, promoted, or enhanced. In the chromatogram obtained by the gel permeation chromatography measurement, when there are multiple refractive index intensity maximum points (peak tops) of the hyaluronic acid derivative, the elution time at the refractive index intensity maximum point (peak top) where the refractive index intensity is the maximum is adopted as the retention time Pt of the refractive index intensity maximum point (peak top) of the hyaluronic acid derivative. The polyacrylic acid used as the standard substance in the gel permeation chromatography measurement is preferably sodium polyacrylate, and more preferably specifically, Polymer Standards Service-USA, ORDER No. PSS-Paa series (sodium polyacrylate).

[0056] The weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is not particularly limited, but it is preferably 110,000 or more, more preferably 110,000 or more and less than 500,000, even more preferably 130,000 or more and less than 300,000, and most preferably 140,000 or more and less than 250,000. When the weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is within the above range, a relatively large amount of hyaluronic acid derivatives with large particle sizes can be included, and when formulated with the medicinal ingredient, a stable association state with the medicinal ingredient can be formed. Thereby, the medicinal ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, the uptake of the medicinal ingredient by immune cells can be improved, promoted, or enhanced. On the other hand, when the weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative exceeds the above upper limit value, the viscosity increases, and it may be difficult to use as a formulation.

[0057] The weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is calculated from the chromatogram obtained by the above gel permeation chromatography measurement, based on the calibration curve prepared using polyacrylic acids (PSS-Paa2k(2kDa), 4k(4kDa), 8k(8kDa), 18k(18kDa), 40k(40kDa), 150k(150kDa) (manufactured by PSS Polymer Standard service GmbH, sodium polyacrylate) with molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa, and 150 kDa as standard substances, according to the following formula: At3+Bt2+Ct+D.

[0058] Specific examples of preferred hyaluronic acid derivatives include, for example, hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter sometimes referred to as "repeating unit (I)").

[0059]

Chemical formula

[0060] (wherein, R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 or more and 30 or less; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -S-R, -CO-Y a -S-R, -O-CO-Y b -S-R, -NR b -CO-Y b -S-R, and -S-S-R, and is a group selected from the group consisting of the groups represented by; R a , R b and R c are each independently selected from the group consisting of a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl and hydroxy C 2-20 alkyl, wherein the alkyl part of the group may have a group selected from the group consisting of -O- and -NR f - inserted therein; R f is a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl and hydroxy C 2-12selected from the group consisting of alkyl, and the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NH- inserted therein; R is a sterol group; Y is C 2-30 alkylene, or -(CH 2 CH 2 O) m -CH 2 CH 2 -, where said alkylene may have a group selected from the group consisting of -O-, -NR g - and -S-S- inserted therein; R g is a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl and hydroxy C 2-20 alkyl, and the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NH- inserted therein; Y a is C 1-5 alkylene; Y b is C 2-8 alkylene or C 2-8 alkenylene; m is an integer of 1 or more and 100 or less.)

[0061] It is preferable that the hyaluronic acid derivative contains one or more repeating units (Ia) represented by the following general formula (Ia) as the repeating unit (I) (hereinafter, may be referred to as "repeating unit (Ia)").

[0062]

Chemical formula

[0063] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; X is a hydrophobic group represented by -NR a -Y-NR b -COO-R; R a and R b are each independently selected from the group consisting of a hydrogen atom and C1-6 alkyl; R is a sterol group; Y is C 2-30 alkylene, or -(CH 2 CH 2 O)m-CH 2 CH 2 -; m is an integer of 1 or more and 100 or less.)

[0064] Here, when the hyaluronic acid derivative contains two or more repeating units (I) or repeating units (Ia), respectively, the repeating units may be the same or different.

[0065] The hyaluronic acid derivative may be modified at positions other than the repeating unit (I) or the repeating unit (Ia). For example, a hydroxy group may be converted to -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 alkylcarbonyl), etc., and a carboxy group may be converted to an amide or an ester, or may form a salt.

[0066] [Repeating unit (I)] The group "-Z-N(R a )Y-X 1 " in the general formula (I) is 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-NR a -Y-NR b-COO-R (wherein mz is an integer of 2 or more and 30 or less, and R 8 is a hydrogen atom or a methyl group, and R and m are as defined hereinbefore.) is preferably selected from the group consisting of groups represented by -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 -S-S-R (wherein mz, R, and m are as defined hereinbefore.) is more preferably selected from the group consisting of

[0067] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 is preferably -NR b -COO-R. Furthermore, in another embodiment, Z may be a peptide linker represented by -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a )-CO-, wherein n is an integer of 2 or more and 30 or less, and Z a each independently represents a substituent in an α-amino acid represented as H 2 N-CH(-Z a )-COOH. The peptide linker is bonded to the carboxy group of the glucuronic acid moiety at the N-terminus and the group -N(-R a )-Y-X 1It binds to. Examples of amino acids that can be used as amino acid residues of the peptide linker include α - amino acids, such as natural - type (L - type) amino acids like 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, valine, and their D - forms, etc. All α - amino acids including synthetic amino acids can be used. That is, Z a Examples of which include, for example, -CH 3 , H 2 NC(NH)NH(CH 2 ) 3 , H 2 NCOCH 2 -, etc. Also, the n Zs may be the same or different. n is an integer of 2 or more and 30 or less, preferably 2 or more and 10 or less, and more preferably 2 or more and 4 or less. Preferred examples of the peptide linker include, for example, -Gly - Phe - Leu - Gly -, -Asn - Phe - Phe -, -Phe - Phe -, Phe - Gly -, etc.

[0068] (Y) In general formula (I), Y is a group selected from the group consisting of -(CH 2 ) n1 - and -(CH 2 CH 2 O) m1 -CH 2 CH 2 -(where n1 is an integer of 2 or more and 20 or less, preferably an integer of 2 or more and 15 or less, more preferably an integer of 2 or more and 12 or less, and even more preferably an integer of 2 or more and 6 or less. m1 is an integer of 1 or more and 4 or less). Specifically, -(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 8 -, -(CH 2 ) 12 -, or -(CH 2 CH2 O) 2 -CH 2 CH 2 - is preferred. Also, from the viewpoint of achieving high solubility in pure water or at low salt concentrations and showing high precipitation-forming ability at physiological salt concentration, Y is -(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 8 - and -(CH 2 ) 12 - selected from the group consisting of is preferred, and -(CH 2 ) 6 - is more preferred.

[0069] Y may be, for example, -CH 2 CH 2 O-CH 2 CH 2 -S-S-CH 2 CH 2 O-CH 2 CH 2 -, -(CH 2 CH 2 O) 2 -CH 2 CH 2 -S-S-CH 2 CH 2 O-CH 2 CH 2 -, -CH 2 CH 2 O-CH 2 CH 2 -S-S-(CH 2 CH 2 O) 2 -CH 2 CH 2 -, -(CH 2 CH 2 O) 2 -CH 2 CH 2 -S-S-(CH 2 CH 2 O) 2 -CH 2 CH 2 - etc. may be used.

[0070] (Ya ) Y a is preferably -CH 2 - or -CH 2 -CH 2 - is preferred.

[0071] (Y b ) Y b is preferably -CH 2 -CH 2 -, -CH(CH 3 )CH 2 -, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl or octa-2,4,6-triene-1,8-diyl, and -CH 2 -CH 2 - or -CH(CH 3 )CH 2 - is more preferred.

[0072] Specific examples of the group “-Z-N(R a )Y-X 1 ” 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-(CH2 ) 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. are mentioned. Preferred group "-Z-N(R a )Y-X 1 " is such that R a , R b and R c are hydrogen atoms, Y is linear C 2-30 alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 -, and Y a is linear C 1-5 alkylene, or Y b is linear C 2-8 alkylene or linear C 2-8 alkenylene.

[0073] [Repeating unit (Ia)] In general formula (Ia), X is -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 is preferred, -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 is more preferred, -NH-(CH 2 ) 6 -NH-COO-cholesteryl is even more preferred.

[0074] In addition to the repeating unit (I), the hyaluronic acid derivative can further contain a repeating unit represented by the general formula (II) (hereinafter sometimes referred to as "repeating unit (II)").

[0075] [Chemical formula]

[0076] (In the formula, R 1a , R 2a , R 3a , and R 4a are each independently selected from the group consisting of a hydrogen atom, C 1-6 alkyl, formyl, and C 1-6 alkylcarbonyl; X ais selected from the group consisting of hydroxy and -O-Q + ; where Q + is a counter cation.)

[0077] Here, when the hyaluronic acid derivative contains two or more repeating units (II), the repeating units may be the same or different. In another embodiment, the hyaluronic acid derivative may be a hyaluronic acid derivative consisting essentially of repeating unit (I), repeating unit (Ia) and repeating unit (II).

[0078] [Repeating unit (II)] In the general formula (II), Q + is not particularly limited as long as it is a counter cation that forms a salt with a carboxy group in water, and in the case of a divalent or higher valence, it forms a salt with a plurality of carboxy groups according to the valence. Examples of the counter cation include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, calcium ion; formula: N + R j R k R l R m (wherein R j , R k , R l and R m are each independently selected from the group consisting of a hydrogen atom and C 1-6 alkyl) and the like, such as ammonium ions represented by. Among them, Q + is preferably a sodium ion, a potassium ion, or a tetraalkylammonium ion (for example, a tetra-n-butylammonium ion, etc.). In the above formula, R j , R k , R l and R m are preferably the same group selected from the group consisting of C 1-6 alkyl, and an n-butyl group is preferred.

[0079] R1 , R 2 , R 3 , and R 4 , and R 1a , R 2a , R 3a , and R 4a are all preferably hydrogen atoms. Also, R a and R b are both preferably hydrogen atoms.

[0080] Among them, the hyaluronic acid derivative is preferably a hyaluronic acid derivative substantially composed of the repeating unit (I) and the repeating unit (II). The hyaluronic acid derivative is, for example, 80% or more, preferably 90% or more, more preferably 95% or more of the repeating units of the disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative are the repeating unit (I) and the repeating unit (II). The hyaluronic acid derivative may be composed only of the repeating unit (I) and the repeating unit (II), or may be composed only of the repeating unit (I).

[0081] In the hyaluronic acid derivative of the present embodiment, the introduction rate of the sterol group (hereinafter, may be simply referred to as "sterol group introduction rate") with respect to the repeating unit of the disaccharide constituting the hyaluronic acid derivative is preferably 15% or more and 60% or less, preferably 20% or more and 60% or less, preferably 30% or more and 60% or less, more preferably 30% or more and 55% or less, further preferably 35% or more and 50% or less, and particularly preferably 35% or more and 45% or less. By the sterol group introduction rate being not less than the above lower limit value, a stable hydrogel can be maintained without precipitation in vivo. On the other hand, by being not more than the above upper limit value, the average particle diameter of the hydrogel can be within the above range.

[0082] The sterol group introduction rate can be measured by 1 H-NMR measurement. That is, the 1The integration value of the peak derived from the sterol group of the hyaluronic acid derivative in the 1H-NMR spectrum and the integration value of the peak (COCH 3 , from 1.6 ppm to 2.0 ppm, 3H) derived from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative can be used to calculate based on the following formula. In the formula, n H represents the number of hydrogen atoms corresponding to the peak. The above-mentioned 1 1H-NMR can be carried out, for example, using a 0.02N DCl DMSO-d 6 / D 2 O mixture (2N DCl D 2 O:DMSO-d 6 =1:99) as the measurement solvent at a measurement temperature of 85°C. Since the peak from 1.6 to 2.0 ppm containing the peak derived from the acetyl group of glucosamine overlaps with the peak (5H) derived from the cholesteryl group, the value calculated by subtracting 5 / 3 of the integration value of the peak (0.7 ppm) derived from the cholesteryl group methyl from the integration value of the peak around 1.6 to 2.0 ppm (that is, integration value (1.6 - 2.0 ppm) - integration value (0.7 ppm) × 5 / 3) can be used as the integration value of the acetyl group derived from hyaluronic acid for the calculation of the introduction rate.

[0083] [Sterol group introduction rate](%) =[(Peak integration value derived from sterol group × 3 / n H ) / (Peak integration value derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0084] <Method for producing hyaluronic acid derivative> The hyaluronic acid derivative can be obtained, for example, by converting the carboxyl group of glucuronic acid into an amide and introducing a sterol group. Also, the introduction rate of the sterol group can be controlled by adjusting the blending amount of the compound having a sterol group to be reacted with the raw material hyaluronic acid or its derivative.

[0085] As a method for converting the carboxy group of glucuronic acid into an amide and introducing a sterol group, specifically, for example, hyaluronic acid or a derivative thereof as a raw material, preferably hyaluronic acid or a derivative thereof composed only of the repeating unit (II), is ion-exchanged with a tetraalkylammonium salt (for example, tetrabutylammonium (TBA) salt), and in the presence of a suitable condensing agent, in a solvent, the hyaluronic acid salt is reacted with an amine having a sterol group (particularly, a cholesteryl 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 -Y-O-COO-R, HNR a -Y-S-R, HNR a -Y-CO-Y a -S-R, HNR a -Y-O-CO-Y b -S-R, HNR a -Y-NR b -CO-Y b -S-R, HNR a -Y-S-S-R, or -Z-NR a -Y-NR b -COO-R (wherein R a , R b , R c , Y, Y a , Y b , Z and R are as defined hereinbefore)".

[0086] The condensing agent that can be used in the above reaction is not particularly limited. For example, 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazol-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazol-1-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), etc. can be mentioned.

[0087] In particular, although not limited, DMT-MM is preferable in that the reaction proceeds with high efficiency even in a mixed solvent of water and an organic solvent. Further, by using DMT-MM as a condensing agent, in a system where a large number of hydroxy groups coexist, amide bond formation between an amino group and a carboxy group can be carried out with high selectivity 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 carboxy group of the hyaluronic acid moiety, and the carboxy group and the hydroxy group simultaneously present in the hyaluronic acid moiety from bonding intramolecularly or intermolecularly to form an undesirable crosslink.

[0088] Examples of the solvent used in the steryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyhydric alcohol, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and a mixed solvent thereof. The polyhydric alcohol may be a divalent alcohol or a trivalent alcohol. Examples of the dihydric alcohol include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, 1,6-hexanediol, and the like. Examples of the trihydric alcohol include glycerin, trimethylolpropane, and the like.

[0089] Alternatively, hyaluronic acid or its derivative as a raw material is ion-exchanged with a tetraalkylammonium salt (for example, tetrabutylammonium (TBA) salt), and in the presence of a suitable condensing agent, the hyaluronate and the spacer moiety are reacted in a solvent (in this case, a protection and deprotection reaction may be performed as necessary), the carboxy group (-COOH) of the raw material hyaluronic acid or its derivative is converted, and then it may be reacted with a suitable reagent. Examples of the combination of the group derived from the carboxy 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 -Y-O-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 (wherein, R a , R b , R c , Y, Y a , Y b , and Z are as defined hereinbefore, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0090] Examples of reaction modes include dehydrohalogenation reaction, condensation reaction, dehydration reaction, nucleophilic addition reactions 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 preferable reaction conditions to carry them out. When the transformant or reactant has a carboxy group, it may be reacted as an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester.

[0091] In addition, a method is provided in which 2-aminoethyl 2-pyridyldisulfide is reacted with the carboxy group of raw material hyaluronic acid or its derivative to prepare a hyaluronic acid derivative into which a spacer having a mercapto group modified with a leaving group at the end is introduced, and then thiocolesterol is subjected to a nucleophilic substitution reaction with this to form a disulfide bond.

[0092] Furthermore, a method is also provided in which a hyaluronic acid or its derivative into which a part of a spacer is introduced into the carboxy group and a sterol group into which a part of a spacer is introduced are prepared and reacted with each other. Although some specific examples have been described above, when -S-S- is inserted into Y, a hyaluronic acid derivative into which a spacer having a mercapto group at the end is introduced into the carboxy group of hyaluronic acid and a sterol group into which a spacer having a mercapto group at the end is introduced are respectively prepared, and these are oxidatively reacted to form a disulfide bond. At this time, one mercapto group can be reacted with 2-mercaptopyridine to form a disulfide and then substituted with the other mercapto group.

[0093] Moreover, after preparing a hyaluronic acid derivative, other substituents may be further introduced. For example, 0.1 mol% or more and 99.5 mol% or less, preferably 40 mol% or more and 65 mol% or less of the carboxy groups in a hyaluronic acid derivative substantially composed of repeating unit (I) and repeating unit (II) are replaced with -CO-X z , [where X z is the following group: -NH-(CH 2 ) p1 -O-CO-C(R 17 )=CH2 ; -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 -CH2 -O-CO-C(R 17 )=CH 2 ; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -O-CO-CH(R 17 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-C(R 17 )=CH 2 ; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-C(=NH)-(CH 2 ) 3 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-(CH 2 ) r -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-CH(R 17 )-CH 2 -S-CH 2 -CH(OH)-CH(OH)-CH 2 -SH; -NH-(CH 2 -CH 2 -O)q -CH 2 -CH 2 -NH-CO-CH(NH 2 )-CH 2 -SH; -NH-(CH 2 -CH 2 -O) q -CH 2 -CH 2 -NH-CO-CH(NH 2 )-(CH 2 ) 2 -SH; -NH-CH(CO 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 (wherein R 17 is a hydrogen atom or a C 1-6 alkyl group, p1 is an integer of 2 or more and 10 or less, q is an integer of 1 or more and 100 or less, and r is an integer of 1 or more and 3 or less, respectively) selected from the group consisting of] By converting to, it is also possible to chemically crosslink intramolecularly or intermolecularly including other molecules to cause gelation.

[0094] When controlling the particle size within a specific range by dialysis, by dialyzing one or more times and nine or fewer times, preferably three or more times and nine or fewer times, more preferably six or more times and nine or fewer times, with a dialysis membrane having an MWCO (molecular weight cut-off) of 300 kDa, a hyaluronic acid derivative having a particle size distribution satisfying an area ratio A1 / A2 of 0.9 or more can be obtained. The number of dialysis times can be appropriately selected by those skilled in the art as any number as long as the area ratio A1 / A2 satisfies 0.9 or more. In some cases, fractionation by dialysis nine or more times may also be performed.

[0095] At this time, the method for obtaining a hyaluronic acid derivative having a particle size distribution satisfying an area ratio A1 / A2 of 0.9 or more is not limited to dialysis only. For example, centrifugal filtration separation using an ultrafiltration membrane or a microfiltration membrane, preparative HPLC, preparative GPC, separation by ultracentrifugation, separation by an ion exchange resin, separation by membrane distillation, membrane separation using an organic or inorganic membrane, adsorption / desorption separation using activated carbon, zeolite, MOF (Metal Organic Frameworks), separation by TFF (Tangential Flow Filtration), liquid feeding using a filter, pressure or vacuum filtration separation, precipitation separation method using salting out, etc. Similarly, by removing a hyaluronic acid derivative having a particle size distribution below that of the standard substance polyacrylic acid (50 kDa, manufactured by Polymer Standards Service-USA, ORDER No. PSS-Paa50k), a hyaluronic acid derivative having a particle size distribution satisfying an area ratio A1 / A2 of 0.9 or more can be obtained.

[0096] The obtained hyaluronic acid derivative may be dried. Examples of the drying method include ventilation drying, drying in a constant temperature bath, vacuum drying, hot air circulation drying, freeze drying, etc. Among them, freeze drying is preferable. When performing freeze drying, from the viewpoint of more effectively suppressing an increase in the particle diameter of the fine particles formed by the hyaluronic acid derivative, it is preferable that the hyaluronic acid derivative further contains a cryoprotectant.

[0097] The cryoprotectant is not particularly limited as long as it is known as a "cryoprotectant" or a "lyoprotectant". Examples thereof include disaccharides, sorbitol, dextran, polyethylene glycol, propylene glycol, glycerin, glycerol, polyvinylpyrrolidone, dimethyl sulfoxide, etc.

[0098] The disaccharide is not particularly limited, and examples thereof include sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobios, melibiose, melibiulose, neolactose, galactosucrose, syrabiose, neohesperidose, rutinose, rutinosulose, bicinose, xylobiose, primeverose, etc. Among them, sucrose, trehalose, maltose, or lactose is preferable because it is widely used as a cryoprotectant. Further, sucrose is more preferable from the viewpoints of the usage record as a pharmaceutical additive and more effectively suppressing the increase in the particle size of the fine particles formed by the hyaluronic acid derivative during freeze-drying.

[0099] The cryoprotectant may be added in a solid state or in a state dissolved in a solvent such as water.

[0100] The addition amount of the cryoprotectant is not particularly limited, but is preferably 20 parts by mass or more with respect to 100 parts by mass of the hyaluronic acid derivative. By setting the addition amount of the cryoprotectant to be equal to or higher than the above lower limit value, a more sufficient effect of suppressing the increase in particle size can be obtained. On the other hand, the upper limit of the addition amount of the cryoprotectant is not particularly limited, but can be, for example, 100,000 parts by mass.

[0101] The apparatus used in freeze-drying is also not particularly limited, and for example, a commercially available freeze-dryer can be used. Among them, from the viewpoint of controlling the degree of vacuum, a freeze-dryer capable of monitoring the degree of vacuum inside the apparatus during freeze-drying is preferable, and from the viewpoint of controlling the product temperature, a shelf-type freeze-dryer is preferable.

[0102] The hyaluronic acid derivative of the second embodiment of the present invention is a hyaluronic acid derivative into which a sterol group has been introduced. In the chromatogram obtained by gel permeation chromatography measurement, the ratio Pt / Pr of the retention time Pt at the refractive index intensity maximum point (peak top) of the hyaluronic acid derivative to the retention time Pr at the refractive index intensity maximum point (peak top) of 50 kDa polyacrylic acid, which is a standard substance, is 0.5 or more and less than 1.0, more preferably 0.7 or more and less than 0.95, and most preferably 0.75 or more and less than 0.92. When the ratio Pt / Pr is within the above range, a relatively large amount of hyaluronic acid derivative having a large particle size can be included, and when formulated with a medicinal ingredient, a stable association state with the medicinal ingredient can be formed. As a result, the medicinal ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and furthermore, the uptake of the medicinal ingredient by immune cells can be improved, promoted, or enhanced.

[0103] The polyacrylic acid used as a standard substance in the gel permeation chromatography measurement is preferably sodium polyacrylate, and more preferably, specifically, Polymer Standards Service-USA, ORDER No. PSS-Paa50k (sodium polyacrylate). The measurement by the gel permeation chromatography can be performed, for example, by the following method. Prepare a 1 mg / mL aqueous solution of the hyaluronic acid derivative and a 2 mg / mL aqueous solution of the polyacrylic acid standard substance, and perform measurement by gel permeation chromatography under the conditions shown below. (Measurement conditions) Apparatus: High-speed GPC (gel permeation chromatography) apparatus Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI (differential refractive index detector) Temperature: 30 °C Regarding other aspects with the same configuration as the hyaluronic acid derivative of the first embodiment, the description thereof will be omitted.

[0104] The hyaluronic acid derivative of the third embodiment of the present invention is a hyaluronic acid derivative into which a sterol group has been introduced. The weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is 110,000 or more and less than 500,000, preferably 130,000 or more and less than 300,000, and more preferably 140,000 or more and less than 250,000. When the weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative satisfies the above range, a relatively large amount of hyaluronic acid derivatives with a large particle size can be included, and when formulated with a medicinal ingredient, a stable association state with the medicinal ingredient can be formed. Thereby, the medicinal ingredient can be stably and efficiently delivered to immune cells (preferably DCs, more preferably cDC1s), and furthermore, the uptake of the medicinal ingredient by immune cells can be improved, promoted, or enhanced. On the other hand, when the weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative exceeds the above upper limit value, the viscosity increases, and it may be difficult to use as a formulation.

[0105] The weight average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is calculated according to the following formula: At3 + Bt2 + Ct + D based on a calibration curve created using polyacrylic acid with molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa, and 150 kDa as standard substances from a chromatogram obtained by gel permeation chromatography measurement. The polyacrylic acid used as the standard substance is preferably sodium polyacrylate, and more preferably specifically the Pоlymer Standards Service-USA, ORDER No. PSS-Paa series (sodium polyacrylate).

[0106] The measurement by the gel permeation chromatography can be performed by the method described in the first or second embodiment. In addition, the description of the same configuration and the like of the hyaluronic acid derivative of the first embodiment and / or the second embodiment is omitted.

[0107] ≪Pharmaceutical composition≫ The hyaluronic acid derivatives of the first to third embodiments can be formulated with a medicinal ingredient and used as a pharmaceutical composition. That is, the pharmaceutical composition of the present embodiment contains the above-described hyaluronic acid derivative and a medicinal ingredient.

[0108] In the pharmaceutical composition of the present embodiment, the above hyaluronic acid derivative forms a complex with the medicinal ingredient (hereinafter sometimes referred to as a "medicinal ingredient-hyaluronic acid derivative complex"). Specifically, it is presumed that a sterol group in the hyaluronic acid derivative and a hydrophobic site of the medicinal ingredient form a complex by hydrophobic interaction, and the medicinal ingredient and the hydrophobic sites such as the sterol group are present in the central part, while the hydrophilic sites such as the site derived from hyaluronic acid in the hyaluronic acid derivative are present in the outer edge part, presenting a core-shell type spherical structure. That is, it is presumed that the medicinal ingredient has a structure encapsulated or included in the hyaluronic acid derivative.

[0109] In the pharmaceutical composition of the present embodiment, the average particle diameter of the spherical structure containing the active ingredient - hyaluronic acid derivative complex can be 20 nm or more and 100 nm or less, can be 20 nm or more and 95 nm or less, and can be 20 nm or more and 90 nm or less. The average particle diameter referred to here is the value represented by the z-average. By the average particle diameter being within the above numerical range, it can exist in a stable structure in the living body and can more easily pass through lymph nodes. The average particle diameter can be measured, for example, by DLS (Dynamic Light Scattering), a nanoparticle tracking particle measuring device, size exclusion chromatography, high performance liquid chromatography, and electron microscopy methods. More specifically, for example, it is measured by diluting with a 10 mM phosphate buffer containing 10 w / v% sucrose so that the hyaluronic acid derivative concentration becomes 1 mg / mL using a DLS device.

[0110] In the pharmaceutical composition of the present embodiment, the weight average molecular weight Mw (in terms of polyacrylic acid) of the hyaluronic acid derivative or the active ingredient - hyaluronic acid derivative complex is preferably 110,000 or more. The weight average molecular weight Mw (in terms of polyacrylic acid) can be calculated by creating a calibration curve using a plurality of polyacrylic acid standards having different molecular weights. The polyacrylic acid standard used is preferably sodium polyacrylate, and more preferably, it is Polymer Standards Service-USA, ORDER No. PSS-Paa series (sodium polyacrylate). By Mw being within the above numerical range, more aggregate sizes excellent in activating costimulatory molecules exist, and by using the pharmaceutical composition of the present embodiment, an active ingredient such as a peptide can be stably and efficiently delivered to immune cells (particularly, cDC1, macrophages), and further, the uptake of the active ingredient into immune cells can be improved, promoted, or enhanced. At the same time, it is considered that the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1) can be improved, promoted, maintained, or enhanced. Mw can be measured, for example, by Dynamic Light Scattering (DLS), size exclusion chromatography, electron microscopy, etc. More specifically, for example, it is measured by diluting with a size exclusion chromatography apparatus so that the hyaluronic acid derivative concentration becomes 1 mg / mL.

[0111] In the pharmaceutical composition of this embodiment, it is preferable that the peak top time in the particle size distribution by size exclusion chromatography of the hyaluronic acid derivative or the drug efficacy component-hyaluronic acid derivative complex is smaller than the peak top time of the standard substance polyacrylic acid (50 kDa). When the peak top time is within the above range, there are more aggregate sizes excellent in activating co-stimulatory molecules. By using the pharmaceutical composition of this embodiment, a drug efficacy component such as a peptide can be stably and efficiently delivered to immune cells (particularly, cDC1, macrophages), and further, the uptake of the drug efficacy component into immune cells can be improved, promoted, or enhanced. At the same time, it is considered that the activity of immune cells (particularly, the expression of co-stimulatory molecules CD80 and CD86 in cDC1) can be improved, promoted, maintained, or enhanced. The peak top time can be measured, for example, by size exclusion chromatography. More specifically, for example, it is measured by diluting with a size exclusion chromatography apparatus so that the hyaluronic acid derivative concentration becomes 1 mg / mL.

[0112] In the pharmaceutical composition of this embodiment, the content of the hyaluronic acid derivative is not particularly limited, but for example, it is preferably 0.01 part by mass or more and 50.00 parts by mass or less, more preferably 0.10 part by mass or more and 25.00 parts by mass or less, and even more preferably 0.20 part by mass or more and 10.00 parts by mass or less with respect to 100 parts by mass of the pharmaceutical composition.

[0113] Next, the components of this embodiment will be described in detail below.

[0114] <Drug efficacy component> The active ingredient is not particularly limited, and examples thereof include antigens (cancer antigens, antigens derived from infectious diseases, autoantigens in immune diseases, etc.), pharmaceutically active peptides or proteins, nucleic acids, low-molecular compounds, medium-molecular compounds, and the like. Among them, an antigen is preferable, at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases is more preferable, and a cancer antigen or an antigen derived from an infectious disease is more preferable.

[0115] That is, the pharmaceutical composition of the present embodiment is preferably a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases, and more preferably a pharmaceutical composition for preventing or treating cancer or an infectious disease. When the applicable disease of the pharmaceutical composition of the present embodiment is cancer, an infectious disease, or an immune disease, it can also be referred to as a vaccine composition.

[0116] [Antigen] (Cancer antigen) A cancer antigen is an antigen that is highly expressed in cancer cells and, in some cases, is expressed only by cancer cells. A cancer antigen can be expressed inside cancer cells or on the surface of cancer cells.

[0117] The antigen proteins that can be used in the pharmaceutical composition of this embodiment include, but are not limited to, ERK1, ERK2, WT1, MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-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, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-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, Epstein-Barr virus-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, c-erbB-2, and the like.

[0118] As the above antigen protein, all of its sequences may be used, or sequences with partial deletions may be used.

[0119] The antigenic peptide that can be used in the pharmaceutical composition of the present embodiment is an antigenic peptide containing one or more epitopes selected from the group consisting of CD8-positive cytotoxic T cell recognition epitopes and CD4-positive helper T cell recognition epitopes among the sequences of antigenic proteins. In one embodiment, from the viewpoint of being loaded onto MHC class I molecules or MHC class II molecules via degradation in antigen-presenting cells, the above antigenic peptide is preferably an antigenic peptide containing two or more epitopes. Specifically, examples of the above antigenic peptide include antigenic peptides containing epitopes of tumor cell antigenic proteins.

[0120] In one embodiment, the above antigenic peptide has, for example, 8 to 120 amino acids, preferably 8 to 80 amino acids, more preferably 15 to 80 amino acids, even more preferably 16 to 80 amino acids, still more preferably 23 to 80 amino acids, even more preferably 23 to 60 amino acids, and particularly preferably 23 to 50 amino acids.

[0121] In one embodiment, from the viewpoint of inducing the activation of cytotoxic T cells (CTL) by helper T cells, the above antigenic peptide is an antigenic peptide containing at least one CD8-positive cytotoxic T cell recognition epitope and at least one CD4-positive helper T cell recognition epitope respectively.

[0122] In one embodiment, when two or more epitopes are included, an amino acid linker may be arranged between the epitopes. The linker has, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, and more preferably 4 to 8 amino acids. Examples of the amino acids used for the linker include glycine (G), tyrosine (Y), leucine (L), tryptophan (W), etc. Preferably, they are tyrosine (Y), leucine (L), and tryptophan (W). Specific examples of the amino acid linker include a linker consisting of four consecutive tyrosines (Y) (4Y), a linker consisting of four consecutive leucines (L) (4L), a linker consisting of four consecutive tryptophans (W) (4W), a linker consisting of six consecutive glycines (G) (6G), a linker consisting of six consecutive tyrosines (Y) (6Y), a linker consisting of six consecutive leucines (L) (6L), a linker consisting of six consecutive tryptophans (W) (6W), a linker consisting of eight consecutive tyrosines (Y) (8Y), a linker consisting of six consecutive leucines (L) (8L), and a linker consisting of eight consecutive tryptophans (W) (8W). Preferably, they are 6Y, 6L, or 6W.

[0123] The cancer antigen may be a tumor-associated antigen, a cancer testicular antigen, a viral antigen, or a tumor-specific antigen (including neoantigen). One cancer antigen may be used, or two or more may be used in combination.

[0124] (Antigen derived from infectious disease) The antigen derived from infectious disease is not particularly limited as long as it is an infectious pathogen and an antigen derived from an infectious pathogen. Examples of the infectious pathogen include virus, bacterium, fungus, nematode, etc. The antigen derived from an infectious pathogen may be an antigen protein or an antigen peptide.

[0125] The diseases caused by the above infectious pathogens are not particularly limited. For example, viral diseases such as those caused by adenovirus, herpes virus (e.g., HSV-I, HSV-II, CMV, VZV), poxvirus (e.g., orthopoxvirus such as smallpox or vaccinia, molluscum contagiosum, etc.), picornavirus (e.g., rhinovirus, enterovirus), orthomyxovirus (e.g., influenza virus), paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)), coronavirus (e.g., SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS-CoV-2), papovavirus (e.g., papillomavirus that causes genital warts, common warts, plantar warts, etc.), hepadnavirus (e.g., hepatitis B virus), flavivirus (e.g., hepatitis C virus, dengue virus), retrovirus (e.g., lentivirus such as HIV), etc.; bacterial diseases such as those caused by bacteria of the genera Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Streptococcus pneumoniae, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, Bordetella, etc.; fungal diseases such as Chlamydia, candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, etc.; malaria, Pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, Trypanosoma infection, etc.

[0126] The structure of the antigen that can be used in the pharmaceutical composition of the present embodiment is not particularly limited as long as it is at least a part of various components constituting the pathogen. For example, live vaccines, inactivated whole particles, parts thereof, protein subunits, proteins, peptides, etc. can be mentioned. Among them, from the viewpoint of complexation with hyaluronic acid derivatives, protein subunits, proteins, or peptides are preferred.

[0127] The above influenza virus is an RNA envelope virus belonging to the family Orthomyxoviridae and having a particle size of about 100 nm in diameter. Based on the antigenicity of the internal protein, it is divided into types A, B, and C. The above influenza virus consists of a core of ribonucleic acid (RNA) associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope having a lipid bilayer structure, and an external glycoprotein. The inner layer of the viral envelope is mainly composed of matrix protein, and the outer layer is mostly composed of host-derived lipid substances. In addition, the RNA of the above influenza virus has a segmented structure. Note that the influenza pandemic worldwide is caused by type A influenza virus, and this type A influenza virus has two types of envelope glycoproteins, hemagglutinin and neuraminidase, and is classified into 16 subtypes for hemagglutinin and 9 subtypes for neuraminidase according to differences in antigenicity.

[0128] As the above antigen derived from an infectious disease, antigens derived from type A and type B influenza viruses are preferably used. Note that the subtypes of the above type A and type B influenza viruses are not particularly limited, and may be subtypes isolated so far or subtypes to be isolated in the future.

[0129] As the influenza virus-derived antigen, there is no particular limitation as long as it is at least a part of various components constituting the above influenza virus. For example, it includes whole virus particles in which purified virus particles are inactivated with an organic solvent / surfactant or other reagents, or virus subunits prepared by removing impurities from the whole virus particles and purifying hemagglutinin and / or neuraminidase. From the viewpoint of immunogenicity, hemagglutinin subunits or whole virus particles are preferred. The above whole virus particles are more preferably those inactivated with formalin or the like. In addition, it is particularly effective for hemagglutinin subunits (split) with few impurities and an adjuvant such as an immunopotentiator being essential.

[0130] The method for preparing the above influenza virus antigen is not particularly limited, and known methods can be used without limitation. For example, there is a method of infecting a virus strain isolated from an influenza-infected animal or an influenza patient into a chicken egg or the like, culturing it by a conventional method, and preparing an antigen from the purified virus stock solution. In addition, an antigen derived from a virus prepared in cultured cells by genetic engineering may also be used.

[0131] (Antigen in immune diseases) The antigen in immune diseases is not particularly limited as long as it contains an epitope of the target protein of the immune disease. The immune diseases are not particularly limited, and examples include psoriasis vulgaris, ankylosing spondylitis, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, bronchial asthma, chronic urticaria, pollinosis, atopic dermatitis, and the like. The target protein is not particularly limited, and examples include IL-17A, DPP4, S100A9, PCSK9, IL-23, IgE, TNFα, IL-12 / 23p40, IL-6, α4β7 integrin, IL-4 / 13, IL-5, BLyS, IL-13, and the like. Reference 2 (International Publication No. 2017 / 164409) describes a peptide derived from IL-17A. The peptide may have epitopes not only of the target protein but also of non-target proteins. It may be a B cell epitope or a T cell epitope. The peptide is an array having an epitope and may have a cyclic structure. The peptide may have a plurality of cyclic structures in the molecule. Furthermore, the peptide may be conjugated to a protein. A therapeutic effect can be expected by the production of antibodies against the administered protein or peptide in the body.

[0132] [Pharmaceutically active peptide or protein] A pharmaceutically active peptide or protein means one that has a positive or beneficial effect on the condition or disease state of a subject when administered in a therapeutically effective amount to the subject. Preferred pharmaceutically active peptides or proteins have a curative or symptomatic nature and are administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Pharmaceutically active peptides or proteins may have a prophylactic nature and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "pharmaceutically active peptide or protein" implies a full-length protein or polypeptide and may also refer to a pharmaceutically active fragment thereof. This term also encompasses pharmaceutically active analogs of the peptide or protein.

[0133] Examples of pharmaceutically active proteins include, but are not limited to, cytokines such as immunologically active compounds and immune system proteins (e.g., interleukins, colony stimulating factors (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, immunoglobulins, antibodies, hormones (insulin, thyroid hormones, catecholamines, gonadotropins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative enzymes, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochrome, adenylate cyclase or guanylaste cyclase, neuramidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone binding protein or growth factor binding protein, etc.), transcription factors and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin, etc.), 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.

[0134] In one embodiment, the pharmaceutically active protein is a cytokine involved in the control of lymphocyte homeostasis, preferably a cytokine involved in and inducing or enhancing one or more selected from the group consisting of T cell development, primary stimulation, amplification, differentiation and survival. In one embodiment, the cytokine is an interleukin. In one embodiment, the pharmaceutically active protein is one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15 and IL-21. In one embodiment, the pharmaceutically active peptide may have a cyclic structure. The peptide may have a plurality of cyclic structures in the molecule.

[0135] [Nucleic acid] Examples of the nucleic acid include DNA, RNA, antisense nucleic acid, decoy nucleic acid, ribozyme, small interfering RNA, nucleic acid aptamer and the like. When the antigen is a peptide or a protein, nucleic acids (such as DNA or mRNA) encoding these antigen peptides or proteins are also preferably used.

[0136] [Small molecule compound] Examples of the small molecule compound include anticancer agents (such as alkylating agents, antimetabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroidal agents, non-steroidal anti-inflammatory agents, etc.), antirheumatic agents, antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfonamides, etc.).

[0137] In addition, as the active ingredient of the drug, those with high hydrophobicity, that is, those with poor water solubility, can also be preferably used because they can sufficiently exert the interaction with the sterol group of the above-mentioned hyaluronic acid derivative. The term "poor water solubility" means that in the 17th revised Japanese Pharmacopoeia, the amount of water required to dissolve 1 g of the solute is 30 mL or more.

[0138] Examples of poorly water-soluble solid active pharmaceutical ingredients include antipyretic analgesics such as acetaminophen, ibuprofen, benzoic acid, ethenzamide, caffeine, camphor, quinine, calcium gluconate, dimethylcaprol, sulfamine, theophylline, theopromine, riboflavin, mephenesin, phenobarbital, aminophylline, thioacetazone, quercetin, rutin, salicylic acid, theophylline sodium salt, pyrapital, quinine hydrochloride, irgapyrin, dicitoxin, glyceofulvin, phenacetin, etc.; nervous system pharmaceuticals, sedative hypnotics, muscle relaxants, blood pressure hardening agents, antihistamines, etc.; antibiotics such as acetylspiramycin, ampicillin, erythromycin, xanthamycin, chloramphenicol, triacetyl oleandomycin, nystatin, colistin sulfate, etc.; steroid hormone agents such as methyltestosterone, methylandrostenediol, progesterone, estradiol benzoate, ethinylestradiol, deoxycorticosterone acetate, cortisone acetate, hydrocortisone, hydrocortisone acetate, prednisolone, etc.; non-steroidal egg yolk hormone agents such as dienoestrol, hexoestrol, diethylstilbestrol, diethylstilbestrol dibromide, chlorotrianisene, etc.; and other fat-soluble vitamins, etc., including active pharmaceutical ingredients of pharmaceuticals described in the "Japanese Pharmacopoeia", "Extra Pharmacopoeia", "USP (United States Pharmacopeia)", "NF (National Formulary)", "EP (European Pharmacopoeia)". One of these active pharmaceutical ingredients can be used, or two or more can be used in combination.

[0139] The active ingredient may be a poorly water-soluble oily or liquid substance. Examples of poorly water-soluble oily or liquid active ingredients include pharmaceuticals and active ingredients described in "Japanese Pharmacopoeia", "External Standards", "USP", "NF", "EP", such as teprenone, indomethacin farnesyl, menatetrenone, phytomenadione, vitamin A oil, phenipentol, vitamins such as vitamin D and vitamin E, higher unsaturated fatty acids such as DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), and fish oil, coenzyme Qs, and oil-soluble flavoring agents such as orange oil, lemon oil, and peppermint oil. Vitamin E has various homologs and derivatives, and is not particularly limited as long as it is liquid at normal temperature. Examples include dl-α-tocopherol, dl-α-tocopherol acetate, d-α-tocopherol, and d-α-tocopherol acetate. One of these active ingredients may be used, or two or more thereof may be used in combination.

[0140] The active ingredient may be a poorly water-soluble semi-solid substance. Examples of poorly water-soluble semi-solid active ingredients include Chinese herbs or crude drug extracts such as earthworm, licorice, cinnamon, peony, button mushroom, cantharides, sansho, ginger, aconite, mao wu, nantenjitsu, aconite root, angelica, campanula, schizonepeta, schizonepeta tenuifolia, lycoris radiata, seneca, baimo, wikyo, eucommia, ourei, gadjutsu, chamomile, gentiana, gou ou, animal bile, shajin, ginger, soudetsu, clove, aconite, byakujutsu, chikusetsu carrot, carrot, kudzu decoction, cinnamon twig decoction, xiangsu powder, bupleurum and cinnamon twig decoction, minor bupleurum decoction, minor bluegreen dragon decoction, ophiopogon decoction, pinellia and magnolia bark decoction, ephedra decoction; oyster extract, propolis and propolis extract, coenzyme Qs, etc. One of these active ingredients may be used, or two or more thereof may be used in combination.

[0141] In the pharmaceutical composition of the present embodiment, the content of the active ingredient can be 0.001% by mass or more and 10,000% by mass or less, preferably 0.1% by mass or more and 1000% by mass or less, more preferably 1.0% by mass or more and 100.0% by mass or less, still more preferably 1.5% by mass or more and 50.0% by mass or less, particularly preferably 3.0% by mass or more and 30.0% by mass or less, and most preferably 5.0% by mass or more and 20.0% by mass or less, although it depends on the structure of the active ingredient. Alternatively, in the pharmaceutical composition of the present embodiment, the content of the active ingredient is more preferably 0.1% by mass or more and 100.0% by mass or less, still more preferably 0.1% by mass or more and 50.0% by mass or less, still more preferably 0.1% by mass or more and 30.0% by mass or less, still more preferably 0.5% by mass or more and 30.0% by mass or less, still more preferably 1.0% by mass or more and 30.0% by mass or less, still more preferably 1.0% by mass or more and 25.0% by mass or less, still more preferably 1.0% by mass or more and 20.0% by mass or less, even more preferably 1.0% by mass or more and 15.0% by mass or less, and particularly preferably 1.0% by mass or more and 10.0% by mass or less, based on the mass of the hyaluronic acid derivative.

[0142] Alternatively, in the pharmaceutical composition of the present embodiment, the content of the active ingredient is preferably 0.0001 part by mass or more and 1.00 part by mass or less, more preferably 0.001 part by mass or more and 0.100 part by mass or less, and still more preferably 0.002 part by mass or more and 0.500 part by mass or less, based on 100 parts by mass of the pharmaceutical composition.

[0143] Alternatively, in the pharmaceutical composition of the present embodiment, the content of the active ingredient is preferably 0.001 part by mass or more and 1.00 part by mass or less, more preferably 0.001 part by mass or more and 0.500 part by mass or less, more preferably 0.001 part by mass or more and 0.200 part by mass or less, and still more preferably 0.005 part by mass or more and 0.100 part by mass or less, based on 100 parts by mass of the pharmaceutical composition.

[0144] By having the content of the medicinal ingredient be at least the above lower limit value, immune cells can be activated more effectively. On the other hand, by being at most the above upper limit value, the medicinal ingredient can be encapsulated in the hyaluronic acid derivative component to form a more stable structure.

[0145] <Adjuvant> When the pharmaceutical composition of the present embodiment is a vaccine composition, in addition to the above medicinal ingredient and the above hyaluronic acid derivative, an adjuvant can be further included. Thereby, immunity can be induced more effectively. Here, the induced immunity may be either humoral immunity or cellular immunity. That is, the pharmaceutical composition of the present embodiment preferably is a pharmaceutical composition that includes the above hyaluronic acid derivative, an antigen (preferably a cancer antigen or an antigen derived from an infectious disease) as the above medicinal ingredient, and further includes an adjuvant.

[0146] Generally, humoral immunity refers to the immune mechanism centered around B cells and antibodies. B cells are stimulated by cytokines produced by helper T cells (Th2 cells), causing B cells to differentiate into plasma cells, produce a large amount of antibodies, and the antibodies circulate in the body fluid and spread throughout the body. Also, a part of the stimulated B cells becomes memory B cells that store antigen information, and upon re-infection, can produce a large amount of antibodies that are more rapid than the initial response and have a higher affinity for the antigen. On the other hand, cellular immunity refers to the immune mechanism in which cells are the main effectors in the elimination of foreign substances such as the pathogen itself, virus-infected cells, and cancer cells. It is an elimination mechanism by immune cells themselves such as macrophages, cytotoxic T cells (CTL, killer T cells), and natural killer cells (NK cells).

[0147] The adjuvant is not particularly limited as long as it is usually used in vaccines. For example, aluminum salts, squalene, ligands for innate immune receptors, etc. can be mentioned.

[0148] As used herein, the "ligand" refers to a substance that specifically binds to a receptor. In particular, a substance that specifically binds to a receptor and exhibits various physiological effects can be used. Such a substance is also referred to as an "agonist".

[0149] Examples of innate immune receptors include toll-like receptor (TLR), RIG-I-like receptor (RLR), NOD-like receptor (NLR), C-type lectin receptor (CLR), and the like.

[0150] As the TLR ligand, for example, those that interact with at least one TLR selected from the group consisting of TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, and TLR-9 can be appropriately selected.

[0151] Examples of the TLR-2 ligand include Pam3CSK4 and the like.

[0152] Examples of the TLR-3 ligand include poly ICLC, polyinosinic:polycytidylic acid (poly I:C), and the like.

[0153] Examples of the TLR-4 ligand include R-type lipopolysaccharide, S-type lipopolysaccharide, paclitaxel, lipid A, monophosphoryl lipid A, and the like.

[0154] Examples of the TLR-5 ligand include flagellin and the like.

[0155] Examples of the TLR-2 and TLR-6 ligands include MALP-2 and the like.

[0156] Examples of TLR-7 and TLR-8 ligands include resiquimod (R848), imiquimod (R837), gardiquimod, loxoribine, and the like.

[0157] Examples of TLR-9 ligands include CpG oligodeoxynucleotides and the like.

[0158] Among them, as the adjuvant, an anionic compound is preferred, and CpG oligodeoxynucleotides are more preferred because they can further improve the antigen-presenting function.

[0159] Examples of CpG oligodeoxynucleotides include CpG-ODN 1826, CpG-K3, and the like.

[0160] <Other additives> The pharmaceutical composition of this embodiment can be administered alone or can be administered according to conventional means together with a pharmaceutically acceptable carrier. When used in combination with a pharmaceutically acceptable carrier, for example, the above-mentioned hyaluronic acid derivative and the above-mentioned active ingredient, and, if necessary, an adjuvant and water or other physiologically acceptable liquids (e.g., physiological saline, aqueous ethanol, phosphate-buffered saline (PBS), etc.) can be mixed, and it can also contain physiologically acceptable buffers, excipients, vehicles, preservatives, stabilizers, binders, lyophilization aids, and the like.

[0161] Examples of buffers include Tris, sodium phosphate, potassium phosphate, histidine, or citric acid, and the like.

[0162] Examples of preservatives include benzalkonium chloride, methyl paraben, propyl paraben, chlorobutanol, sorbic acid, alkyl polyaminoethyl glycine, and the like.

[0163] Examples of the stabilizer include sodium edetate hydrate, polyvinylpyrrolidone (povidone), polysorbate 80, and the like.

[0164] As the pharmaceutical composition of this embodiment, a formulated one may be used. The form of the preparation can be in a solid, semi-solid or liquid form. In the case of a solid, forms such as powder, granule, pill, pellet, tablet, capsule, etc. can be mentioned. Among them, as the solid, a lyophilized powder is preferable. In the case of a semi-solid, forms such as gel can be mentioned. In the case of a liquid, forms such as a suspension in which a powder is diluted or suspended with a buffer solution such as water or phosphate buffered saline (PBS) can be mentioned.

[0165] <Method for manufacturing a pharmaceutical composition> The pharmaceutical composition of this embodiment can be produced by appropriately mixing the above-mentioned hyaluronic acid derivative and the above-mentioned medicinal ingredient, but it is preferably produced by the method shown below.

[0166] The method for manufacturing the pharmaceutical composition of this embodiment is a method for manufacturing a pharmaceutical composition containing a hyaluronic acid derivative and a medicinal ingredient, and includes the following steps. A preparation step of dissolving the medicinal ingredient in an organic solvent or water containing an organic solvent to prepare an oil phase containing the medicinal ingredient; and, A mixing step of mixing the oil phase and the aqueous phase containing the hyaluronic acid derivative so that the mixing ratio is 20:100 to 0.01:100 by volume ratio.

[0167] Conventionally, depending on the ratio of the oil phase to the aqueous phase, the hyaluronic acid derivative has difficulty maintaining the particle size because the interaction between sterol groups is inhibited by the oil phase, resulting in the particles falling apart or some of them aggregating. Therefore, the pharmaceutical composition contained only a complex of a hyaluronic acid derivative with a relatively small size in which the particles had fallen apart or a hyaluronic acid derivative containing partially aggregated particles and a medicinal ingredient. In addition, since the pharmaceutical composition was produced using the dialysis method, it was difficult to control the weight ratio of the hyaluronic acid derivative to the medicinal ingredient, and it was left to chance. Furthermore, a concentration step was also required to achieve the target medicinal ingredient concentration.

[0168] In contrast, the method for producing the pharmaceutical composition of the present embodiment does not use the dialysis method. By mixing such that the above configuration, particularly the mixing ratio of the oil phase to the aqueous phase, is within the above numerical range, a complex with the medicinal ingredient can be formed while maintaining the particle size distribution of the hyaluronic acid derivative before the production of the pharmaceutical composition, and the weight ratio of the hyaluronic acid derivative to the medicinal ingredient can also be controlled to be constant. As a result, a medicinal ingredient-hyaluronic acid derivative complex can be obtained with a stable structure. As a result, a pharmaceutical composition excellent in deliverability to immune cells in lymph nodes and the ability to activate the immune cells can be obtained.

[0169] Next, each step of the method for producing the pharmaceutical composition of the present embodiment will be described in detail below.

[0170] [Preparation step] In the preparation step, the medicinal ingredient is dissolved in an organic solvent or water containing an organic solvent to prepare an oil phase containing the medicinal ingredient.

[0171] Since it is dissolved in an organic solvent or water containing an organic solvent, poorly water-soluble substances can preferably be used as the medicinal ingredient. Specifically, those exemplified in the above "medicinal ingredient" can be used as the medicinal ingredient.

[0172] Examples of the organic solvent for dissolving the active ingredient include known ones commonly used in the production of pharmaceutical compositions. Specifically, for example, dimethyl sulfoxide (DMSO), methanol, ethanol, tetrahydrofuran, acetone, acetonitrile, ethyl acetate, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, methyl ethyl ketone, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl isobutyl ketone, dimethylformamide, etc. can be mentioned.

[0173] [Mixing step] In the mixing step, the oil phase obtained in the preparation step and the aqueous phase containing the hyaluronic acid derivative are mixed so that the mixing ratio is 20:100 to 0.01:100 by volume ratio.

[0174] The hyaluronic acid derivative is dissolved or dispersed in water or the like in advance so as to have a desired concentration. At this time, the pH of the aqueous phase containing the hyaluronic acid derivative is preferably 6.00 or more, and preferably 6.00 or more and 11.00 or less. When the pH is at or above the lower limit value, a complex with the active ingredient can be formed while more effectively suppressing the aggregation of the hyaluronic acid derivative, and thus, the active ingredient-hyaluronic acid derivative complex can be obtained in a more stable structure. When the pH is at or below the upper limit value, the drug can be complexed while maintaining the dispersion stability of the hyaluronic acid derivative. Also, since the charge of the drug tends to be negative, there is also an aspect that the hydrophobic part of the hyaluronic acid derivative and the drug interact more easily. On the other hand, when it is at or below the upper limit value, the main chain decomposition of the hyaluronic acid derivative can be more suppressed.

[0175] In the mixing step, due to the hydrophobic interaction between the steric group of the hyaluronic acid derivative and the active ingredient, a structure is formed in which the steric group and the active ingredient are present inside, while the hydrophilic site derived from hyaluronic acid is present at the outer edge, and an active ingredient-hyaluronic acid derivative complex is formed. Depending on the structure of the active ingredient, the complex can be formed not only by the above hydrophobic interaction but also by interactions such as electrostatic interaction and hydrogen bond.

[0176] In the mixing step, the mixing ratio of the oil phase to the aqueous phase is 20:100 to 0.01:100 by volume ratio, preferably 10:100 to 0.05:100, more preferably 5:100 to 0.1:100, and even more preferably 2.5:100 to 0.5:100. When the mixing ratio of the oil phase to the aqueous phase is within the above range, a sufficient amount of the active ingredient can be encapsulated in the hyaluronic acid derivative to form an active ingredient-hyaluronic acid derivative complex, and the particles of the hyaluronic acid derivative can be effectively prevented from aggregating due to the inhibition of the hydrophobic interaction between the steric groups by the oil phase. However, it is not limited to the hydrophobic interaction only, and the disintegration of interactions that can contribute to the structural stability such as electrostatic interaction and hydrogen bond can also be effectively suppressed.

[0177] In the mixing step, the concentration of the active ingredient dissolved in the oil phase is not particularly limited, but it is preferably 0.1 part by mass or more and 50 parts by mass or less, more preferably 0.2 part by mass or more and 25 parts by mass or less, and even more preferably 0.5 part by mass or more and 10 parts by mass or less with respect to 100 parts by mass of the oil phase.

[0178] In the mixing step, the concentration of the hyaluronic acid derivative dissolved in the aqueous phase is not particularly limited, but it is preferably 0.01 part by mass or more and 10 parts by mass or less, more preferably 0.1 part by mass or more and 10 parts by mass or less, and even more preferably 0.5 part by mass or more and 5 parts by mass or less with respect to 100 parts by mass of the aqueous phase.

[0179] In the mixing step, from the viewpoint of slightly exposing the hydrophobic part of the active ingredient and facilitating the interaction with the hyaluronic acid derivative, the temperature is preferably 20°C or higher and 65°C or lower, more preferably 23°C or higher and 55°C or lower, and even more preferably 25°C or higher and 40°C or lower. Alternatively, the optimal temperature can be appropriately set according to the structure and properties of the active ingredient.

[0180] In the mixing process, the method is not particularly limited. For example, an oil phase containing a medicinal ingredient can be added to the aqueous phase containing the hyaluronic acid derivative in any ratio, or an aqueous phase containing the hyaluronic acid derivative can be added to the oil phase containing the medicinal ingredient in any ratio. By passing through a mixing process where the final mixing ratio of the oil phase to the aqueous phase is 20:100 to 0.01:100 by volume, the particle size of the hyaluronic acid derivative can be maintained.

[0181] The mixing process is not limited to the batch method (batch process), and the oil phase and the aqueous phase may be mixed by a flow method (continuous process). By setting the mixing ratio of the oil phase to the aqueous phase at that time to 20:100 to 0.01:100 by volume, the particle size of the hyaluronic acid derivative can be maintained.

[0182] For the material and shape of the reactor in the flow method, materials and shapes applicable to the manufacturing method of the present invention may be selected and are not particularly limited. For example, the inner diameters of the pipes through which the oil phase and the aqueous phase pass may be different diameters.

[0183] For the material and shape of the tube in the flow method, materials and shapes applicable to the manufacturing method of the pharmaceutical composition of the present embodiment may be selected and are not particularly limited. For example, tubes made of Teflon (registered trademark), stainless steel tubes, glass tubes, plastic tubes, etc. may be mentioned.

[0184] In the mixing process, the time is not particularly limited. For example, it can be 30 minutes or more and 90 hours or less, and it can be 1 hour or more and 80 hours or less.

[0185] In the mixing process, after the medicinal ingredient is complexed with the hyaluronic acid derivative, there may be a step of removing the organic solvent in which the medicinal ingredient is dissolved by tangential flow filtration (TFF), or by a plurality of TFFs in centrifugal filtration, ultrafiltration / diafiltration (UFDF) membranes, etc.

[0186] [Sterilization process] After the mixing step, a sterilization step of sterilizing the obtained solution containing the active ingredient - hyaluronic acid derivative complex to obtain a sterilized preparation may be performed. Examples of the sterilization method for the preparation include filtration sterilization using a sterilizing filter, gas sterilization, γ-ray sterilization, or electron beam sterilization. Filtration sterilization using a sterilizing filter is most preferred.

[0187] [Drying step] After the mixing step, a drying step of drying the obtained solution containing the active ingredient - hyaluronic acid derivative complex to obtain a dried product may be performed. Examples of the drying method include the same methods as those exemplified in the above "Method for Producing Hyaluronic Acid Derivative".

[0188] [Administration method] The subjects to which the pharmaceutical composition of this embodiment is administered include animals classified as mammals including humans (such as monkeys, marmosets, mice, rats, cows, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.).

[0189] The administration route can be carried out by methods known to those skilled in the art, for example, intrathecal injection, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, transpulmonary, intramuscular, transdermal, or oral. Among them, when the pharmaceutical composition of this embodiment is a vaccine composition, subcutaneous injection or intramuscular injection is preferred.

[0190] In the pharmaceutical composition of this embodiment, when administered parenterally, the dosage can be appropriately selected considering the type of the administration subject (including age, gender, etc.). Generally, for example, in humans (assuming a body weight of 60 kg), the amount of the active ingredient (preferably the antigen) per administration can be 0.01 μg or more and 5 mg or less, 0.1 μg or more and 500 μg or less, or 1 μg or more and 100 μg or less.

[0191] The number of administrations may be a single administration of the above-described dosage, or the above-described dosage may be administered multiple times, such as once a week, two weeks, three weeks, four weeks, one month, two months, three months, or six months, or more than two times. Alternatively, the drug may be administered at two or more sites in a single administration.

[0192] ≪Other Embodiments≫ In one embodiment, the present invention provides a method for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases, which comprises administering an effective amount of the above pharmaceutical composition to a patient or a diseased animal. Examples of the infectious diseases include those exemplified in the "antigen derived from infectious diseases" of the above "antigen". Here, the "effective amount" includes an amount effective for prevention or treatment, that is, an amount suitable for preventing the onset or treating the above diseases.

[0193] In one embodiment, the present invention provides a composition for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases, which comprises the above active ingredient - hyaluronic acid derivative complex.

[0194] In one embodiment, the present invention provides the use of the above active ingredient - hyaluronic acid derivative complex for producing a pharmaceutical composition. The pharmaceutical composition is preferably an infectious disease vaccine, a cancer vaccine, or a pharmaceutical composition for immune diseases.

[0195] In one embodiment, the present invention further provides a pharmaceutical composition characterized by comprising a composition containing a hyaluronic acid derivative and an antigen, and lymphocytes expressing an immune receptor for the antigen. By combining the composition containing a hyaluronic acid derivative and an antigen with antigen-specific lymphocytes, an antitumor effect can be exerted more strongly, more efficiently, more sustainably, and / or over a wider range.

[0196] Provided is a pharmaceutical composition for use in combination administration with a composition containing a hyaluronic acid derivative and an antigen, which contains lymphocytes expressing an immunoreceptor for the antigen.

[0197] The pharmaceutical composition, which is characterized by comprising a combination of a composition containing a hyaluronic acid derivative and an antigen and antigen-specific lymphocytes, can also be in the form of a kit. The pharmaceutical composition can be, for example, a cancer treatment kit containing a composition containing a hyaluronic acid derivative and an antigen and antigen-specific lymphocytes.

[0198] In a preferred embodiment of the present invention, the composition containing a hyaluronic acid derivative and an antigen is preferably administered before the administration of antigen-specific lymphocytes. In a preferred embodiment of the present invention, when the administration of the antigen-specific lymphocytes is performed once after the composition containing a hyaluronic acid derivative and an antigen has been administered once, it is preferred that the composition containing a hyaluronic acid derivative and an antigen be administered at least once after 1 unit or 2 units of administration.

[0199] In a preferred embodiment of the present invention, the interval between the first administration and the second administration of the composition containing a hyaluronic acid derivative and an antigen can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more. Also, the interval can be, for example, 28 days or less, 24 days or less, 21 days or less, 17 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, or 2 days or less.

[0200] In one embodiment, the present invention provides that the composition containing a hyaluronic acid derivative and an antigen may be administered in combination with one or more antibodies used for cancer treatment. The antibody is, for example, an antibody that inhibits immunosuppressive signals caused by tumors, or one or more antibodies that activate co-stimulatory signals of immune cells, preferably an antibody that inhibits immunosuppressive signals caused by tumors or an antibody that activates co-stimulatory signals of immune cells. Specifically, it is one or more antibodies selected from the group consisting of anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-OX40, and anti-4-1BB antibody.

[0201] When administered in combination, the dosage of the composition containing the hyaluronic acid derivative and the antigen is, for example, 0.01 to 100 mg per administration, preferably 0.1 to 50 mg per administration, more preferably 0.1 to 20 mg per administration. The dosage of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, more preferably 1 to 40 mg / kg body weight.

[0202] The antibody may be administered at the same timing as the composition containing the hyaluronic acid derivative and the antigen (including the case where the antibody is included in the formulation), or at different timings. When administered at different timings, it is preferable to administer the other one, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours, after administering one of them.

[0203] The composition containing the hyaluronic acid derivative and the antigen may be administered in combination with both the above adjuvant and the antibody. In that case, the dosage of the composition containing the hyaluronic acid derivative and the antigen is, for example, 0.01 to 100 mg / time, preferably 0.1 to 50 mg / time, more preferably, for example, 0.1 to 20 mg / time; the dosage of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight; and the dosage of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, more preferably 1 to 40 mg / kg body weight. The adjuvant and the antibody may be administered at the same timing as the composition containing the hyaluronic acid derivative and the antigen (including the case where the adjuvant is included in the formulation), or at different timings. When administered at different timings, it is preferable to administer all of the vaccine formulation, the adjuvant, and the antibody, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours.

[0204] In one embodiment, the present invention provides a composition for improving, promoting, or enhancing the uptake of a medicinal ingredient by immune cells, the composition comprising the above medicinal ingredient-hyaluronic acid derivative complex.

[0205] In one embodiment, the present invention provides a composition for improving, promoting, or enhancing the activity of immune cells, the composition comprising the above medicinal ingredient-hyaluronic acid derivative complex.

[0206] In one embodiment, the present invention provides a composition for improving, promoting, maintaining, or enhancing the expression of co-stimulatory molecules (particularly CD80 and CD86) of DC (particularly cDC1), the composition comprising the above medicinal ingredient-hyaluronic acid derivative complex.

[0207] In one embodiment, the present invention provides a method for improving, promoting, or enhancing the uptake of a medicinal ingredient by immune cells in vivo or in vitro, the method comprising administering a composition comprising the above medicinal ingredient-hyaluronic acid derivative complex.

[0208] In one embodiment, the present invention provides a method for improving, promoting, or enhancing the activity of immune cells in vivo or in vitro, which comprises administering a composition containing the above-mentioned active ingredient - hyaluronic acid derivative complex.

[0209] In one embodiment, the present invention provides a method for improving, promoting, maintaining, or enhancing the expression of co-stimulatory molecules (particularly CD80 and CD86) of DC (particularly cDC1) in vivo or in vitro, which comprises administering a composition containing the above-mentioned active ingredient - hyaluronic acid derivative complex.

Example

[0210] 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.

[0211] <Synthesis of hyaluronic acid derivative> [Synthesis Example 1-1] The hyaluronic acid derivative was prepared according to the following steps 1 to 3.

[0212] 1. Step 1 (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following Step 1-1, followed by Step 1-2.

[0213] (1) Step 1-1 To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice-cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and after stirring for 30 minutes under ice-cooling as it was, the temperature was raised 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 the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:4), and the fractions of the target product were combined and the solvent was distilled off under reduced pressure.

[0214] (2) Step 1-2 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 at room temperature (about 25 °C) overnight. The resulting precipitate was collected 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-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0215] 2. Step 2 (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 and then Step 2-2.

[0216] (1) Step 2-1 DOWEX (registered trademark) 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed about 3 times with ultrapure water by decantation. An aqueous solution of 40 wt% tetrabutylammonium hydroxide (TBA-OH) (manufactured by Aldrich) was added in an approximately 1.5-fold molar equivalent to the cation exchange capacity of the resin, and the mixture was stirred at room temperature (about 25 °C) for 30 minutes. After removing the excess TBA-OH solution by decantation, the resin was further washed with excess ultrapure water to obtain a TBA-chlorinated cation exchange resin.

[0217] (2) Step 2-2 Sodium hyaluronate salt (HA-Na) with a weight-average molecular weight (absolute molecular weight) of 10,000 (10 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. A suspension of the cation exchange resin chlorinated with TBA in “(1) Step 2-1” was added in a 5-fold molar equivalent based on the ion exchange capacity of the resin with respect to the number of moles of HA units (unit molecular weight 401.3). After stirring at room temperature (about 25 °C) for 15 minutes, filtration was performed using a 0.45 μm filter, and the filtrate was lyophilized to obtain hyaluronic acid TBA salt (HA-TBA) as a white solid.

[0218] 3. Step 3 An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in “2. (2) Step 2-2” was prepared. Then, choline hydrochloride was added so that the addition amount with respect to the disaccharide repeating unit (HA unit) present in the HA-TBA synthesized in “1. Step 1” was 44 / 100 in molar ratio. Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added so that the addition amount with respect to the HA unit was 48 / 100 in molar ratio, and the mixture was stirred overnight at room temperature (about 25 °C). The reaction solution was dialyzed using a Spectra / Por 7 dialysis membrane (manufactured by Spectrum Laboratories, fractionation molecular weight (MWCO): 3,500) in the order of 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water. The obtained dialysate was lyophilized to obtain the target product (HA-C 6 -Chol) as a white solid.

[0219] The cholesterol introduction rate in the obtained white solid was calculated by the following method. As the measurement solvent, a 0.02N DCl DMSO-d 6 / D 2 O mixture (2N DCl D 2 O:DMSO-d 6 =1:99) was used, and measurement was performed at 85 °C using a JNM-ECS400 NMR apparatus (manufactured by JEOL Ltd.) to obtain the 1 1H-NMR spectrum of the white solid. The obtained 1In the 1H-NMR spectrum, the 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), and the peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%.

[0220] The freeze-dried hyaluronic acid derivative (10k HA-C 6 -Chol-44%) was dissolved in water for injection at 1 mg / mL, and measurement by gel permeation chromatography was performed under the conditions shown below. Figure 3A shows the obtained chromatogram.

[0221] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Temperature: 30 °C

[0222] [Synthesis Example 1-2] A hyaluronic acid derivative was synthesized in the same procedure as in Synthesis Example 1-1. Of the product 1 In the 1H-NMR spectrum, the 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), and the peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. Measurement by gel permeation chromatography was performed under the conditions shown in Synthesis Example 1-1 above, and the obtained chromatogram is shown in Figure 3B.

[0223] [Synthesis Example 1-3] A hyaluronic acid derivative was synthesized in the same procedure as in Synthesis Example 1-1. Of the product 1 In the 1H-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), and a peak derived from the methyl group in the cholesteryl group (CH 3 , 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. Measurement was performed by gel permeation chromatography under the conditions shown in Synthesis Example 1-1 above, and the obtained chromatogram is shown in FIG. 3C.

[0224] [Example 1-1] The fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was carried out as follows. The freeze-dried hyaluronic acid derivative (10k HA-C 6 -Chol-44%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Float-A-Lyzer G2, MWCO: 300,000, Kadota Trading Co., Ltd.), and dialyzed 9 times against 10 mM phosphate buffer pH 7.4. The obtained hyaluronic acid derivative in the dialyzed internal solution was measured by gel permeation chromatography. FIG. 4A is the obtained chromatogram. FIGS. 21 and 22 are diagrams showing the chromatograms of the hyaluronic acid derivative obtained in Example 1-1 and polyacrylic acid, which is a standard substance, superimposed to explain the calculation methods of the area ratio A1 / A2 and the distance ratio Da / Db described later. The filtrate was concentrated with an ultrafiltration concentrator (Vivaspin 20, MWCO: 10,000, Sartorius) to the desired concentration.

[0225] [Example 1-2] The fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-2 was carried out in the same procedure as in Example 1-1. The obtained hyaluronic acid derivative in the dialyzed internal solution was measured by gel permeation chromatography. FIG. 4B is the obtained chromatogram.

[0226] [Example 1-3] In the same procedure as in Example 1-1, fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-3 was performed. The hyaluronic acid derivative in the obtained dialysis internal solution was measured by gel permeation chromatography. Figure 4C shows the obtained chromatogram.

[0227] [Comparative Example 1-1] Fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was performed as follows. The freeze-dried hyaluronic acid derivative (10k HA-C 6 -Chol-44%) was dissolved in water for injection at 10 mg / mL and diluted with 100 mM phosphate buffer to 10 mM phosphate buffer (pH 7.4). Then, it was filtered through an ultrafiltration membrane (Vivaspin (registered trademark) 20, MWCO: 300,000, Sartorius), and the hyaluronic acid derivative in the obtained filtrate was measured by gel permeation chromatography. Figure 5A shows the obtained chromatogram. The filtrate was concentrated with an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius) to the desired concentration.

[0228] [Comparative Example 1-2] In the same procedure as in Comparative Example 1-1, fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-2 was performed. The hyaluronic acid derivative in the obtained filtrate was concentrated with an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius) to the desired concentration. Then, it was diluted with water for injection to 1 mg / mL and measured by gel permeation chromatography. Figure 5B shows the obtained chromatogram.

[0229] [Comparative Example 1-3] In the same procedure as in Comparative Example 1-2, fractionation of the hyaluronic acid derivative obtained in Synthesis Example 1-3 was performed. The hyaluronic acid derivative in the obtained filtrate was concentrated with an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius) to the desired concentration. Then, it was diluted with water for injection to 1 mg / mL and measured by gel permeation chromatography. Figure 5C shows the obtained chromatogram.

[0230] [Comparative Examples 1-4] The freeze-dried product of cholesteryl-modified pullulan (CHP: manufactured by NOF Corporation, product number CHP-80T) was dissolved in water for injection to a concentration of 1 mg / mL, and measurement was performed by gel permeation chromatography. Fig. 5D shows the obtained chromatogram.

[0231] [Evaluation of Particle Size Distribution of Hyaluronic Acid Derivative: Area Ratio A1 / A2 and Distance Ratio Da / Db] From the gel permeation chromatograms of the hyaluronic acid derivative obtained in Example 1-1 and polyacrylic acid as a standard substance, the area ratio A1 / A2 and the distance ratio Da / Db were calculated. As the standard substance polyacrylic acid (50 kDa), Polyacrylic acid sodium (ORDER No. PSS-Paa50k) manufactured by Polymer Standards Service-USA was used. As the standard substance polyacrylic acid (150 kDa), Polyacrylic acid sodium (ORDER No. PSS-Paa150k) manufactured by Polymer Standards Service-USA was used. The powder of the standard substance polyacrylic acid was dissolved in water for injection to a concentration of 2 mg / mL. All of the polyacrylic acid as the standard substance described hereinafter was of the type manufactured by Polymer Standards Service-USA (sodium polyacrylate).

[0232] Next, by the method shown below, areas A1 and A2 were calculated from the gel permeation chromatogram shown in Fig. 21, and the area ratio A1 / A2 was calculated.

[0233] (i) First, the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid as the standard substance to the baseline B and the chromatogram of the hyaluronic acid derivative was defined as Ub, and the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the baseline was defined as Bb. Here, the elution time at the refractive index intensity maximum point Kb was 7.41 minutes. (ii) Next, in the chromatogram of the hyaluronic acid derivative, the area value enclosed by the curve from Ub to the end point, the perpendicular line drawn from the maximum refractive index intensity point Kb to the baseline B, and the baseline is defined as A2, and the area value enclosed by the curve from the starting point to Ub, the perpendicular line drawn from the maximum refractive index intensity point Kb to the baseline B, and the baseline in the chromatogram of the hyaluronic acid derivative is defined as A1.

[0234] Next, the distances Da and Db were calculated from the gel permeation chromatogram shown in Fig. 22 by the method shown below, and the ratio of the distances Da / Db was calculated.

[0235] (i) First, a perpendicular line was drawn from the maximum refractive index intensity point Ka on the chromatogram of 150 kDa polyacrylic acid, which is a standard substance, to the baseline B, the intersection point with the baseline was designated as Ba, and the length between the maximum refractive index intensity point Ka and Ba was designated as La. Here, the elution time at the maximum refractive index intensity point Ka was 6.46 minutes. (ii) Next, among the two points on the chromatogram where the refractive index intensity becomes La / 20, the point with the earlier elution time was designated as point R1, and the point with the later elution time was designated as point S1. Here, the elution time at point R1 was 5.74 minutes, and the elution time at point S1 was 7.68 minutes. (iii) Next, the intersection point of the perpendicular line drawn from the maximum refractive index intensity point Kb on the chromatogram of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B and the baseline was designated as Bb. Here, the elution time at the maximum refractive index intensity point Kb is as described above. (iv) Next, the intersection point of the straight line D1 connecting the point R1 and the point S1 and the perpendicular line drawn from the maximum refractive index intensity point Ka to the baseline B was designated as Ta, and the intersection point of the perpendicular line drawn from the maximum refractive index intensity point Kb to the baseline B and the straight line D1 was designated as Tb. (v) Finally, the distance between the point R1 and the Ta was designated as Da, and the distance between the Ta and the Tb was designated as Db.

[0236] In the same manner as the hyaluronic acid derivative obtained in Example 1-1, the area ratio A1 / A2 and the distance ratio Da / Db were calculated from the gel permeation chromatograms of each hyaluronic acid derivative obtained in Synthesis Examples 1-1 to 1-3, Examples 1-2 to 1-3, and Comparative Examples 1-1 to 1-4 and the standard substance polyacrylic acid.

[0237] The calculated area ratio A1 / A2 and distance ratio Da / Db are shown in Table 1 below. In Table 1, the distance ratio Da / Db of cholesteryl-modified pullulan (CHP) in Comparative Example 3 had a small RI value and could not be calculated.

[0238]

Table 1

[0239] As shown in Table 1, hyaluronic acid derivatives of Examples 1-1 to 1-3 with a high area ratio A1 / A2 were obtained compared to the hyaluronic acid derivatives of Synthesis Examples 1-1 to 1-3 before purification.

[0240] <Manufacture of Pharmaceutical Composition> [Example 2-1] The hyaluronic acid derivative obtained in Example 1-1 was diluted with 10 mM phosphate buffer pH 7.4 to a concentration of 5 mg / mL. On the other hand, in another container, a fluorescently labeled peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL. As the fluorescently labeled peptide, a fluorescently labeled peptide having the following amino acid sequence with fluorescein bound to the N-terminus manufactured by Biolika was used.

[0241] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0242] At room temperature (about 25 °C), the above aqueous solution of hyaluronic acid derivative and the DMSO solution of fluorescently labeled peptide were mixed at a volume ratio of 100:1 and stirred at room temperature (about 25 °C) for 24 hours to obtain a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex.

[0243] Thereafter, solid purified sucrose (manufactured by FUJIFILM Wako Pure Chemical Corporation, for exclusive manufacturing use, product number 198-18385) was added to a concentration of 10% by mass of sucrose with respect to the total mass of the composition containing the fluorescently labeled peptide - hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25°C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the concentration of the peptide was about 0.3 mg / mL, and sterilized by filtration using a 0.2 μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mm Φ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing the fluorescently labeled peptide - hyaluronic acid derivative complex. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0317 parts by mass.

[0244] When measurement was carried out by gel permeation chromatography after diluting the hyaluronic acid derivative to a concentration of 1 mg / mL by the method described in Synthesis Example 1-1, no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after peptide encapsulation (Figure 6A). At this time, the peptide concentration was 0.1 mg / mL, and it was also confirmed that 100% of the charged peptide was encapsulated.

[0245] [Example 2-2] Using the hyaluronic acid derivative obtained in Example 1-2, a pharmaceutical composition containing the fluorescently labeled peptide - hyaluronic acid derivative complex was obtained by the same procedure as in Example 2-1. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0478 parts by mass. When measurement was carried out by gel permeation chromatography by the method described in Synthesis Example 1-1, no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after peptide encapsulation (Figure 6B).

[0246] [Example 2-3] Using the hyaluronic acid derivatives obtained in Examples 1-3, a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex was obtained in the same procedure as in Example 2-1. The content of the fluorescently labeled peptide relative to 100 parts by mass of the pharmaceutical composition was 0.0303 part by mass. When measurement by gel permeation chromatography was carried out by the method described in Synthesis Example 1-1, no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after peptide encapsulation (Figure 6C).

[0247] [Comparative Example 2-1] The hyaluronic acid derivative obtained in Comparative Example 1-1 was diluted with 10 mM phosphate buffer pH 7.4 to a concentration of 5 mg / mL. On the other hand, in a separate container, it was dissolved in DMSO so that the concentration of the fluorescently labeled peptide became 50 mg / mL.

[0248] At room temperature (about 25 °C), the above aqueous hyaluronic acid derivative solution and the dimethyl sulfoxide solution of the fluorescently labeled peptide were mixed at a volume ratio of 100:1 and stirred at room temperature (about 25 °C) for 24 hours to obtain a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex.

[0249] Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198-18385) was added so as to be 10% by mass of sucrose with respect to the total mass of the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25 °C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the concentration of the peptide became 0.3 mg / mL, and sterilization filtration was carried out with a 0.2 μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mmΦ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide relative to 100 parts by mass of the pharmaceutical composition was 0.0308 part by mass.

[0250] [Comparative Example 2-2] Using the hyaluronic acid derivatives obtained in Comparative Examples 1-2, a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex was obtained in the same procedure as in Comparative Example 2-1. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0332 part by mass.

[0251] [Comparative Example 2-3] Using the hyaluronic acid derivative obtained in Comparative Example 1-3, a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex was obtained in the same procedure as in Comparative Example 2-1. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0348 part by mass.

[0252] [Comparative Example 3] The freeze-dried product of CHP (manufactured by NOF Corporation, product number CHP-80T) was dissolved in 6M urea-containing phosphate buffered saline pH 7.4 to a concentration of 10 mg / mL. On the other hand, in another container, the fluorescently labeled peptide was dissolved in dimethyl sulfoxide so that the concentration became 50 mg / mL.

[0253] At room temperature (about 25 °C), the above 6M urea-containing CHP aqueous solution and the dimethyl sulfoxide solution of the fluorescently labeled peptide were mixed at a volume ratio of 100:1 and stirred at room temperature (about 25 °C) for 24 hours to obtain a composition containing a fluorescently labeled peptide-CHP complex.

[0254] Thereafter, it was transferred to a dialysis cassette (manufactured by Thermo, Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL, No. 87724) and dialyzed against 0.6M urea-containing phosphate buffered saline pH 7.4. Subsequently, dialysis was carried out in the order of 0.06M urea-containing phosphate buffered saline pH 7.4 and phosphate buffered saline pH 7.4. Further, it was concentrated with an ultrafiltration concentrator (Vivaspin20, MWCO: 10,000, Sartorius) to the desired concentration.

[0255] Finally, it was sterilized and filtered with a 0.2-μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mm Φ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-CHP complex. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0420 part by mass.

[0256] [Comparative Example 4-1] The lyophilized product of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added so as to have a concentration of 5 mg / mL, and it was stirred overnight to be sufficiently dissolved. On the other hand, in another container, it was dissolved in DMSO so that the concentration of the fluorescently labeled peptide became 50 mg / mL.

[0257] At room temperature (about 25°C), the above aqueous solution of the hyaluronic acid derivative and the dimethyl sulfoxide solution of the fluorescently labeled peptide were mixed at a volume ratio of 100:1 and stirred at room temperature (about 25°C) for 24 hours to obtain a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex.

[0258] Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for production use only, product number 198-18385) was added so as to be 10% by mass of sucrose with respect to the total mass of the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose, and it was stirred at room temperature (about 25°C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 25 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the concentration of the peptide became 0.3 mg / mL, and it was sterilized and filtered with a 0.2-μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mm Φ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0316 part by mass.

[0259] [Comparative Example 4-2] Using the lyophilized product of the hyaluronic acid derivative obtained in Synthesis Example 1-2, a composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex was obtained in the same procedure as in Comparative Example 4-1.

[0260] Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for exclusive manufacturing use, product number 198 - 18385) was added so as to be 10% by mass sucrose with respect to the total mass of the composition containing the fluorescently labeled peptide - hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25°C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 30 mM phosphate buffer pH 7.4 containing 10% by mass sucrose so that the concentration of the peptide was 0.33 mg / mL, and sterilization - filtered with a 0.2 μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mm Φ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing the fluorescently labeled peptide - hyaluronic acid derivative complex. The content of the fluorescently labeled peptide with respect to 100 parts by mass of the pharmaceutical composition was 0.0358 part by mass.

[0261] [Confirmation of Fluorescently Labeled Peptide Concentration] By high - performance liquid chromatography measurement under the following conditions, in the pharmaceutical compositions containing the fluorescently labeled peptide - hyaluronic acid derivative complexes of Examples 2 - 1 to 2 - 3 and Comparative Examples 2 - 1 to 2 - 3, 3, 4 - 1, and 4 - 2, it was confirmed that the fluorescently labeled peptide was complexed with the hyaluronic acid derivative and solubilized, and the concentration of the fluorescently labeled peptide in each pharmaceutical composition was provided.

[0262] (Measurement Conditions) HPLC device: JASCO HPLC - EXTREMA Column: PLRP - S 1000A 8μm, length 50 mm × inner diameter 4.6 mm (manufactured by Agilent Technologies, product number: PL1512 - 1802) Column temperature: 40°C Mobile phase: (A) 0.1 v / v% trifluoroacetic acid / acetonitrile (B) 0.1 v / v% trifluoroacetic acid / water Flow rate: 2 mL / min Injection volume: 30 μL Detector: UV (215 nm) Gradient program: Shown in Table 2 below. In Table 2, “%” means “v / v%”.

[0263]

Table 2

[0264] [Test Example 1-1] (Analysis of Peptide Uptake and Activation in Myeloid Cells within Lymph Nodes) 1. Materials Adjuvant: CpG Oligo DNA 1668 (purchased from Ajinomoto Bio-Pharma Services Gene Design) Administered compositions: Pharmaceutical compositions of Example 2-1, Comparative Example 2-1, Comparative Example 3, and Comparative Example 4-1.

[0265] The fluorescently labeled antibodies used for evaluation are as follows. All were purchased from Biolegend. Brilliant Violet 421-labeled anti-mouse F4 / 80 antibody (clone BM8); Brilliant Violet 510-labeled anti-mouse CD11c antibody (clone N418); APC-Cy7-labeled anti-mouse XCR1 antibody (clone ZET); APC-labeled anti-mouse CD80 antibody (clone 16-10A1); APC-labeled anti-mouse CD86 antibody (clone GL-1); Purified CD16 / 32 antibody (clone 93).

[0266] Note that the combinations of antibodies used in the FACS analysis described below are as follows.

[0267]

Table 3

[0268] 2. Analysis Method The pharmaceutical compositions of Example 2-1, Comparative Example 2-1 and Comparative Example 4-1 were diluted with 10% by mass sucrose and 10 mM phosphate buffer pH 7.4 so that the concentration of the fluorescent (fluorescein) - labeled peptide was about 0.3 mg / mL. Also, the pharmaceutical composition of Comparative Example 3 was diluted with phosphate - buffered saline pH 7.4. Then, the diluted solutions of each pharmaceutical composition were subcutaneously administered to the right posterior back of BALB / c mice at 60 μg each as peptides. At the same time, as an adjuvant, 50 μg of CpG oligo DNA 1668 (Ajinomoto BioPharma Services Gene Design) dissolved in PBS was administered.

[0269] Twenty hours after administration, the draining lymph nodes (right inguinal lymph nodes) of the administration site were collected. After grinding the lymph nodes using a slide glass, the cells were suspended in RPMI 1640 medium. At this time, the cells from two animals in one group were pooled. After centrifugation (400×g, 5 minutes, 4°C), the supernatant was removed, and the cells were washed twice with RPMI 1640 medium and then suspended in RPMI 1640 medium containing 10 v / v% FBS. The cell number was measured using a cell counter, and the cell concentration was adjusted to 1.2×10 7 cells / mL.

[0270] In a 96 - well V - bottom microplate (Nunc, Thermo Fisher Scientific), 6×10 5To make the number of cells reach a certain value, 50 μL of the cell suspension was added. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4 °C). After removing the supernatant, the cells were washed twice with 200 μL of staining buffer (PBS containing 0.5 v / v% FBS) and then resuspended. After centrifuging to remove the supernatant, 50 μL of a solution containing anti-mouse CD16 / CD32 antibody diluted 50-fold was added, and the mixture was left standing in the dark at 4 °C for 10 minutes. 150 μL of staining buffer was added, and after centrifuging to remove the supernatant, the cells were further washed twice with 200 μL of staining buffer. According to the recommended usage concentrations of each antibody, 50 μL of a solution containing the first, second, and third antibodies was added in the combination shown in Table 3 above, and after mixing, the mixture was left standing in the dark at 4 °C for 15 minutes. 150 μL of staining buffer was added, and after centrifuging to remove the supernatant, the cells were further washed twice with 200 μL of staining buffer. After centrifuging to remove the supernatant, the cells were resuspended in 200 μL of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a flow cytometer, FACS Canto II (BD Biosciences), and the attached analysis software (FACSDiva).

[0271] Dendritic cells (DC) were detected as a population positive for CD11c, macrophages (Mph) were detected as a population positive for F4 / 80, and conventional type 1 DC (cDC1) were detected as a population positive for both CD11c and XCR1. For each cell type, FITC (fluorescein)-positive cells were detected as peptide-uptaking cells. The expression of co-stimulatory molecules CD80 and CD86 was analyzed for FITC-positive cells in each cell type. The FACS analysis method is shown in FIGS. 7A to 7F. Also, the analysis results are shown in Tables 4-1 to 4-3. For example, in Table 4-1, "peptide + CD80 + DC" means "peptide-uptaking dendritic cells (DC) expressing CD80", "peptide + CD86 + DC" means "peptide-uptaking dendritic cells (DC) expressing CD86", and "peptide + DC" means "peptide-uptaking dendritic cells (DC)".

[0272]

Table 4-1

[0273]

Table 4-2

[0274]

Table 4-3

[0275] As shown in Tables 4-1 to 4-3, in the mouse group administered with the pharmaceutical composition of Example 2-1, higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) was confirmed than in the mouse groups administered with the pharmaceutical compositions of Comparative Example 2-1 and Comparative Example 4-1 (pre-fractionated hyaluronic acid derivative). Also, in DCs (especially cDC1), higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) was confirmed in the mouse group administered with the pharmaceutical composition of Example 2-1 than in the mouse group administered with the pharmaceutical composition of Comparative Example 3, which is a derivative of another polysaccharide (pullulan).

[0276] [Test Example 1-2] Using the pharmaceutical compositions of Example 2-2, Comparative Example 2-2, and Comparative Example 4-2, except that they were diluted with 10% by mass sucrose 10 mM phosphate buffer pH 7.4 so that the concentration of the fluorescent (fluorescein) - labeled peptide was about 0.2 mg / mL and subcutaneously administered at 40 μg each as peptides, the same method as in Test Example 1-1 was carried out. The analysis results are shown in Tables 5-1 to 5-3.

[0277]

Table 5-1

[0278]

Table 5-2

[0279]

Table 5-3

[0280] Similar to Test Example 1-1, in the group of mice administered with the pharmaceutical composition of Example 2-2, higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) was confirmed than in the groups of mice administered with the pharmaceutical compositions of Comparative Example 2-2 and Comparative Example 4-2 (pre-fractionated hyaluronic acid derivative). Although Comparative Example 4-2 had relatively high expression of CD80 and CD86 in cDC1, the expression of CD80 and CD86 in macrophages was low, and it was inferior to the pharmaceutical composition of Example 2-2.

[0281] [Test Example 1-3] Using the pharmaceutical composition of Example 2-3, except for subcutaneous administration at 40 μg each as peptides, the same method as Test Example 1-1 was carried out. The analysis results are shown in Tables 6-1 to 6-3.

[0282]

Table 6-1

[0283]

Table 6-2

[0284]

Table 6-3

[0285] Similar to Test Example 1-1 and Test Example 1-2, in the group of mice administered with the pharmaceutical composition of Example 2-3, higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) was confirmed.

[0286] Here, it has been reported that when the expression of CD86 on macrophages is reduced, it functions or is functioning in immunosuppression (Reference 3: Taams LS et al., “Modulation of monocyte / macrophage function by human CD4+CD25+ regulatory T cells.”, Hum Immunol., Vol. 66, Issue 3, pp. 222-230, 2005.; Reference 4: Sansom DM et al., “What's the difference between CD80 and CD86?”, Trends in Immunology., Vol. 24, Issue 6, pp. 313-318, 2003.). According to the reported content of the above Reference 3 and the results of Test Examples 1-1 to 1-3 above, the pharmaceutical compositions of Example 2-1, Example 2-2, and Example 2-3 in which CD86 is strongly expressed not only in DCs but also in macrophages are expected to induce stronger immunity.

[0287] From these results, by using the pharmaceutical composition of the present embodiment, a pharmaceutically active ingredient such as a peptide can be stably and efficiently delivered to immune cells (particularly, cDC1, macrophages), and further, the uptake of the pharmaceutically active ingredient by immune cells can be improved, promoted, or enhanced. At the same time, it is presumed that the activity of immune cells (particularly, the expression of co-stimulatory molecules CD80 and CD86 in cDC1) is improved, promoted, maintained, or enhanced.

[0288] <Examination of manufacturing conditions of pharmaceutical composition> [Reference Example 1] Using the hyaluronic acid derivative obtained in Synthesis Example 1-1, a pharmaceutical composition containing a peptide-hyaluronic acid derivative complex was prepared.

[0289] As the peptide, the gp100RP2RP1_6Y peptide having the following amino acid sequence manufactured by Biolika was used.

[0290] SVYDFFVWLYYYYYYTWHRYHLLYYYYYYEGSRNQDWL (SEQ ID NO: 2)

[0291] Specifically, first, a lyophilized product of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added so as to obtain the concentrations shown in Table 7 below, followed by stirring overnight to dissolve it sufficiently. On the other hand, in another container, it was dissolved in DMSO so that the peptide concentration became 50 mg / mL. After confirming the dissolution, a 1 mol / L aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the above-mentioned aqueous hyaluronic acid derivative solution according to Table 7 below.

[0292] At room temperature (about 25°C), the above-mentioned aqueous hyaluronic acid derivative solution and the dimethyl sulfoxide solution of the peptide were mixed at the volume ratios described in Table 7 below, and stirred at room temperature (about 25°C) for 24 hours to obtain a composition containing a peptide-hyaluronic acid derivative complex. The pH of the solution at this time was also measured.

[0293] (Stability of peptide-hyaluronic acid derivative complex: Appearance of aqueous solution) The state of the composition (aqueous solution) containing the peptide-hyaluronic acid derivative complex after 24 hours was visually judged to verify whether it was turbid. The results are shown in Table 7 below.

[0294] [Table 7]

[0295] Subsequently, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198 - 18385) was added to be 10% by mass of sucrose based on the total mass of the composition containing the peptide - hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25°C) for 1 hour or more. Subsequently, it was diluted with 25 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the theoretical concentration of the peptide was 0.3 mg / mL, and sterilization - filtered with a 0.2 - μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mmΦ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing the peptide - hyaluronic acid derivative complex. The peptide concentration in the pharmaceutical composition containing the peptide - hyaluronic acid derivative complex at this time was quantified by the same method as in Example 2 - 1 above, and the results are shown in Table 7 above.

[0296] (Average particle diameter) Regarding the composition containing the peptide - hyaluronic acid derivative complex produced by the method shown below, measurement was performed by the dynamic light scattering method (DLS) under the following conditions to obtain the z - average particle diameter. The results are shown in Table 7 above.

[0297] (Measurement conditions) DLS apparatus: manufactured by Otsuka Electronics Co., Ltd., ELSZ2000 Cell: Micro - particle size cell Temperature: 25°C Concentration of the peptide - hyaluronic acid derivative complex: 4.95 mg / mL

[0298] As shown in Table 7 above, in the production of the pharmaceutical composition, by adjusting the pH of the mixed solution within a specific range, the particle diameter of the complex of the drug - effective component such as the peptide and the hyaluronic acid derivative can be maintained within a certain range, and it was clarified that the stability of the complex in the solution can be made good.

[0299] [Reference Examples 2 - 1 to 2 - 3] Using the hyaluronic acid derivative obtained in Synthesis Example 1 - 1 and a peptide different from that in Reference Example 1, a pharmaceutical composition containing the peptide - hyaluronic acid derivative complex was prepared.

[0300] As the peptide, a peptide having the following amino acid sequence manufactured by Biolojic was used.

[0301] GSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 3)

[0302] Specifically, first, a lyophilized product of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added so as to have the concentration shown in Table 8 below, and the mixture was stirred overnight to be sufficiently dissolved. On the other hand, in another container, it was dissolved in DMSO so that the peptide concentration became 50 mg / mL. After confirming the dissolution, a 1 mol / L aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the above hyaluronic acid derivative aqueous solution according to Table 8 below.

[0303] At room temperature (about 25°C), the above hyaluronic acid derivative aqueous solution and the DMSO solution of the peptide were mixed at a volume ratio according to Table 8 below, and stirred at room temperature (about 25°C) for 24 hours to obtain a composition containing a peptide-hyaluronic acid derivative complex. The pH of the solution at this time was also measured. Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198-18385) was added so as to be 10% by mass sucrose with respect to the total mass of the composition containing the peptide-hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25°C) for 1 hour or more. Subsequently, it was sterilization-filtered with a 0.2 μm PES (polyethersulfone) (manufactured by Pall Corporation, Acrodisc syringe filter, 25 mm Φ) to obtain a pharmaceutical composition containing a peptide-hyaluronic acid derivative complex. The peptide concentration in the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex at this time was quantified by the same method as in Example 2-1 above, and the average particle diameter of the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex was measured by the same method as in Reference Example 1 above. The results are shown in Table 8 below.

[0304]

Table 8

[0305] As shown in Table 8 above, even in the production of a pharmaceutical composition using a peptide different from that of Reference Example 1, by adjusting the pH of the mixed solution within a specific range, the particle size of the complex of a drug ingredient such as a peptide and a hyaluronic acid derivative can be maintained within a certain range, the stability of the complex in the solution can be made good, and it has been clarified that the peptide concentration in the pharmaceutical composition can be increased.

[0306] <Manufacturing conditions for maintaining an appropriate particle size distribution> [Test Examples 2-1 to 2-8] An aqueous solution with a concentration of 5 mg / mL was prepared for the hyaluronic acid derivative fractionated by the same method as in Example 1-1, and a detailed study was conducted on the mixing ratio of the aqueous phase and the oil phase containing the hyaluronic acid derivative. To 400 μL of a 5 mg / mL hyaluronic acid derivative, DMSO as the oil phase was mixed according to Table 9 below. After thorough mixing, water for injection was added according to Table 9 below so that the final hyaluronic acid derivative concentration was constant. Further, after incubating at room temperature (25°C) for 24 hours, measurement was performed by gel permeation chromatography under the conditions shown below, and the area ratio A1 / A2 was calculated by the same method as in Example 1-1 (Table 9). The obtained gel permeation chromatogram is shown in Figure 8.

[0307] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Temperature: 30°C

[0308]

Table 9

[0309] [Test Examples 3-1 to 3-6] Verification of the mixing ratio for maintaining an appropriate particle size distribution was carried out in the same manner as in Test Examples 2-1 to 2-8, except that ethanol was used instead of DMSO for the oil phase. Measurement was performed by gel permeation chromatography, and the area ratio A1 / A2 was calculated in the same manner as in Example 1-1, and the results are shown in Table 10 below. The obtained gel permeation chromatogram is shown in Figure 9.

[0310]

Table 10

[0311] From the results of Test Examples 2-1 to 2-8 and 3-1 to 3-6, it was found that by passing through a mixing step in which the mixing ratio of the oil phase and the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100 by volume ratio, the particle size of the hyaluronic acid derivative with excellent drug efficacy can be maintained.

[0312] [Comparative Example 5-1] Powder of hyaluronic acid (manufactured by Kikkoman Corporation) with a weight average molecular weight Mw (absolute molecular weight) of 980 kDa was dissolved in water for injection to a concentration of 1 mg / mL, and measurement by gel permeation chromatography was carried out under the same conditions as shown in Synthesis Example 1-1. The obtained chromatogram is shown in Figure 10.

[0313] Furthermore, powder of hyaluronic acid with a weight average molecular weight Mw (absolute molecular weight) of 980 kDa was dissolved in water for injection to a concentration of 10 mg / mL, and then diluted with water for injection to a concentration of 5 mg / mL to obtain an aqueous hyaluronic acid solution. On the other hand, in another container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL to obtain a DMSO solution of the antigen peptide. As the antigen peptide, a peptide having the following amino acid sequence manufactured by Biológica was used.

[0314] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0315] At room temperature (about 25 °C), the above hyaluronic acid aqueous solution and the DMSO solution of the antigen peptide were mixed at a volume ratio of 100:1, and stirred at room temperature (about 25 °C) for 24 hours to attempt to prepare an antigen peptide-hyaluronic acid complex. As confirmed by the photograph described as "hyaluronic acid (980 kDa)" in Fig. 11, the obtained mixed solution was turbid, suggesting that the antigen peptide was not dissolved and a large amount of peptide was not encapsulated.

[0316] Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198-18385) was added so as to be 10% by mass sucrose with respect to the total mass of the composition containing the peptide-hyaluronic acid complex and sucrose, and stirred at room temperature for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass sucrose so that the concentration of the peptide was about 0.30 mg / mL, and sterilization filtered with a 0.2 μm PES (polyethersulfone) (Pall Corporation, Acrodisc syringe filter, 25 mm Φ) to obtain a pharmaceutical composition containing a peptide-hyaluronic acid complex. The peptide content in the pharmaceutical composition was quantified by high performance liquid chromatography measurement in the same manner as in Example 2-1. As a result, the peptide content in the pharmaceutical composition was below the detection limit.

[0317] [Comparative Example 5-2] A powder of hyaluronic acid with a weight average molecular weight Mw (absolute molecular weight) of 650 kDa (manufactured by Kewpie Corporation) was dissolved in water for injection so as to be 1 mg / mL, and measurement by gel permeation chromatography was performed under the same conditions as shown in Synthesis Example 1-1. The obtained gel permeation chromatogram is shown in Fig. 12.

[0318] A powder of hyaluronic acid with a weight average molecular weight Mw (absolute molecular weight) of 650 kDa was dissolved in water for injection so as to be 10 mg / mL, and diluted with water for injection so as to be 5 mg / mL to obtain an aqueous hyaluronic acid solution. On one hand, in another container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of the antigen peptide such that the concentration of the antigen peptide was 50 mg / mL. As the antigen peptide, a peptide having the following amino acid sequence manufactured by Biolika was used.

[0319] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0320] At room temperature (about 25 °C), the above hyaluronic acid aqueous solution and the DMSO solution of the antigen peptide were mixed at a volume ratio of 100:1, and stirred at room temperature (about 25 °C) for 24 hours to attempt to prepare an antigen peptide-hyaluronic acid complex. As confirmed by the photograph described as "Hyaluronic acid (640 kDa)" in Fig. 11, the resulting mixture was turbid, suggesting that the antigen peptide was not dissolved and the peptide was not encapsulated.

[0321] Thereafter, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198 - 18385) was added so as to be 10% by mass sucrose with respect to the total mass of the composition containing the peptide-hyaluronic acid complex and sucrose, and stirred at room temperature (about 25 °C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass sucrose so that the concentration of the peptide was about 0.30 mg / mL, and sterile filtered with a 0.2 μm PES (polyethersulfone) (Acrodisc syringe filter, 25 mm Φ, manufactured by Pall Corporation) to obtain a pharmaceutical composition containing the peptide-hyaluronic acid complex. The content of the peptide in the pharmaceutical composition was quantified by high performance liquid chromatography measurement in the same manner as in Example 2-1. As a result, the peptide content in the pharmaceutical composition was below the detection limit.

[0322] From the results of Comparative Examples 5-1 and 5-2, in high molecular weight hyaluronic acid with an area ratio A1 / A2 of 0.9 or more, it is difficult to encapsulate peptides, and it is difficult to stably and efficiently deliver medicinal components such as peptides to immune cells (particularly cDC1 and macrophages), and further improve, promote, or enhance the uptake of the medicinal components by immune cells.

[0323] <Functional evaluation as an infectious disease vaccine (evaluation of antibody production against exogenous antigen) and evaluation of in vivo antibody production function for the treatment of immune diseases> [Test Example 4]

[0324] [Comparative Example 6-1] A hyaluronic acid derivative was synthesized in the same manner as in Synthesis Example 1-1. Fractionation was not performed, and it was used as it was. The lyophilized product of the obtained hyaluronic acid derivative was weighed, and water for injection was added to a concentration of 5 mg / mL, and stirred overnight at room temperature to dissolve it sufficiently. The above aqueous solution of the hyaluronic acid derivative was diluted 5-fold with water for injection to a concentration of 1 mg / mL, and measured by gel permeation chromatography. FIG. 13 is the obtained gel permeation chromatogram. In addition, the particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1 etc., and the results are shown in Table 11 below.

[0325] [Example 6-1] The hyaluronic acid derivative synthesized in the same manner as in Synthesis Example 1-1 was fractionated in the same manner as in Example 1-1 to obtain a hyaluronic acid derivative. The obtained hyaluronic acid derivative in the dialysis internal solution was measured by gel permeation chromatography. FIG. 14 is the obtained gel permeation chromatogram. In addition, the particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1 etc., and the results are shown in Table 11 below.

[0326]

Table 11

[0327] <Production of pharmaceutical composition> [Example 6-1-1] When the concentration of the hyaluronic acid derivative in the aqueous solution of the hyaluronic acid derivative obtained in Example 6-1 was calculated from the area value of the hyaluronic acid derivative with a known concentration, it was 4.28 mg / mL. In another container, ovalbumin (OVA, low endotoxin product manufactured by Fujifilm Wako Pure Chemical Corporation) was dissolved in PBS (pH 7.4) (manufactured by Fujifilm Wako Pure Chemical Corporation, product number: 166-23555) so that the concentration became 5 mg / mL to obtain an OVA / PBS solution. After mixing in the order of water for injection, the aqueous solution of the hyaluronic acid derivative, and the OVA / PBS solution, sucrose was added as a solid so as to obtain a 10% sucrose isotonic solution, and it was sufficiently stirred at room temperature until a clear solution was obtained. The OVA concentration in the preparation was 0.15 mg / mL, and the hyaluronic acid derivative concentration was 3.0 mg / mL, and it was a 10% sucrose solution containing 10 mM PB (pH 7.4). It was incubated under heating conditions of 75°C for 1 hour to obtain a composition containing an OVA-hyaluronic acid derivative complex.

[0328] [Comparative Example 6-1-1] Using the aqueous solution of the hyaluronic acid derivative obtained in Comparative Example 6-1, OVA was encapsulated in the hyaluronic acid derivative in the same manner as in Example 6-1-1 to obtain a composition containing an OVA-hyaluronic acid derivative complex.

[0329] [Antibody production test] The compositions containing the OVA - hyaluronic acid derivative complex prepared in Example 6 - 1 - 1 and Comparative Example 6 - 1 - 1 were subcutaneously administered to BALB / c mice at 0 day and 7 day so that the amount of OVA was 20 μg, and sera were collected on day 10. At the time of administering the OVA - hyaluronic acid derivative complex, 50 μg of CpG1826 (manufacturer: INVIVOGEN, product code: tlrl - 1826 - 1), which is an adjuvant, was subcutaneously administered. The antibody titers against OVA were measured using an ELISA kit (manufactured by Chondrex, Mouse Anti - OVA IgG Antibody Assay Kit, Mouse Anti - OVA IgG1 Antibody Assay Kit, Mouse Anti - OVA IgG2a Antibody Assay Kit) according to the attached protocol. The dilution factors of the sera were 10, 50, 250, 1250, 6250, and 31250.

[0330] The IgG antibody titer (OD) calculated using the Mouse Anti - OVA IgG Antibody Assay Kit is shown in Figure 15, the IgG1 antibody titer (OD) calculated using the Mouse Anti - OVA IgG1 Antibody Assay Kit is shown in Figure 16, and the IgG2a antibody titer (OD) calculated using the Mouse Anti - OVA IgG2a Antibody Assay Kit is shown in Figure 17. The antibody titers at a dilution factor of 10 are shown in Figure 18A (IgG antibody titer), Figure 18B (IgG1 antibody titer), and Figure 18C (IgG2a antibody titer).

[0331] When the serum dilution is 1250 - fold or more, there is no difference in the antibody titer. However, when the dilution factor is lower than that, it was confirmed that the composition containing the OVA - hyaluronic acid derivative complex of Example 6 - 1 - 1 has a higher antibody titer than the composition containing the OVA - hyaluronic acid derivative complex of Comparative Example 6 - 1 - 1. Thereby, it became clear that the hyaluronic acid derivative (A1 / A2 = 2.03) of Example 6 - 1 has a higher antibody - producing ability against foreign antigens than the hyaluronic acid derivative (A1 / A2 = 0.81) of Comparative Example 6 - 1, and it was also shown that the in - vivo antibody - producing function for therapeutic antibodies is high.

[0332] It has been reported that mature dendritic cells to which an antigen has been added enhance antigen-specific IgG2a production via induction of IFN-γ-producing type I helper T cells (Reference 5: Shigeharu Fujita et al., “Regulatory dendritic cells protect against allergic airway inflammation in a murine asthmatic model.”, J Allergy Clin Immunol 2008;121:95-104.). According to the report content of the above Reference 5 and the results of the above Test Example 4, it is presumed that the pharmaceutical composition containing the OVA-hyaluronic acid derivative of Example 6-1-1 activated the co-stimulatory molecules of DCs, strongly expressed CD80 and CD86, and then enhanced the induction of antigen-specific T cells and IFN-γ-producing ability, and as a result, enhanced antigen-specific IgG2a production. Therefore, the pharmaceutical composition of Example 6-1-1 is expected to induce not only humoral immunity but also cellular immunity and induce a stronger immune response as an infectious disease vaccine.

[0333] By using the pharmaceutical composition of the present embodiment, not only peptides but also pharmaceutical active ingredients of protein antigens can be protected from heating conditions, stably and efficiently delivered to immune cells (particularly, cDC1, macrophages) while maintaining an appropriate three-dimensional structure, and further, the uptake of the pharmaceutical active ingredients by immune cells can be improved, promoted, or enhanced. At the same time, it is presumed that the activity of immune cells (particularly, the expression of co-stimulatory molecules CD80 and CD86 in cDC1) is improved, promoted, maintained, or enhanced. It is presumed that by promoting the activation of helper T cells and further promoting the activation and increase of B cells, antigens against epitopes are produced.

[0334] By using the hyaluronic acid derivatives of the first to third embodiments, it is considered that a vaccine against foreign antigens (mainly infectious disease vaccines) superior to those using conventional hyaluronic acid derivatives can be provided. At the same time, it is considered that pharmaceuticals containing excellent therapeutic antibody-inducing antigens can be provided.

[0335] <Functional evaluation as a cancer vaccine (Cancer vaccine antitumor test using an administration composition containing a hyaluronic acid derivative and an antigen)> [Test Example 5] To clarify the antitumor effect of cancer vaccines with hyaluronic acid derivatives having different area ratios A1 / A2, using immunocompetent checkpoint inhibitor (ICI)-resistant fibrosarcoma cell line CMS5a-bearing mice, a long-chain peptide antigen-loaded hyaluronic acid derivative cancer vaccine was prepared by complexing a long-chain peptide antigen containing the CD8 epitope of the mutant ERK2 antigen (mERK2) expressed in CMS5a with various hyaluronic acid derivatives, and the therapeutic effect was examined.

[0336] Materials and methods are shown below. (1) Cells RPMI1640 medium (added with 2-mercaptoethanol) was purchased from the Institute of Cell Science. Fetal bovine serum (FBS) was purchased from Gibco. The mouse fibrosarcoma CMS5a cell line was obtained through a transfer from the Memorial Sloan Kettering Cancer Institute and was subcultured at Tohoku University. The mouse fibrosarcoma CMS5a cell line expresses the mutant ERK2 protein. A peptide (QYIHSANVL (SEQ ID NO: 4)) containing the mutated part of the mutant ERK2 protein is recognized by CD8-positive cytotoxic T cells of BALB / c mice. A T cell receptor (TCR) that recognizes this peptide has been isolated, and transgenic mice (DUC18 mice) into which the TCR has been introduced have been created.

[0337] (2) Test animals Female BALB / c mice (CD90.2 positive) aged 6 to 8 weeks were purchased from Japan SLC. Both types of mice were housed in the animal experiment facility of the Tohoku University Advanced Science Research Support Center. The protocol for the animal experiment was approved by the Ethics Committee of the Tohoku University School of Medicine.

[0338] (3) Long-chain peptide antigen The synthetic long-chain peptide antigen was purchased from Sigma Genosis. The sequence was as follows. NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1). This sequence contains a 9 - amino acid sequence from Q at position 16 to L at position 24 (QYIHSANVL (SEQ ID NO: 4)) as the CD8 - positive cytotoxic T - cell recognition epitope sequence of the mutant ERK2, and a 17 - amino acid sequence from R at position 13 to K at position 29 (RGLQYIHSANVLHRDLK (SEQ ID NO: 5)) as the CD4 - positive helper T - cell recognition epitope sequence. The long - chain peptide antigen - loaded hyaluronic acid derivative obtained by complexing the long - chain peptide antigen with various hyaluronic acid derivatives may also be referred to as "long - chain peptide antigen - loaded HA nanogel cancer vaccine" or "HA nanogel cancer vaccine".

[0339] [Example 7 - 1] The hyaluronic acid derivative obtained in Comparative Example 6 - 1 was fractionated as follows. The freeze - dried hyaluronic acid derivative (10k HA - Chol - 41.3%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Flow - Thru Dialyzer G2, MWCO: 300,000, Kada Trading Co., Ltd.), and dialyzed against 10 mM phosphate buffer pH 7.4. The hyaluronic acid derivative in the obtained dialysis internal solution was measured by gel permeation chromatography. Figure 19 is the obtained gel permeation chromatogram. The particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1 - 1 etc. The results are shown in Table 12. 6 - Chol - 41.3%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Flow - Thru Dialyzer G2, MWCO: 300,000, Kada Trading Co., Ltd.), and dialyzed against 10 mM phosphate buffer pH 7.4. The hyaluronic acid derivative in the obtained dialysis internal solution was measured by gel permeation chromatography. Figure 19 is the obtained gel permeation chromatogram. The particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1 - 1 etc. The results are shown in Table 12.

[0340] [Table 12]

[0341] [Manufacture of Pharmaceutical Composition] [Example 7 - 1 - 1] When the concentration of the hyaluronic acid derivative in the aqueous solution of the hyaluronic acid derivative obtained in Example 7 - 1 was calculated from the area value of the hyaluronic acid derivative with a known concentration, it was 3.8 mg / mL. On the other hand, in a separate container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL to obtain a DMSO solution of the antigen peptide. As the antigen peptide, a peptide having the following amino acid sequence manufactured by Biolika was used.

[0342] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0343] At room temperature (about 25°C), the above-mentioned aqueous solution of the hyaluronic acid derivative and the DMSO solution of the antigen peptide were mixed at a ratio of 10 parts by mass of the peptide to 100 parts by mass of the hyaluronic acid derivative, and stirred at room temperature (about 25°C) for 2 hours to attempt to prepare an antigen peptide-hyaluronic acid derivative complex. When the state of the solution at this time was visually confirmed, a clear solution was obtained as confirmed by the photograph described as "hyaluronic acid derivative" in Fig. 11. From this, it was confirmed that the peptide was indeed encapsulated.

[0344] Subsequently, solid purified sucrose (manufactured by Fujifilm Wako Pure Chemical Corporation, for manufacturing use only, product number 198-18385) was added to 10% by mass of sucrose based on the total mass of the composition containing the peptide-hyaluronic acid derivative complex and sucrose, and stirred at room temperature (about 25 °C) for 1 hour or more. After confirming that the sucrose was sufficiently dissolved, it was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the concentration of the peptide was about 0.30 mg / mL, and sterilization filtered with a 0.2 μm PES (polyethersulfone) (Pall Corporation, Acrodisc syringe filter, 25 mm Φ) to obtain a pharmaceutical composition containing the peptide-hyaluronic acid derivative complex. The peptide content in the pharmaceutical composition was quantified by high performance liquid chromatography measurement in the same manner as in Example 2-1. As a result, the content of the peptide relative to 100 parts by mass of the pharmaceutical composition was 0.383 parts by mass. Further, the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex was diluted with 10 mM phosphate buffer pH 7.4 containing 10% by mass of sucrose so that the peptide concentration was 250 μg / mL to obtain a pharmaceutical composition of the administration solution. Regarding the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex, measurement by gel permeation chromatography was performed under the measurement conditions shown below by the method described in Example 1-1. Figure 20 is the obtained gel permeation chromatogram.

[0345] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Temperature: 30 °C

[0346] [Comparative Example 7-1-1] The peptide used in Example 7-1-1 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL. It was diluted with 10 mM phosphate buffer pH 7.4 containing 10% sucrose so that the peptide concentration became 0.25 mg / mL.

[0347] [Test Example 6] Tumor growth test Using a T75 culture flask (Corning), the mouse fibrosarcoma CMS5a cell line was cultured in RPMI 1640 medium containing 10% FBS. The cultured cells were detached using PBS containing 0.5% trypsin and suspended in RPMI 1640 medium containing 10% FBS. After centrifuging the suspension (400×g, 5 minutes, 4°C), the supernatant was removed. Then, it was washed twice with RPMI 1640 medium and suspended in RPMI 1640 medium at a concentration of 1×10 6 cells / 100 μL. It was subcutaneously transplanted into the right anterior dorsal part of BALB / c mice at a dose of 100 μL / individual (5 mice per group).

[0348] When administering the long-chain peptide antigen-loaded HA nanogel cancer vaccine, 50 μg of the long-chain peptide antigen-loaded HA nanogel cancer vaccine was subcutaneously administered to the right posterior dorsal part of the mice together with 50 μg of CpG oligodeoxynucleotide (Ajinomoto BioPharma Services Gene Design) dissolved in PBS 7 days, 10 days, 13 days, and 16 days after tumor transplantation. Then, the tumor area was measured over time. When administering the long-chain peptide antigen of Comparative Example 7-1-1, 50 μg of the long-chain peptide was subcutaneously administered to the right posterior dorsal part of the mice together with 50 μg of CpG oligodeoxynucleotide (Ajinomoto BioPharma Services Gene Design) dissolved in PBS 7 days, 10 days, 13 days, and 16 days after tumor transplantation. Then, the tumor area was measured over time.

[0349] The tumor area values (average values) over time are shown in Figure 23. The pharmaceutical composition combining the long-chain peptide antigen-loaded HA nanogel cancer vaccine obtained by complexing the hyaluronic acid derivative of Example 7-1 (A1 / A2 = 6.62) with the long-chain peptide antigen and CpG oligonucleotide DNA was found to suppress the growth of CMS5a tumors more than the pharmaceutical composition combining the long-chain peptide antigen and CpG oligonucleotide DNA of Comparative Example 7-1. From this, it is considered that an excellent cancer vaccine can be provided by using a hyaluronic acid derivative with A1 / A2 = 6.62.

[0350] <Effect of retention time of peak top (maximum refractive index intensity point) by size exclusion chromatography (SEC) on immune cell activation and vaccine function)> [Test Example 7] [Retention time ratio of peak top in particle size distribution of hyaluronic acid derivative] For the hyaluronic acid derivatives obtained in Synthesis Examples 1-1 to 1-3, Examples 1-1 to 1-3, 6-1, 7-1, and Comparative Examples 1-1 to 1-3, 6-1, 7-1, 7-2, and polyacrylic acid (50 kDa) as a standard substance, samples were prepared by the method described in Example 1-1, and measurements were performed by gel permeation chromatography under the conditions shown below.

[0351] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Temperature: 30 °C

[0352] The peak top retention times Pt of the hyaluronic acid derivatives obtained in Synthesis Examples 1-1 to 1-3, Examples 1-1 to 1-3, 6-1, 7-1, and Comparative Examples 1-1 to 1-3, 6-1, 7-1, 7-2, and the peak top retention time Pr of polyacrylic acid (50 kDa) as a standard substance are shown in Table 13 below. Also, Pt / Pr, which is the ratio of the peak top retention time Pt of the hyaluronic acid derivative to the peak top retention time Pr of polyacrylic acid (50 kDa) as a standard substance, was calculated and described in Table 13 below.

[0353]

Table 13

[0354] When the peak top retention time Pt of the hyaluronic acid derivative is shorter than the peak top retention time Pr in polyacrylic acid (50 kDa) as a standard substance, there are more aggregate sizes that are excellent in activating costimulatory molecules. By using the pharmaceutical composition of the present embodiment, the medicinal components of peptides and proteins can be stably and efficiently delivered to immune cells (particularly, cDC1, macrophages), and furthermore, the uptake of the medicinal components by immune cells can be improved, promoted, or enhanced. At the same time, it is presumed that the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1) is improved, promoted, maintained, or enhanced.

[0355] <Influence of the average molecular weight (in terms of polyacrylic acid) by GPC-SEC on immune cell activation and vaccine function> [Test Example 8] The weight average molecular weight (Mw) and number average molecular weight (Mn) (in terms of polyacrylic acid) of the hyaluronic acid derivatives obtained in Synthesis Examples 1-1 to 1-3, Examples 1-1 to 1-3, 6-1, 7-1, and Comparative Examples 1-1 to 1-3, 6-1 were measured under the following conditions. Using polyacrylic acid as a standard substance, a calibration curve was created, and the molecular weights of the hyaluronic acid derivatives obtained in Synthesis Examples 1-1 to 1-3, Examples 1-1 to 1-3, 6-1, 7-1, and Comparative Examples 1-1 to 1-3, 6-1 were calculated based on polyacrylic acid. As the polyacrylic acid standard substances, PSS-Paa2k (2 kDa), 4k (4 kDa), 8k (8 kDa), 18k (18 kDa), 40k (40 kDa), 150k (150 kDa) (manufactured by PSS Polymer Standard service GmbH (sodium polyacrylate)) were used. The results are shown in Table 14.

[0356] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Sample concentration: The hyaluronic acid derivatives obtained in the synthesis examples, examples, and comparative examples were 1 mg / ml, and the polyacrylic acid standard substances were 2 mg / ml Calibration curve conditions: As an approximation formula, the cubic equation At3 + Bt2 + Ct + D was used.

[0357]

Table 14

[0358] When the weight average molecular weight Mw (in terms of polyacrylic acid) of the hyaluronic acid derivative calculated using polyacrylic acid as a standard substance satisfies 110,000 or more, there are more aggregate sizes excellent in activating costimulatory molecules. By using the pharmaceutical composition of the present embodiment, a pharmaceutical active ingredient such as a peptide can be stably and efficiently delivered to immune cells (particularly, cDC1, macrophages), and further, the uptake of the pharmaceutical active ingredient into immune cells can be improved, promoted, or enhanced. At the same time, it is presumed that the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1) is improved, promoted, maintained, or enhanced.

[0359] <Fractionation method by SEC (size exclusion chromatography)> [Example 8]

[0360] The fractionation method is not limited to only the fractionation method using a dialysis membrane. Dissolve the hyaluronic acid derivative obtained in Synthesis Example 1-1 at 20 mg / mL in water for injection, and perform fractionation by gel permeation chromatography under the conditions shown below. For example, fractionate every 0.1 minute.

[0361] (Measurement conditions) Apparatus: HITACHI, CHROMASTER Column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 mm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Guard column: TSKGEL GUARDCOLUMN SWXL (6.0 mm I.D. X 4 CM) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 100 mL Detector: RI

[0362] After fractionation, perform GPC analysis by the method described in Synthesis Example 1-1, confirm that the fraction has an area ratio A1 / A2 of 0.90 or more, and obtain the target hyaluronic acid derivative.

[0363] <Manufacturing conditions of a pharmaceutical composition maintaining an appropriate particle size distribution> [Examples 9-1 to 9-4, Comparative Examples 9-1 to 9-4] An aqueous solution with a concentration of 5 mg / mL was prepared from the hyaluronic acid derivative fractionated by the same method as in Example 1-1, and a detailed study was conducted on the mixing ratio of the aqueous phase and the oil phase containing the hyaluronic acid derivative. Cyclosporine (manufactured by Tokyo Chemical Industry Co., Ltd., product number: C2408), a peptide drug, was weighed into a 6 mL clean vial and dissolved in DMSO (manufactured by Fujifilm Wako Pure Chemical Corporation, product number: 045-24511) so that the concentration of the peptide was 50 mg / mL. Subsequently, 4 μL of 50 mg / mL cyclosporine, which is the oil phase containing the drug, was added to 400 μL of the 5 mg / mL hyaluronic acid derivative. Then, the oil phase DMSO was mixed according to Table 15 below. After thorough mixing, water for injection was added according to Table 15 below so that the final concentration of the hyaluronic acid derivative was constant. Furthermore, after incubating at room temperature (25°C) for 24 hours, measurement was performed by gel permeation chromatography under the conditions shown below, and the area ratio A1 / A2 was calculated by the same method as in Example 1-1 (Table 15).

[0364] (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) Eluent: 10 mM phosphate buffer (pH 7.4) Flow rate: 1 mL / min Injection volume: 50 μL Detector: RI Temperature: 30°C

[0365]

Table 15

[0366] From the results of Examples 9-1 to 9-4 and Comparative Examples 9-1 to 9-4, it was found that by passing through a mixing step in which the mixing ratio of the oil phase and the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100 by volume, it is possible to maintain the particle size of the hyaluronic acid derivative having excellent drug efficacy.

Industrial Applicability

[0367] According to the hyaluronic acid derivative of the present embodiment, it is possible to provide a hyaluronic acid derivative having excellent delivery ability to immune cells in lymph nodes and activation ability of the immune cells when formulated with a drug efficacy component. The pharmaceutical composition of the present embodiment contains the hyaluronic acid derivative and has excellent delivery ability to immune cells in lymph nodes and activation ability of the immune cells. The method for producing the pharmaceutical composition of the present embodiment uses the hyaluronic acid derivative, and a pharmaceutical composition having excellent delivery ability to immune cells in lymph nodes and activation ability of the immune cells can be obtained.

Claims

1. A hyaluronic acid derivative into which a sterol group has been introduced, wherein the ratio A1 / A2 of the areas A1 and A2 calculated by the following method from the chromatogram obtained by gel permeation chromatography measurement is 0.90 or more. (i) Let the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb on the chromatogram of 50 kDa polyacrylic acid, which is a standard substance, to the baseline B and the chromatogram of the hyaluronic acid derivative be Ub; (ii) The area value enclosed by the curve from Ub to the end point on the chromatogram of the hyaluronic acid derivative, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A2, and the area value enclosed by the curve from the start point to Ub on the chromatogram of the hyaluronic acid derivative, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A1.

2. The hyaluronic acid derivative according to claim 1, wherein the ratio Da / Db of the distances Da and Db calculated by the following method from the chromatogram obtained by gel permeation chromatography measurement is greater than 0.00 and less than or equal to 1.

20. (i) Draw a perpendicular line from the refractive index intensity maximum point Ka on the chromatogram of 150 kDa polyacrylic acid, which is a standard substance, to the baseline B, let the intersection with the baseline be Ba, and the length between the refractive index intensity maximum point Ka and Ba be La; (ii) Of the two points on the chromatogram where the refractive index intensity is La / 20, let the point with the earlier elution time be point R1 and the point with the later elution time be point S1; (iv) Let the intersection of the straight line D1 connecting the point R1 and the point S1 and the perpendicular line drawn from the refractive index intensity maximum point Ka to the baseline B be Ta, and the intersection of the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the straight line D1 be Tb; (v) Let the distance between the point R1 and Ta be Da, and the distance between Ta and Tb be Db.

3. The hyaluronic acid derivative according to claim 1 or 2, wherein the ratio A1 / A2 of the areas A1 and A2 is 1.70 or more.

4. A hyaluronic acid derivative into which a sterol group has been introduced, A hyaluronic acid derivative having a ratio Pt / Pr of the retention time Pt at the refractive index intensity maximum point of the hyaluronic acid derivative to the retention time Pr at the refractive index intensity maximum point of 50 kDa polyacrylic acid, which is a standard substance, calculated from a chromatogram obtained by gel permeation chromatography measurement, of 0.5 or more and less than 1.

0.

5. The hyaluronic acid derivative according to claim 1 or 4, wherein the hyaluronic acid derivative has one or more repeating units represented by the following general formula (I). 【Chemical 1】 (wherein, R １ , R ２ , R ３ , and R ４ are each independently selected from the group consisting of a hydrogen atom, C １－６ alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of two or more and 30 or less arbitrary amino acid residues; X １ is given by the following formula: -NR ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NR ｃ -R, -COO-R, -O-COO-R, -S-R, -CO-Y ａ -S-R, -O-CO-Y ｂ -S-R, -NR ｂ -CO-Y ｂ -S-R, and -S-S-R, is a group selected from the group consisting of groups represented by; R ａ , R ｂ and R ｃ are each independently selected from the group consisting of a hydrogen atom, C １－２０ alkyl, amino C ２－２０ alkyl and hydroxy C ２－２０ alkyl, wherein the alkyl moiety of said group may have a group selected from the group consisting of -O- and -NR ｆ - inserted therein; R ｆ is selected from the group consisting of a hydrogen atom, C １－１２ alkyl, amino C ２－１２ alkyl, and hydroxy C ２－１２ alkyl, and the alkyl moiety of the group may have a group selected from the group consisting of -O- and -NH- inserted therein; R is a sterol group; Y is C ２－３０ alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, where the alkylene may have a group selected from the group consisting of -O-, -NR ｇ - and -S-S- inserted therein; R ｇ is selected from the group consisting of a hydrogen atom, C １－２０ alkyl, amino C ２－２０ alkyl, and hydroxy C ２－２０ alkyl, and a group selected from the group consisting of -O- and -NH- may be inserted into the alkyl moiety of the group; Y ａ is an alkylene C １－５ ; Y ｂ is C ２－８ alkylene or C ２－８ alkenylene; m is an integer of 1 or more and 100 or less. )

6. The hyaluronic acid derivative according to claim 1 or 4, wherein the sterol group is a cholesteryl group.

7. The hyaluronic acid derivative according to claim 1 or 4, wherein the introduction rate of the sterol group with respect to the repeating unit of the disaccharide constituting the hyaluronic acid derivative is 30% or more and 60% or less.

8. The hyaluronic acid derivative according to claim 1 or 4, wherein the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 4,000 or more and 1,000,000 or less.

9. The hyaluronic acid derivative according to claim 8, wherein the weight average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 5,000 or more and 25,000 or less.

10. A pharmaceutical composition containing the hyaluronic acid derivative according to claim 1 or 4 and a medicinal ingredient.

11. The pharmaceutical composition according to claim 10, which is for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases.

12. The pharmaceutical composition according to claim 10, wherein the medicinal ingredient contains at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases, and further contains an adjuvant.

13. The pharmaceutical composition according to claim 12, wherein the medicinal ingredient contains a cancer antigen or an antigen derived from an infectious disease.

14. A method for producing a pharmaceutical composition containing the hyaluronic acid derivative according to claim 1 or 4 and a medicinal ingredient, A preparation step of dissolving the medicinal ingredient in an organic solvent or water containing an organic solvent to prepare an oil phase containing the medicinal ingredient, A mixing step of mixing such that a mixing ratio of the oil phase and the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100 by volume ratio, A method for producing a pharmaceutical composition, comprising:

15. The method for producing a pharmaceutical composition according to claim 14, wherein the pH of the aqueous phase containing the hyaluronic acid derivative is 6.00 or more and 11.00 or less.

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