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

Abstract

The hyaluronic acid derivative into which a steroid group has been introduced according to the present invention has an area (A1) / (A2) ratio of 0.90 or more, said area (A1) / (A2) being calculated by the following method from a chromatogram obtained by gel permeation chromatography measurement. Program from intersection (Ub) of vertical line demarcated from point (Kb) of maximum refractive index intensity on chromatogram of reference substance 50kDa polyacrylic acid to baseline (B) and chromatogram of hyaluronic acid derivative to end point of chromatogram of hyaluronic acid derivative, vertical line demarcated from point (Kb) of maximum refractive index intensity to baseline (B), and baseline (B) the value of the enclosed area is A2; a value of an area enclosed by the baseline (B) and a perpendicular line drawn from the refractive index intensity maximum point (Kb) to the baseline (B) in a curve from a start point of the chromatogram of the hyaluronic acid derivative to the intersection point (Ub) is set as A1.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid derivative, a pharmaceutical composition and a method for preparing the pharmaceutical composition. This application claims priority based on Japanese Patent Application No. 2022-088995 filed on May 31, 2022, the contents of which are incorporated herein by reference. Background Art

[0002] In recent years, biopharmaceuticals with proteins, peptides, and nucleic acids as active ingredients have been put into practical use, and the number continues to increase every year. Biopharmaceuticals can meet the insufficient medical needs that cannot be met by existing low-molecular drugs. However, in addition to being difficult to absorb from the digestive tract or mucous membranes, there are also problems of instability in the body and short half-life in the blood. Therefore, biopharmaceuticals need to be frequently administered by injection, which is a heavy burden for patients and medical personnel. Therefore, a drug substrate (sustained-release drug delivery system substrate) that can slowly release active ingredients in vivo by encapsulating biopharmaceuticals without damaging pharmacological activity is sought.

[0003] From such background, a sustained-release drug delivery system substrate consisting of a hyaluronic acid derivative having excellent safety is proposed in patent documentation 1. The hyaluronic acid derivative associates spontaneously in aqueous solution, and medicine, particularly biopharmaceuticals, can be efficiently enclosed in the state maintaining its biological activity, condenses (or can also be dispersed in the concentration of physiological saline) at normal saline concentration, and retention is good in blood in addition. Especially when biopharmaceuticals are used as active ingredients, the hyaluronic acid derivative can be used as the carrier that can efficiently enclose many medicines in the state maintaining pharmacological activity and the blood of excellent blood sustained-release carrier and targeting carrier, and can also be set to the local (such as subcutaneous etc.) sustained-release carrier that can continue sustained-release drugs.

[0004] Furthermore, there has been reported a cancer vaccine using the above-mentioned hyaluronic acid derivative as a carrier (see, for example, Patent Document 2, etc.). Prior art literature Patent Literature

[0005] Patent Document 1: International Publication No. 2010 / 053140 Patent Document 2: International Publication No. 2020 / 158771 Summary of the invention Problem that the invention aims to solve

[0006] However, the hyaluronic acid derivatives used in Patent Document 2 and the like have a wide and non-uniform particle size distribution, and no specific studies have been conducted on the correlation between the particle size distribution of the hyaluronic acid derivatives and the delivery properties to immune cells in lymph nodes and the activation capacity of the immune cells, so there is still room for improvement.

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

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

[0009] [Chemistry 1]

[0010] (Where R 1 , R 2 , R 3 and R 4 Each independently from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 Select from the group consisting of alkylcarbonyl; Z represents a direct bond or a peptide linker composed of 2 or more and 30 or less arbitrary amino acid residues; X 1 is a group selected from the group consisting of groups represented by the following formulae, -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b-CO-Y b -SR and -SSR; R a , R b and R c Each independently from the hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group is selected from the group consisting of alkyl groups, wherein the alkyl portion of the group may be interrupted by -O- and -NR f - A group selected from the group consisting of; R f It can be obtained from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The alkyl group is selected from the group consisting of -O- and -NH-; the alkyl part of the group may be inserted with a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, wherein the alkylene group may be interrupted by -O-, -NR g - and -SS- group selected from the group consisting of; R g From hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group may be selected from the group consisting of -O- and -NR f - A group selected from the group consisting of; Y a C 1-5 Alkylene; Y b C 2-8 Alkylene or C 2-8 alkenylene; m is an integer greater than or equal to 1 and less than or equal to 100.)

[0011] [7] The hyaluronic acid derivative according to any one of [1] to [6], wherein the steryl group is a cholesteryl group. [8] The hyaluronic acid derivative according to any one of [1] to [7], wherein the introduction rate of the steryl group is 30% or more and 60% or less relative to the repeating unit of the disaccharide constituting the hyaluronic acid derivative. [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 5000 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 used 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] , which contains at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases as the active ingredient, and further contains an adjuvant.

[14] The pharmaceutical composition according to any one of

[11] to

[13] , comprising a cancer antigen or an antigen derived from an infectious disease as the medicinal ingredient, and further comprising an adjuvant.

[15] A method for producing a pharmaceutical composition, This is a method for producing a pharmaceutical composition comprising the hyaluronic acid derivative described in any one of [1] to

[10] and a medicinal ingredient. The method comprises the steps 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 of the oil phase and the aqueous phase containing the hyaluronic acid derivative becomes 20:100 to 0.01:100 in terms of volume ratio.

[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. Effects of the Invention

[0012] According to the hyaluronic acid derivative of the above-mentioned embodiment, a hyaluronic acid derivative having excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells when formulated with a pharmaceutical ingredient can be provided. The pharmaceutical composition of the above-mentioned embodiment contains the above-mentioned hyaluronic acid derivative, and has excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells. The manufacturing method of the pharmaceutical composition of the above-mentioned embodiment uses the above-mentioned hyaluronic acid derivative to obtain a pharmaceutical composition having excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [ Figure 1 ] is an example of a gel permeation chromatogram of the hyaluronic acid derivative according to the present embodiment. [ Figure 2 ] is an example of a gel permeation chromatogram of the hyaluronic acid derivative according to the present embodiment. [ Figure 3A ] is a gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-1. [ Figure 3B ] is the gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-2. [ Figure 3C ] is the gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-3. [ Figure 4A ] is the gel permeation chromatogram of the hyaluronic acid derivative in Example 1-1. [ Figure 4B ] is the gel permeation chromatogram of the hyaluronic acid derivative in Example 1-2. [ Figure 4C ] is the gel permeation chromatogram of the hyaluronic acid derivatives in Examples 1-3. [ Figure 5A ] is a gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-1. [ Figure 5B ] is the gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-2. [ Figure 5C ] is the gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-3. [ Figure 5D ] is the gel permeation chromatogram of cholesteryl-modified pullulan (CHP) in Comparative Example 1-4. [ Fig. 6A ] is a gel permeation chromatogram of the hyaluronic acid derivative in Example 1-1 and the hyaluronic acid derivative in the pharmaceutical composition in Example 2-1. [ Figure 6B ] is a gel permeation chromatogram of the hyaluronic acid derivative in Example 1-2 and the hyaluronic acid derivative in the pharmaceutical composition in Example 2-2. [ Figure 6C ] is a gel permeation chromatogram of the hyaluronic acid derivatives in Examples 1-3 and the hyaluronic acid derivatives in the pharmaceutical compositions in Examples 2-3. [ Fig. 7A ] is a diagram showing an example of FACS analysis for calculating the ratio (%) of peptide-incorporated dendritic cells (DCs) expressing CD80 relative to peptide-incorporated DCs in Test Example 1-1. [ Figure 7B ] is a diagram showing an example of FACS analysis for calculating the ratio (%) of peptide-incorporated DCs expressing CD86 relative to peptide-incorporated DCs in Test Example 1-1. [ Figure 7C] is a diagram showing an example of FACS analysis for calculating the ratio (%) of standard type 1 dendritic cells (cDC1) expressing CD80 and incorporated with the peptide relative to cDC1 incorporated with the peptide in Test Example 1-1. [ Fig.7D ] is a diagram showing an example of FACS analysis for calculating the ratio (%) of peptide-incorporated cDC1 expressing CD86 to peptide-incorporated cDC1 in Test Example 1-1. [ Fig. 7E ] is a diagram showing an example of FACS analysis for calculating the ratio (%) of CD80-expressing peptide-incorporated macrophages (Mph) to peptide-incorporated Mph in Test Example 1-1. [ Figure 7F ] is a diagram showing an example of FACS analysis when calculating the ratio (%) of the Mph of the incorporated peptide expressing CD86 to the Mph of the incorporated peptide in Test Example 1-1. [ Figure 8 ] is a gel permeation chromatogram of the hyaluronic acid derivatives in Test Examples 2-1 to 2-8. [ Fig. 9 ] is a gel permeation chromatogram of the hyaluronic acid derivatives in Test Examples 3-1 to 3-6. [ Fig.10 ] is the gel permeation chromatogram of hyaluronic acid in Comparative Example 5-1. [ Fig.11 ] are photographs of the mixed solution when attempting to prepare the antigen peptide-hyaluronic acid complex in Comparative Examples 5-1 and 5-2, and a photograph of the solution when preparing the antigen peptide-hyaluronic acid derivative complex in Example 7-1-1. [ Fig.12 ] is the gel permeation chromatogram of hyaluronic acid in Comparative Example 5-2. [ Fig.13 ] is the gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 6-1. [ Fig.14 ] is the gel permeation chromatogram of the hyaluronic acid derivative in Example 6-1. [ Fig.15 ] is a graph showing the IgG antibody titer (OD value) in Example 6-1-1 and Comparative Example 6-1-1. [ Fig.16 ] is a graph showing the IgG1 antibody titer (OD value) in Example 6-1-1 and Comparative Example 6-1-1. [ Fig.17 ] is a graph showing the IgG2a antibody titer (OD value) in Example 6-1-1 and Comparative Example 6-1-1. [ Fig.18A] represents the 10-fold dilution ratio (titer 10 -1 ) at 4 ℃ and 7 ℃. [ Fig.18B ] represents the 10-fold dilution ratio (titer 10 -1 ) at 4 % IgG1 antibody titer (OD value). [ Fig.18C ] represents the 10-fold dilution ratio (titer 10 -1 ) at IgG2a antibody titer (OD value). [ Fig.19 ] is the gel permeation chromatogram of the hyaluronic acid derivative in Example 7-1. [ Fig. 20 ] is a gel permeation chromatogram of the pharmaceutical composition containing the antigen peptide-hyaluronic acid derivative complex in Example 7-1-1. [ Fig.21 ] is a gel permeation chromatogram of the hyaluronic acid derivative obtained in Example 1-1. [ Fig. 22 ] is a gel permeation chromatogram of the hyaluronic acid derivative obtained in Example 1-1. [ Fig.23 ] is a graph showing the tumor area value (average value) over time in Test Example 6. DETAILED DESCRIPTION

[0014] Hereinafter, an embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail, but the present invention is not limited thereto and various modifications can be made without departing from the gist of the invention.

[0015] The following describes the terms used in this specification.

[0016] The “C 1-20 The term "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms, including, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, etc. 1-4 "Alkyl" further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, etc. 1-20 The alkyl group also includes a C 1-12 Alkyl, C having 1 or more and 6 or less carbon atoms 1-6 alkyl.

[0017] The “C 1-6 The term "alkylcarbonyl" refers to the C 1-6 The alkylcarbonyl of the alkyl group includes, for example, acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc. 1-4 "Alkylcarbonyl".

[0018] The term "amino C" used in this specification 2-20 The term "alkyl" refers to a linear or branched alkyl group having 2 or more and 20 or less carbon atoms and having an amino group as a substituent. For example, the amino group may be located at the carbon atom at the terminal of the alkyl group. 2-20 The alkyl group also includes amino groups having 2 or more and 12 carbon atoms. 2-12 alkyl.

[0019] The term "hydroxyl C 2-20 The term "alkyl" refers to a linear or branched alkyl group having 2 or more and 20 or less carbon atoms and having a hydroxyl group as a substituent. For example, the hydroxyl group may be located on the carbon atom at the terminal of the alkyl group. 2-20 The alkyl group also includes a hydroxyl group having 2 or more and 12 carbon atoms. 2-12 alkyl.

[0020] The “C 2-30 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 or more and 30 carbon atoms, including ethylene, propylene, etc., including C 2-20 Alkylene, C 2-8 Alkylene, group "-(CH2) n -"(wherein n is greater than or equal to 2 and less than or equal to 30, preferably greater than or equal to 2 and less than or equal to 20, and more preferably greater than or equal to 2 and less than or equal to 15).

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

[0022] The term "C 2-8"Alkenylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 or more and 8 carbon atoms and containing one or more double bonds, such as -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hept-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomers exist, various isomers and mixtures thereof are also included.

[0023] 《Hyaluronic acid derivatives》 The hyaluronic acid derivative according to the first embodiment of the present invention is a hyaluronic acid derivative into which a steryl group is introduced, and the hyaluronic acid derivative obtained by gel permeation chromatography is Figure 1 In the chromatogram shown, the ratio A1 / A2 of the areas A1 and A2 calculated by the method shown below is greater than 0.90. (i) The intersection point of a vertical line drawn from the maximum refractive index intensity point Kb on the chromatogram of the standard substance 50 kDa polyacrylic acid to the baseline B and the chromatogram of the above-mentioned hyaluronic acid derivative is set as Ub, and the intersection point of a vertical line drawn from the maximum refractive index intensity point Kb to the baseline B and the baseline is set as Bb; (ii) On the chromatogram of the above-mentioned hyaluronic acid derivative, let the curve from the above-mentioned Ub to the end point, the vertical line drawn from the above-mentioned point of maximum refractive index intensity Kb to the above-mentioned baseline B and the area enclosed by the baseline be A2; on the chromatogram of the above-mentioned hyaluronic acid derivative, let the curve from the above-mentioned starting point to the above-mentioned Ub, the vertical line drawn from the above-mentioned point of maximum refractive index intensity Kb to the above-mentioned baseline B and the area enclosed by the baseline be A1.

[0024] Furthermore, the hyaluronic acid derivative of this embodiment is preferably: obtained from gel permeation chromatography Figure 2 The ratio Da / Db of the distance Da to Db calculated by the method shown below in the chromatogram shown is greater than 0.00 and less than 1.20. (i) a vertical line is drawn from the maximum refractive index intensity point Ka on the chromatogram of the standard substance 150 kDa polyacrylic acid to the baseline B, and the intersection of the vertical line and the baseline is defined as Ba, and the length between the maximum refractive index intensity point Ka and Ba is defined as La; (ii) Of the two points on the chromatogram with a refractive index intensity of La / 20, the point with an earlier dissolution time is defined as point R1, and the point with a later dissolution time is defined as point S1; (iii) The intersection point of a vertical line drawn from the maximum refractive index intensity point Kb on the chromatogram of the standard substance 50 kDa polyacrylic acid to the baseline B and the baseline is set as Bb; (iv) The intersection point of the straight line D1 connecting the point R1 and the point S1 and the vertical line drawn from the point Ka with the maximum refractive index intensity to the baseline B is defined as Ta, and the intersection point of the vertical line drawn from the point Kb with the maximum refractive index intensity to the baseline B and the straight line D1 is defined as Tb; (v) Let the distance between the point R1 and Ta be Da, and let the distance between Ta and Tb be Db. In (ii), when there are three or more points with a refractive index intensity of La / 20 on the chromatogram, the intersection point closest to the starting point is set as R1, and the intersection point closest to the end point is set as S1.

[0025] The hyaluronic acid derivative of the present embodiment is different from the existing hyaluronic acid derivatives in that the particle size distribution contains more specific particle sizes and the particle size distribution is controlled to be narrow. The inventors formulated the hyaluronic acid derivative with controlled particle size distribution together with the medicinal ingredient and found that, as in the examples described later, the delivery property to the immune cells in the lymph nodes and the activation ability of the immune cells are significantly superior compared with the hyaluronic acid derivative with uncontrolled particle size distribution or the cholesterol-modified pullulan (CHP) that has been used as a carrier, thereby completing the present invention.

[0026] The immune cells referred to herein are preferably myeloid cells, more preferably macrophages or dendritic cells (DC), further preferably DC, and particularly preferably standard 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, responsible for regulating the proliferation and function of T cells and natural killer cells in lymphoid and non-lymphoid tissues. There are many subtypes of dendritic cells. Since cDC1 specifically expresses the chemokine receptor XCR1 and selectively expresses the C-type lectin endocytosis receptor CLEC9A, it has high cross-presentation ability and can efficiently carry antigens on 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.). By simultaneously expressing costimulatory molecules, T cells (cytotoxic T cells (CTL)) that attack cells infected with viruses and bacteria and cancer cells can be activated. That is, improving the antigen delivery to macrophages or DCs (preferably cDC1) will enhance the effect of preventing or treating cancer or infection.

[0028] In addition, macrophages and DCs can carry antigens onto MHC class II molecules, which activates helper T cells and promotes antibody production by B cells. That is, improving the antigen delivery to macrophages or DCs (preferably cDC1) will enhance the effect of preventing or treating infections and treating immune diseases.

[0029] In addition, as the activation ability of immune cells, it refers to the property of the activity of the cell as mentioned above, wherein, it is preferably the property of the expression of the costimulatory molecules in the DC (preferably cDC1) to be promoted, promoted or maintained. As costimulatory molecules, CD80, CD86 etc. can be enumerated. By promoting, promoting or maintaining the expression of these costimulatory molecules, T cells (cytotoxic T cells (CTL)) can be significantly induced. Therefore, the hyaluronic acid derivatives of the present embodiment, when formulated with pharmaceutical ingredients, are preferably the substances that can promote, promote or maintain the expression of CD80 and CD86 in DC (preferably cDC1).

[0030] The hyaluronic acid derivative of the present embodiment can form a stable association state with the medicinal ingredient when it is formulated with the medicinal ingredient, and can stably and efficiently deliver the medicinal ingredient to immune cells (preferably DC, more preferably cDC1), and further can enhance, promote or maintain the incorporation of the medicinal ingredient into immune cells. Accordingly, it can be inferred that the activity of immune cells is enhanced, promoted or maintained. In addition, even if the desired effect is achieved by a mechanism different from the above mechanism, it is also included in the technical scope.

[0031] That is, the hyaluronic acid derivative of this embodiment can also be called a delivery enhancer, delivery accelerator or delivery enhancer of the medicinal ingredient to immune cells. Alternatively, it can also be called an incorporation enhancer, accelerator or enhancer of the medicinal ingredient to immune cells. Furthermore, the pharmaceutical composition described below comprising the hyaluronic acid derivative of this embodiment and a medicinal ingredient can also be referred to as a composition for activating immune cells or a composition for increasing, promoting or maintaining the expression of costimulatory molecules in DC (preferably cDC1).

[0032] The particle size distribution of the hyaluronic acid derivative of this embodiment can be measured by gel permeation chromatography, and the particle size distribution controlled as described above can be expressed by the ratio A1 / A2 of the areas A1 and A2 calculated as follows 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 specifically, ORDER No. PSS-Paa series (sodium polyacrylate) manufactured by Polymer Standards Service-USA is more preferred.

[0033] The particle size distribution of the hyaluronic acid derivative can be measured by the following method based on gel permeation chromatography. A 1 mg / mL aqueous solution of a hyaluronic acid derivative and a 2 mg / mL aqueous solution of a polyacrylic acid standard substance were prepared and measured by gel permeation chromatography under the following conditions. (Measurement conditions) Device: High-efficiency GPC (gel permeation chromatography) device Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (differential refractive index detector) Temperature: 30℃

[0034] Figure 1 is an example of a gel permeation chromatogram of the hyaluronic acid derivative according to the present embodiment. Figure 1 At the same time, the calculation method of areas A1 and A2 is described in detail below.

[0035] (i) Figure 1 The gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment and the gel permeation chromatogram of the standard substance 50kDa polyacrylic acid are recorded in. First, the intersection of the vertical line drawn to the baseline B from the maximum point Kb of the refractive index intensity on the chromatogram of the standard substance 50kDa polyacrylic acid and the chromatogram of the above-mentioned hyaluronic acid derivative is set to Ub, and the intersection of the vertical line drawn to the baseline B from the above-mentioned maximum point Kb of the refractive index intensity and the baseline is set to Bb. At this time, the refractive index intensity when starting to measure is set to 0, and the line horizontally drawn from here is set to the baseline. For example, before starting to measure, the increase and decrease of the refractive index intensity is limited in the scope of ± 0.5mV, and the increase and decrease of the refractive index intensity is adjusted to be limited to below 0.5mV in 5 minutes. For example, the initial point that the refractive index intensity increase exceeds the amount equivalent to 5 times of noise value 3 times is set to the " starting point " of the chromatogram, and the dissolution time is set to 0 minute. For example, the point where the refractive index intensity reaches 1 / 1000 of the maximum refractive index intensity is set as the "end point" of the chromatogram, and when the refractive index intensity does not reach 1 / 1000 of the maximum refractive index intensity, "Tlim" is set as the "end point". "Tlim" is set as the dissolution time at which the maximum refractive index intensity appears when measuring 2 kDa polyacrylic acid. In this device, the refractive index intensity is calculated every 0.00167 minutes.

[0036] (ii) Next, on the chromatogram of the hyaluronic acid derivative, let the area value enclosed by the curve from the intersection Ub to the end point, the vertical line drawn from the point of maximum refractive index intensity Kb to the baseline B (the straight line from the intersection Ub to the intersection Bb), and the baseline B be A2; on the chromatogram of the above hyaluronic acid derivative, let the area value enclosed by the curve from the starting point to the above Ub, the vertical line drawn from the above point of maximum refractive index intensity Kb to the above baseline B (the straight line from the intersection Ub to the intersection Bb), and the baseline B be A1. At this time, each area value was calculated using the analysis application of the GPC workstation EcoSEC Elite-WS. When calculating each area value, peaks derived from the developing solvent used in gel permeation chromatography and the like, and pseudo peaks derived from baseline fluctuations of the column and apparatus used were excluded.

[0037] Figure 1 The ratio A1 / A2 of the areas A1 and A2 shown is 0.90 or more, more preferably 1.00 or more, more preferably 1.10 or more, more preferably 1.20 or more, more preferably 1.30 or more, still more preferably 1.40 or more, 1.50 or more, more preferably 1.60 or more, particularly preferably 1.70 or more. Since the ratio A1 / A2 of the areas A1 and A2 is above the above lower limit, more hyaluronic acid derivatives with large particle sizes can be contained, and when the hyaluronic acid derivatives are formulated with the active ingredients, a stable association state can be formed with the active ingredients. Therefore, the active ingredients can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and the incorporation of the active ingredients into immune cells can be further enhanced, promoted or maintained.

[0038] On the other hand, as for the upper limit value of A1 / A2, the higher the ratio of area A1 to area A2, the more preferable it is. Figure 1 In the above, the particle size becomes larger as it approaches the left side of the baseline (the dissolution time becomes shorter), and the particle size becomes smaller as it approaches the right side (the dissolution time becomes longer), so the ratio of the substance with a large particle size is higher than that of the substance with a small particle size. Therefore, the upper limit 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 the present embodiment is not particularly limited, but the maximum point (peak) of the refractive index intensity of more than 2 hyaluronic acid derivatives can be provided. Although not particularly limited, on both sides (left and right) of the vertical line drawn from the maximum point Kb of the refractive index intensity on the chromatogram of the standard substance 50kDa polyacrylic acid to the baseline B, the maximum point (peak) of the refractive index intensity of more than 1 hyaluronic acid derivative can be provided respectively. At this time, compared to the maximum point (peak) of the refractive index intensity of the hyaluronic acid derivative on the right side of the vertical line drawn from the maximum point Kb of the refractive index intensity to the baseline B, the maximum point (peak) of the refractive index intensity of the hyaluronic acid derivative on the left side of the vertical line drawn from the maximum point Kb of the refractive index intensity to the baseline B can be larger. In addition, the refractive index intensity at the maximum point (peak) on the left side is not particularly limited relative to the refractive index intensity at the maximum point (peak) on the right side, but can be more than 1.0 times, more than 2.0 times, more than 3.0 times, more than 5.0 times or more than 10.0 times, and the upper limit can be large, for example, it can be less than 1000 times, less than 100 times, less than 50.0 times, less than 20.0 times, less than 10.0 times, less than 2.0 times or less than 1.5 times.

[0040] Furthermore, the hyaluronic acid derivative of the present embodiment has a controlled particle size distribution as described above, which can be defined by the ratio Da / Db between the distances Da and Db as shown below.

[0041] Figure 2 is an example of a gel permeation chromatogram of the hyaluronic acid derivative according to the present embodiment. Figure 2 The gel permeation chromatograms of the hyaluronic acid derivatives shown and the gel permeation chromatograms of the standard substance 50 kDa polyacrylic acid are compared with Figure 1 However, in Figure 2 In the above, the gel permeation chromatogram of the standard substance 150 kDa polyacrylic acid is added, which is similar to the above in terms of the definition of various intersection points, etc. Figure 1 Different. The following reference Figure 2 At the same time, the calculation method of distances Da and Db is described in detail.

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

[0043] (ii) Next, of the two points on the chromatogram where the refractive index intensity is La / 20, the point with an earlier dissolution time is defined as point R1, and the point with a later dissolution time is defined as point S1.

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

[0045] (iv) Next, the intersection point of the straight line D1 connecting the above-mentioned point R1 and the above-mentioned point S1 and the vertical line drawn from the above-mentioned point Ka with the maximum refractive index intensity to the baseline B is set as Ta, and the intersection point of the vertical line drawn from the above-mentioned point Kb with the above-mentioned maximum refractive index intensity to the baseline B and the above-mentioned straight line D1 is set as Tb.

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

[0047] Figure 2 The ratio Da / Db of the distance Da shown to Db is preferably more than 0.00, 1.20 or less, more preferably more than 0.10, 1.00 or less, more preferably more than 0.20, 0.8 or less, more preferably more than 0.30, 0.95 or less, more preferably more than 0.40, 0.94 or less, particularly preferably more than 0.50, 0.93 or less. By making the ratio Da / Db of the distance Da to Db within the above range, a hyaluronic acid derivative having a particle size greater than the standard substance 150kDa polyacrylic acid can be contained, and when it is formulated with the active ingredient, a stable association state can be formed with the active ingredient. Accordingly, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and the incorporation of the active ingredient into immune cells can be further enhanced, promoted or maintained.

[0048] In the gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment, the horizontal axis represents the dissolution time, and the vertical axis represents the refractive index intensity obtained using a differential refractometer, and there may be multiple maximum refractive index points. The gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment preferably has a particle size distribution with 1 to 5 maximum points, and more preferably has a particle size distribution with 1 to 3 maximum points. In addition, it may or may not have a minimum point. Furthermore, in the gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment, the chromatogram expressed by the refractive index intensity and the dissolution time obtained using a differential refractometer may be bilaterally asymmetric or symmetric.

[0049] The hyaluronic acid derivative of the present embodiment is a substance in which the steryl groups in the hyaluronic acid derivative self-associate in water to form a nano-sized hydrogel through the association of a single molecule or a plurality of molecules.

[0050] In the hyaluronic acid derivative, the steryl group may be directly bonded to the hyaluronic acid or may be bonded without a linker.

[0051] " connexon" referred to herein, any peptide connexon or synthetic compound connexon that can be introduced by gene engineering can be used, but in hyaluronic acid derivatives, preferably peptide connexon. The length of peptide connexon is not particularly limited, and those skilled in the art can be appropriately selected as needed, but the preferred length is more than 2 amino acids (the upper limit is not particularly limited, but is generally less than 30 amino acids, preferably less than 20 amino acids), particularly preferably 15 amino acids. The peptide connexon included in the hyaluronic acid derivative can use peptide connexons that are all of the same length, or peptide connexons of different lengths can be used.

[0052] [Steryl] For the term of " steryl " used in this specification, as long as there is a group of steroid skeleton, there is no particular limitation. At this moment, as steroid, specifically include: cholesterol, cholestanol, campesterol, ergostanol, stigmasterol, stigmasterol, sitosterol, lanosterol, ergosterol, sagostanol, bile acid, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycorticosterone etc. As steryl, include: cholesteryl, stigmasteryl, lanosteryl, ergosteryl etc., wherein, preferably cholesteryl (particularly cholest-5-ene-3β-base).

[0053] The weight average molecular weight (absolute molecular weight) of hyaluronic acid derivatives is not particularly limited, but from the viewpoint of increasing the number of steryl introductions per 1 molecule of hyaluronic acid derivatives, forming a complex with a pharmaceutical ingredient, in addition, from the viewpoint of improving the entanglement of molecules, improving the retention in the blood, hyaluronic acid derivatives with larger molecular weights are preferred. As the weight average molecular weight (absolute molecular weight) of such hyaluronic acid derivatives, preferably 4000 (4k) or more, 1,000,000 (1,000k) or less, more preferably 5k or more, 500k or less, more preferably 6k or more, 500k or less, more preferably 7k or more, 300k or less, more preferably 7k or more, 100k or less, more preferably 7k or more, 50k or less, more preferably 7k or more, 25k or less, particularly preferably 8k or more, 15k or less. Or as the weight average molecular weight (absolute molecular weight) of hyaluronic acid derivatives, more preferably 5k or more, 25k or less. By making the weight average molecular weight (absolute molecular weight) of hyaluronic acid derivatives more than the above-mentioned lower limit, the entanglement of molecules can be further promoted, and retention in blood can be improved. On the other hand, by making the weight average molecular weight (absolute molecular weight) of hyaluronic acid derivatives below the above-mentioned upper limit, the rise of viscosity can be suppressed, and the hyaluronic acid derivatives can be dissolved in the pharmaceutical composition at a higher concentration. The weight average molecular weight (absolute molecular weight) of hyaluronic acid derivatives can usually be regulated by using a raw material with corresponding molecular weight.

[0054] The "molecular weight (absolute molecular weight) of the hyaluronic acid derivative" referred to herein is a weight average molecular weight (absolute molecular weight) determined by size exclusion chromatography and multi-angle light scattering detector (SEC-MALS).

[0055] In the chromatogram obtained by the above-mentioned gel permeation chromatography, the retention time Pt ​​at the maximum point (peak) of the refractive index intensity of the hyaluronic acid derivative relative to the retention time Pr (dissolution time Bb) at the maximum point (peak) of the refractive index intensity of the standard substance 50kDa polyacrylic acid is not particularly limited, but Pt / Pr is preferably more than 0.5, less than 1.0, more preferably more than 0.7, less than 0.95, and more preferably more than 0.75, less than 0.92. When Pt / Pr is within the above range, it is possible to contain more hyaluronic acid derivatives with large particle size, and when it is formulated with the active ingredient, a stable association state can be formed with the active ingredient. Therefore, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and the incorporation of the active ingredient into immune cells can be further promoted, promoted or enhanced. In addition, when there are multiple maximum refractive index intensity points (peak tops) of the hyaluronic acid derivative on the chromatogram obtained by the above-mentioned gel permeation chromatography, the dissolution time at the maximum refractive index intensity point (peak top) with the largest refractive index intensity is used as the retention time Pt ​​at the maximum refractive index intensity point (peak top) of the above-mentioned hyaluronic acid derivative. Furthermore, the polyacrylic acid used as a standard substance in the gel permeation chromatography measurement is preferably sodium polyacrylate, and more specifically, ORDER No. PSS-Paa series (sodium polyacrylate) manufactured by Polymer Standards Service-USA is more preferred.

[0056] The weight average molecular weight (converted as polyacrylic acid) of hyaluronic acid derivatives is not particularly limited, but the weight average molecular weight (converted as polyacrylic acid) of hyaluronic acid derivatives is preferably more than 110,000, more preferably more than 110,000, less than 500,000, more preferably more than 130,000, less than 300,000, most preferably more than 140,000, less than 250,000. When the weight average molecular weight (converted as polyacrylic acid) of hyaluronic acid derivatives is the situation within the above range, it is possible to contain more hyaluronic acid derivatives with large particle size, and when it is formulated with an active ingredient, a stable association state can be formed with the active ingredient. Therefore, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and the incorporation of the active ingredient into immune cells can be further promoted, promoted or enhanced. On the other hand, when the weight average molecular weight (calculated as polyacrylic acid) of the hyaluronic acid derivative exceeds the above upper limit, the viscosity may increase, making it difficult to use the hyaluronic acid derivative as a preparation.

[0057] From the chromatogram obtained by the above-mentioned gel permeation chromatography, based on the calibration curve prepared with polyacrylic acid (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 2kDa, 4kDa, 8kDa, 18kDa, 40kDa and 150kDa as standard substances, the weight average molecular weight (converted into polyacrylic acid) of the above-mentioned hyaluronic acid derivative is calculated according to the following formula: At3+Bt2+Ct+D.

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

[0059] [Chemistry 2]

[0060] (Where R 1 , R 2 , R 3 and R 4 Each independently from the hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 Select from the group consisting of 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 A group selected from the group consisting of groups represented by the following formulae: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b-SR, -NR b -CO-Y b -SR and -SSR; R a , R b and R c Each independently from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group may be selected from the group consisting of -O- and -NR f - A group selected from the group consisting of; R f It can be obtained from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The alkyl group is selected from the group consisting of -O- and -NH-; the alkyl part of the group may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, in this case, the alkylene group may be inserted with -O-, -NR g - and -SS- group selected from the group consisting of; R g It can be obtained from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group is selected from the group consisting of -O- and -NH-; the alkyl part of the group may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a C 1-5 Alkylene; Y b C 2-8 Alkylene or C 2-8 alkenylene; M is an integer greater than or equal to 1 and less than or equal to 100.)

[0061] The hyaluronic acid derivative preferably contains one or more repeating units represented by the following general formula (Ia) (hereinafter sometimes referred to as "repeating units (Ia)") as the repeating unit (I).

[0062] [Chemistry 3]

[0063] (Where R 1 , R 2 , R3 and R 4 Each independently from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 Select from the group consisting of alkylcarbonyl; X is -NR a -Y-NR b - A hydrophobic group represented by COO-R; R a and R b Each independently from the hydrogen atoms and C 1-6 Select from the group consisting of alkyl groups; R is a steryl group; Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, m is an integer greater than or equal to 1 and less than or equal to 100.)

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

[0065] The hyaluronic acid derivative may be modified at a position other than the repeating unit (I) or the repeating unit (Ia), for example, the hydroxyl group may be converted to -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 The carboxyl group may be converted into an amide or an ester, or may form a salt.

[0066] [Repeating unit (I)] The group "-ZN(R a )YX 1 " is preferably from the following formula: -NH-(CH2) mz -NH-R; -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; -NH-(CH2) mz -COO-R; -NH-(CH2CH2O) m -CH2CH2-COO-R, -NH-(CH2) mz -O-COO-R; -NH-(CH2CH2O) m -CH2CH2-O-COO-R, -NH-(CH2) mz -SR; -NH-(CH2CH2O) m -CH2CH2-SR; -NH-(CH2) mz -O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -SSR; and -Z-NR a -Y-NR b -COO-R Select from the group consisting of the groups represented by (wherein, mz is an integer greater than or equal to 2 and less than or equal to 30, R 8 is a hydrogen atom or a methyl group, and R and m are as defined in the present specification. ), More preferably, -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -SSR (wherein mz, ​​R and m are as defined in this specification.) are selected from the group consisting of

[0067] (Z) In the general formula (I), Z is preferably a direct bond. In other embodiments, when Z is a peptide linker, X 1 Preferably -NR b -COO-R. Furthermore, in other embodiments, Z may be -NH-[CH(-Z a )-CONH] n-1-CH(-Z a )-CO-, wherein n is an integer of 2 to 30, and Z a Each independently represents H2N-CH(-Z a )-COOH in an α-amino acid. The peptide linker is bonded to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R a )-YX 1 As examples of amino acids that can be used as amino acid residues of the peptide linker, α-amino acids include natural (L-type) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, and their D-types. All α-amino acids including synthetic amino acids can be used. That is, as Z a , for example, -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, etc. In addition, n Zs may be the same or different. N is an integer of 2 to 30, but is preferably 2 to 10, more preferably 2 to 4. Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, etc.

[0068] (Y) In the general formula (I), Y is -(CH2) n1 - and -(CH2CH2O) m1 -CH2CH2- (wherein n1 is an integer of 2 to 20, preferably an integer of 2 to 15, more preferably an integer of 2 to 12, and further preferably an integer of 2 to 6. m1 is an integer of 1 to 4). Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12 In addition, from the viewpoint of achieving high solubility in pure water or at low salt concentrations and showing high precipitation forming ability at physiological salt concentrations, Y is preferably selected from -(CH2)2-, -(CH2)6-, -(CH2)8- and -(CH2) 12 -, more preferably -(CH2)6-.

[0069] Y can be, for example, -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, or -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-.

[0070] (Y a ) As Y a , preferably -CH2- or -CH2-CH2-.

[0071] (Y b ) As Y b , preferably -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hept-2,4-diene-1,6-diyl or octa-2,4,6-triene-1,8-diyl, more preferably -CH2-CH2- or -CH(CH3)CH2-.

[0072] As the group "-ZN(R a )YX 1 Specific examples of "" include: -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3 -NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-NH-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-cholesteryl, etc. Preferred groups include "-ZN(R a )YX 1 ”, R a , R b and R cis a hydrogen atom, and Y is a straight-chain C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-,Y a A straight chain C 1-5 Alkylene or Y b A straight chain C 2-8 Alkylene or straight chain C 2-8 Alkenylene.

[0073] [Repeating unit (Ia)] In the general formula (Ia), X is preferably -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl, -NH-(CH2) 12 -NH-COO-cholesteryl or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl, more preferably -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl, further preferably -NH-(CH2)6-NH-COO-cholesteryl.

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

[0075] [Chemistry 4]

[0076] (Where R 1a , R 2a , R 3a and R 4a Each independently from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 Select from the group consisting of alkylcarbonyl; X a From hydroxyl and -OQ + In this case, Q + is the counter cation.)

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

[0078] [Repeating unit (II)] In the general formula (II), Q + There are no particular limitations on the counter cation as long as it forms a salt with a carboxyl group in water. When the counter cation has a valency of two or more, it forms a salt with a plurality of carboxyl groups depending on the valency. Examples of counter cations include metal ions such as lithium ions, sodium ions, rubidium ions, cesium ions, magnesium ions, and calcium ions; + R j R k R l R m (Where R j , R k , R l and R m Each independently from the hydrogen atoms and C 1-6 An ammonium ion represented by (selected from the group consisting of alkyl groups) and the like. Among them, Q + Preferred are sodium ions, potassium ions, or tetraalkylammonium ions (eg, tetra-n-butylammonium ions). In the above formula, R j , R k , R l and R m Preferably, C 1-6 The same group selected from the group consisting of alkyl groups is preferably n-butyl.

[0079] R 1 , R 2 , R 3 and R 4 and R 1a , R 2a , R 3a and R 4a Preferably, all of them are hydrogen atoms. a and R b Both are preferably hydrogen atoms.

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

[0081] In the hyaluronic acid derivative of the present embodiment, the introduction rate of the steryl group relative to the repeating unit of the disaccharide constituting the hyaluronic acid derivative (hereinafter referred to as "steryl group introduction rate") 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, 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 making the steryl introduction rate above the lower limit, a stable hydrogel can be maintained without precipitation in the body. On the other hand, by making it below the upper limit, the average particle size of the hydrogel can be within the above range.

[0082] Available through 1 The steryl introduction rate can be measured by H-NMR measurement. 1 The integral value of the peak of the steryl group of the hyaluronic acid derivative in the H-NMR spectrum and the integral value of the peak of the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH3, 1.6 ppm or more, 2.0 ppm or less, 3H) are calculated based on the following formula. H Indicates the number of hydrogen atoms corresponding to the peak. Above 1 H-NMR can be performed, for example, using a 0.02N DCl DMSO-d6 / D2O mixed solution (2N DCl D2O:DMSO-d6=1:99) as a measurement solvent at a measurement temperature of 85°C. In addition, since the peak near 1.6 to 2.0 ppm included in the peak of acetyl groups derived from glucosamine overlaps with the peak derived from cholesteryl (5H), the value calculated by subtracting 5 / 3 of the integrated value of the peak derived from cholesteryl methyl (0.7 ppm) from the integrated value of the peak near 1.6 to 2.0 ppm (i.e., integrated value (1.6 to 2.0 ppm) - integrated value (0.7 ppm) × 5 / 3) can be used as the integrated value of acetyl groups derived from hyaluronic acid for calculating the introduction rate.

[0083] [Steryl introduction rate] (%) = [(peak integral value derived from steryl group × 3 / n H ) / (peak integration value of acetyl group derived from N-acetyl-D-glucosamine)]×100

[0084] <Method for producing hyaluronic acid derivative> Hyaluronic acid derivatives can be obtained, for example, by converting the carboxyl group of glucuronic acid into amide and introducing a steryl group. In addition, the steryl group introduction rate can be controlled by adjusting the mixing amount of a compound having a steryl group that can react with the raw material hyaluronic acid or its derivative.

[0085] As a method for converting the carboxyl group of glucuronic acid into an amide to introduce a steryl group, for example, there can be mentioned the following methods: ion exchange the raw material hyaluronic acid or its derivative, preferably the hyaluronic acid or its derivative consisting of only repeating unit (II), with a tetraalkylammonium salt (e.g., tetrabutylammonium salt (TBA) salt); in the presence of a suitable condensing agent, the hyaluronic acid salt and the introduced steryl group of the formula: "HNR a -Y-NR b -R, NHR a -Y-NR b -COO-R, HNR a -Y-NR b -COO-R, HNR a -Y-NR b -CO-R, HNR a -Y-NR b -CO-NR c -R, HNR a -Y-COO-R, HNR a -YO-COO-R, HNR a -YSR, HNR a -Y-CO-Y a -SR, HNR a -YO-CO-Y b -SR, HNR a -Y-NR b -CO-Y b -SR, HNR a -YSSR or -Z-NR a -Y-NR b -COO-R (where R a , R b , R c , Y, Y a , Y b A method of reacting an amine of a steryl group (particularly a cholesteryl group) represented by "(Z)", wherein Z and R are as defined in the present specification".

[0086] The condensing agent that can be used in the above reaction is not particularly limited, and examples thereof include: 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium boron tetrafluoride 4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazol-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), etc.

[0087] DMT-MM is not particularly limited, but from the viewpoint of efficiently reacting, it is preferably carried out in a mixed solvent of water and an organic solvent. In addition, by using DMT-MM as a condensing agent, in a system where many hydroxyls coexist, while suppressing the formation of an ester bond, an amide bond utilizing an amino group and a carboxyl group can be formed with high selectivity. By using this condensing agent, for example, it is possible to prevent the alcohol as a solvent from reacting with the carboxyl group of the hyaluronic acid part or the carboxyl group and the hydroxyl group that are simultaneously present in the hyaluronic acid part from forming an undesirable crosslink in the molecule or intermolecular bonding.

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

[0089] Alternatively, the raw material hyaluronic acid or its derivatives can also be subjected to ion exchange with tetraalkylammonium salts (e.g., tetrabutylammonium salts (TBA) salts), and in the presence of a suitable condensing agent, the hyaluronate is reacted with a spacer portion in a solvent (protection and deprotection reactions can also be performed as required), the carboxyl group (-COOH) of the raw material hyaluronic acid or its derivatives is transformed, and then reacted with a suitable reagent. Examples of the combination of the group derived from the carboxyl group and the reaction reagent are shown below. -CONR a -Y-NRb H+Hal-R; -CONR a -Y-NR b H+Hal-COOR; -CONR a -Y-NR b H+HOCO-R; -CONR a -Y-NR b H+Hal-CO-R; -CONR a -Y-NR b -COOH+HNR c -R; -CONR a -Y-NR b -CO-NR c H+Hal-R; -CONR a -Y-NR b H+HOCO-NR c -R; -CONR a -Y-NR b H+Hal-CO-NR c -R; -CONR a -Y-COOH+HO-R; -CONR a -Y-OH+Hal-COO-R; -CONR a -Y-OCOOH+HO-R; -CONR a -Y-OCOOH+Hal-R; -CONR a -Y-OCO-Hal+HO-R; -CONR a -Y-SH+Hal-R; -CONR a -Y-Hal+HS-R; -CONR a -Y-CO-Y a -Hal+HS-R; -CONR a -Y-CO-Y a -SH+Hal-R; -CONR a -Y-O-CO-CH=CH2+HS-R; -CONR a -Y-NR b -CO-CH(CH3)=CH2+HS-R; -CONR a -Y-SH+HS-R; -COZ-OH+HNR a -Y-NR b -COO-R; -COZ-NR a -Y-NR b H+Hal-COO-R (Where R a , R b , R c , Y, Y a , Y b and Z are as defined in the present specification, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0090] As the reaction form, there can be mentioned: dehydrohalogenation reaction, condensation reaction, dehydration reaction, nucleophilic addition reaction such as Michael addition, oxidative disulfide formation reaction, etc. These are well-known reactions, and those skilled in the art can make appropriate selections to find preferred reaction conditions. In the case where the conversion product or reactant has a carboxyl group, it can also be reacted as N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester.

[0091] Another method that can be mentioned is to react the carboxyl group of a raw material hyaluronic acid or a derivative thereof with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative having a spacer group having a thiol group modified with a leaving group at the terminal, and then subjecting the hyaluronic acid derivative to an affinity substitution reaction with thiol cholesterol to form a disulfide bond.

[0092] Further, also can enumerate: prepare the material of part spacer group introduced to the carboxyl of hyaluronic acid or its derivative and the material of part spacer group introduced to steryl, and make them react method.A part of specific example is as mentioned above, further, when inserting the situation of -SS- in Y, also can enumerate: prepare respectively on the carboxyl of hyaluronic acid, introduce the hyaluronic acid derivative with the spacer group of sulfydryl at the terminal and introduce the steryl with the spacer group of sulfydryl at the terminal, make them produce oxidation reaction, form the method for disulfide bond.At this moment, also can make a sulfydryl react with 2-thiopyridine and generate disulfide, make it and other sulfydryl replace.

[0093] In addition, other substituents may be further introduced after the hyaluronic acid derivative is prepared. For example, more than 0.1 mol % and less than 99.5 mol %, preferably more than 40 mol % and less than 65 mol % of the carboxyl groups in the hyaluronic acid derivative substantially composed of repeating units (I) and repeating units (II) may be converted into -CO-X z , so that the chemical cross-linking within the molecule or between molecules containing other molecules leads to gelation [Wherein, X z From the following groups: -NH-(CH2) p1 -O-CO-C(R 17 )=CH2; -NH-(CH2) p1 -O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -SH; -NH-(CH2) p1 -NH-CO-C(R 17 )=CH2; -NH-(CH2) p1 -NH-C(=NH)-(CH2)3-SH; -NH-(CH2) p1 -NH-CO-(CH2) r -SH; -NH-(CH2) p1 -NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-(CH2)2-SH; -NH-NH-CO-(CH2)4-CO-NH-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-CH(R 17)-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-(CH2) r -SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-(CH2)2-SH; -NH-CH(CO2H)-(CH2)-SH; -NH-CH(CO2H)-(CH2)2-SH; and -NH-CH(CO2H)-(CH2)2-CONH-CH(CONH-CH2-CO2H)-CH2-SH (Among them, R 17 A hydrogen atom or C 1-6 alkyl, p1 represents an integer of 2 or more and 10 or less, q represents an integer of 1 or more and 100 or less, and r represents an integer of 1 or more and 3 or less) Select from the group formed].

[0094] When the particle size is controlled within a specific range by dialysis, the dialysis membrane using MWCO (molecular weight cut-off) 300kDa can be used to perform dialysis more than 1 time, less than 9 times, preferably more than 3 times, less than 9 times, more preferably more than 6 times, less than 9 times, thereby obtaining a hyaluronic acid derivative having an area ratio A1 / A2 that satisfies a particle size distribution of more than 0.9. As long as the area ratio A1 / A2 is more than 0.9, those skilled in the art can appropriately select the number of dialysis times of any number. Depending on the situation, it is also possible to intercept by dialysis more than 9 times.

[0095] At this time, as the method for obtaining the hyaluronic acid derivative having an area ratio A1 / A2 satisfying the particle size distribution of more than 0.9, it is not limited to dialysis. For example, by utilizing the centrifugal filtration separation of ultrafiltration membrane or precision filtration membrane, preparing purification HPLC, preparing purification GPC, utilizing ultracentrifugal separation, utilizing ion exchange resin separation, utilizing membrane distillation separation, utilizing organic or inorganic membrane membrane separation, utilizing activated carbon, zeolite, MOF (Metal Organic Frameworks, metal organic framework) adsorption and desorption separation, utilizing TFF (Tangential Flow Filtration, tangential flow filtration) separation, using the liquid feeding of filter, using pressurized or decompressed filtration separation, the precipitation separation method of salting out, using the separation of hyaluronic acid receptors, etc., also similarly removing the hyaluronic acid derivative below the particle size distribution of standard substance polyacrylic acid (50kDa, Polymer Standards Service-USA company manufacture, ORDER No.PSS-Paa50k), thereby obtaining the hyaluronic acid derivative having an area ratio A1 / A2 satisfying the particle size distribution of more than 0.9.

[0096] The obtained hyaluronic acid derivative can be dried. As the drying method, for example, ventilation drying, drying in a thermostatic bath, reduced pressure drying, hot air circulation drying, freeze drying, etc. can be mentioned. Among them, freeze drying is preferred. When freeze drying, the hyaluronic acid derivative preferably further contains a cryoprotectant from the viewpoint of more effectively suppressing the increase in the particle size of the microparticles formed by the hyaluronic acid derivative.

[0097] The cryoprotectant is not particularly limited as long as it is a substance known as a "cryoprotectant" or a "freeze-drying protectant", and examples thereof include disaccharides, sorbitol, dextran, polyethylene glycol, propylene glycol, glycerol, glycerin, polyvinyl pyrrolidone, dimethyl sulfoxide, and the like.

[0098] As disaccharides, there is no particular limitation, and for example, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorobiose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiodiulose, mannobiose, melibiose, plantagobiose, neolactose, galactosucrose, scilla biose, neohesperidose, rutinose, rutinulose, podose, xylobiose, primrose etc. are mentioned. Wherein, from the viewpoint widely used as cryoprotectant, sucrose, trehalose, maltose or lactose are preferably used. In addition, from the viewpoint that the particle size of the microparticles formed by the hyaluronic acid derivative during freeze drying is increased in the actual use situation as a pharmaceutical additive and more effectively suppressing freeze drying, sucrose is more preferably used.

[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 relative to 100 parts by mass of the hyaluronic acid derivative. By making the addition amount of the cryoprotectant above the lower limit, a more sufficient effect of suppressing the increase of the particle size can be obtained. On the other hand, the upper limit of the addition amount of the cryoprotectant is not particularly limited, and for example, can be set to 100,000 parts by mass.

[0101] The device used in freeze drying is not particularly limited either, and for example, a commercially available freeze drying machine can be used. Among them, from the viewpoint of controlling the vacuum degree, a freeze drying machine that can monitor the vacuum degree in the device during freeze drying is preferred, and from the viewpoint of controlling the product temperature, a shelf-type freeze drying machine is preferred.

[0102] The hyaluronic acid derivative of the second embodiment of the present invention is a hyaluronic acid derivative having a steryl group introduced. On the chromatogram obtained by gel permeation chromatography, the retention time Pt ​​of the maximum point (peak) of the refractive index intensity of the hyaluronic acid derivative relative to the retention time Pr of the maximum point (peak) of the refractive index intensity of the standard substance 50kDa polyacrylic acid is more than 0.5, less than 1.0, more preferably more than 0.7, less than 0.95, most preferably more than 0.75, less than 0.92. By making the ratio Pt / Pr to the above range, it is possible to contain more hyaluronic acid derivatives with large particle size, and when it is formulated with an active ingredient, a stable association state can be formed with the active ingredient. Accordingly, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, it is possible to enhance, promote or enhance the incorporation of the active ingredient into immune cells.

[0103] The polyacrylic acid used as a standard substance in the gel permeation chromatography measurement is preferably sodium polyacrylate, and more specifically, ORDER No. PSS-Paa50k (sodium polyacrylate) manufactured by Polymer Standards Service-USA is more preferred. The above-mentioned measurement by gel permeation chromatography can be performed, for example, by the following method. A 1 mg / mL aqueous solution of a hyaluronic acid derivative and a 2 mg / mL aqueous solution of a polyacrylic acid standard substance were prepared and measured by gel permeation chromatography under the following conditions. (Measurement conditions) Device: High-efficiency GPC (gel permeation chromatography) device Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI (differential refractive index detector) Temperature: 30℃ In addition, description of the same configurations and the like as those of the hyaluronic acid derivative according to the first embodiment will be omitted.

[0104] The hyaluronic acid derivative of the third embodiment of the present invention is a hyaluronic acid derivative having a steryl group introduced therein, and the weight-average molecular weight (converted as polyacrylic acid) of the hyaluronic acid derivative is more than 110,000 and less than 500,000, preferably more than 130,000 and less than 300,000, more preferably more than 140,000 and less than 250,000. When the weight-average molecular weight (converted as polyacrylic acid) of the hyaluronic acid derivative meets the above-mentioned range, it is possible to contain more hyaluronic acid derivatives with large particle size, and when formulated with an active ingredient, a stable association state can be formed with the active ingredient. Thus, the active ingredient can be stably and efficiently delivered to immune cells (preferably DC, more preferably cDC1), and further, it is possible to enhance, promote or enhance the incorporation of the active ingredient into immune cells. On the other hand, when the weight average molecular weight (calculated as polyacrylic acid) of the hyaluronic acid derivative exceeds the above upper limit, the viscosity may increase, making it difficult to use the hyaluronic acid derivative as a preparation.

[0105] The weight average molecular weight of the above-mentioned hyaluronic acid derivative (converted into polyacrylic acid) is calculated based on the chromatogram obtained from the gel permeation chromatography measurement, and a calibration curve is prepared using polyacrylic acid 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. The polyacrylic acid used as the standard substance is preferably sodium polyacrylate, and more specifically, ORDER No. PSS-Paa series (sodium polyacrylate) manufactured by Polymer Standards Service-USA is more preferred.

[0106] The measurement by gel permeation chromatography can be performed by the method described in the first or second embodiment. Other than this, description of the same configurations as those of the hyaluronic acid derivative of the first embodiment and / or the second embodiment will be omitted.

[0107] <Pharmaceutical Composition> The hyaluronic acid derivatives according to 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-mentioned hyaluronic acid derivative and a medicinal ingredient.

[0108] In the pharmaceutical composition of the present embodiment, the above-mentioned hyaluronic acid derivative forms a complex with the medicinal ingredient (hereinafter referred to as "medical ingredient-hyaluronic acid derivative complex"). Specifically, it is presumed that the steryl group in the hyaluronic acid derivative and the hydrophobic part of the medicinal ingredient form a complex through hydrophobic interaction, and the hydrophobic part of the medicinal ingredient and the steryl group etc. are present in the central part, and on the other hand, the hydrophilic part such as the part of the hyaluronic acid derived from the hyaluronic acid derivative is present in the outer edge, presenting a spherical structure of a core-shell type. That is, it is presumed that the medicinal ingredient is enclosed or enclosed in the hyaluronic acid derivative.

[0109] In the pharmaceutical composition of the present embodiment, the average particle size of the spherical structure comprising the pharmaceutical ingredient-hyaluronic acid derivative complex can be set to more than 20nm and less than 100nm, can be set to more than 20nm and less than 95nm, can be set to more than 20nm and less than 90nm. The average particle size referred to herein is the value represented by z average. By making the average particle size in the above-mentioned numerical range, it is possible to exist in a stable structure in vivo and to pass through lymph nodes more easily. For average particle size, for example, it can be measured by DLS (Dynamic Light Scattering, dynamic light scattering), nano-tracking particle measuring device, size exclusion chromatography, high performance liquid chromatography and electron microscopy. More specifically, for example, in a DLS device, 10mM phosphate buffer containing 10w / v% sucrose is diluted to a hyaluronic acid derivative concentration of 1mg / mL for measurement.

[0110] In the pharmaceutical composition of the present embodiment, the weight average molecular weight Mw (converted as polyacrylic acid) of hyaluronic acid derivatives or medicinal ingredient-hyaluronic acid derivative complex is preferably more than 110,000. For weight average molecular weight Mw (converted as polyacrylic acid), a calibration curve can be calculated by using a plurality of polyacrylic acid standards with different molecular weights. The polyacrylic acid standards used are preferably sodium polyacrylate. Specifically, more preferably ORDER No.PSS-Paa series (sodium polyacrylate) manufactured by Polymer Standards Service-USA company. It is believed that by making the Mw within the above-mentioned numerical range, there will be more complex sizes that are excellent for the activation of co-stimulatory molecules. By using the pharmaceutical composition of this embodiment, it is possible to stably and efficiently deliver pharmaceutical ingredients such as peptides to immune cells (especially cDC1, macrophages). Furthermore, while being able to enhance, promote or enhance the incorporation of the pharmaceutical ingredients into immune cells, it is possible to enhance, promote, maintain or enhance the activity of immune cells (especially the expression of co-stimulatory molecules CD80 and CD86 in cDC1). Mw can be measured, for example, by DLS (Dynamic Light Scattering), size exclusion chromatography, electron microscopy, etc. More specifically, for example, the hyaluronic acid derivative is diluted to a concentration of 1 mg / mL using a size exclusion chromatography apparatus for measurement.

[0111] In the pharmaceutical composition of the present embodiment, the peak time on the particle size distribution in the size exclusion chromatography of hyaluronic acid derivatives or active ingredient-hyaluronic acid derivative complex is preferably less than the peak time of standard substance polyacrylic acid (50kDa). It is believed that by making the peak time in the above range, there can be more the association size of the activation excellence of costimulatory molecules, by using the pharmaceutical composition of the present embodiment, it is possible to make the active ingredients such as peptides stably and efficiently delivered to immune cells (especially cDC1, macrophages), further, while being able to promote, promote or enhance the active ingredient to the incorporation of immune cells, it is possible to promote, promote, maintain or enhance the activity of immune cells (especially the expression of co-stimulatory molecules CD80 and CD86 in cDC1). The peak time can be measured, for example, by size exclusion chromatography. More specifically, it is measured by, for example, being diluted to hyaluronic acid derivative concentration with a size exclusion chromatography device to become 1mg / mL.

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

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

[0114] <Medicinal ingredients> The pharmaceutical ingredients are 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 weight compounds, medium molecular weight compounds, etc. Among them, antigens are preferred, and at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases is more preferred, and cancer antigens or antigens derived from infectious diseases are more preferred.

[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 is more preferably a pharmaceutical composition for preventing or treating cancer or infectious diseases. When the applicable disease is cancer, infectious disease or immune disease, the pharmaceutical composition of the present embodiment can be a vaccine composition.

[0116] [antigen] (Cancer Antigen) Cancer antigens are antigens that are expressed more frequently in cancer cells, and in some cases, are expressed only by cancer cells. Cancer antigens may be expressed within cancer cells or on the surface of cancer cells.

[0117] The antigenic proteins that can be used in the pharmaceutical composition of the present embodiment are not limited, but include: ERK1, ERK2, WT1, MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal related antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate specific membrane antigen (PSMA), T cell receptor Body / 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, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel1 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), cytosolic protein, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, etc.

[0118] As the antigen protein, the entire sequence may be used, or a sequence in which part of the sequence is deleted may be used.

[0119] The antigenic peptide that can be used in the pharmaceutical composition of this embodiment is: in the sequence of the antigenic protein, an antigenic peptide containing one or more epitopes selected from a group consisting of CD8 positive cell killer T cell recognition epitopes and CD4 positive helper T cell recognition epitopes. In one embodiment, from the viewpoint of being carried on MHC class I molecules or MHC class II molecules after decomposition in antigen presenting cells, the above-mentioned antigenic peptide is preferably an antigenic peptide containing two or more epitopes. Specifically, as the above-mentioned antigenic peptide, an antigenic peptide containing an antigenic protein epitope of a tumor cell can be cited.

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

[0121] In one embodiment, from the viewpoint of inducing activation of cytotoxic T cells (CTLs) by helper T cells, the antigenic peptide is an antigenic peptide containing one or more CD8-positive cytotoxic T cell recognition epitopes and CD4-positive helper T cell recognition epitopes respectively.

[0122] In one embodiment, when containing more than two epitopes, an amino acid linker can be configured between the epitopes. The linker has, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, more preferably 4 to 8 amino acids. As amino acids for the linker, glycine (G), tyrosine (Y), leucine (L), tryptophan (W) and the like can be cited. Preferably, tyrosine (Y), leucine (L), tryptophan (W). Specific examples of amino acid linkers include: a linker (4Y) composed of four consecutive tyrosines (Y), a linker (4L) composed of four consecutive leucines (L), a linker (4W) composed of four consecutive leucines (W), a linker (6G) composed of six consecutive glycines (G), a linker (6Y) composed of six consecutive tyrosines (Y), a linker (6L) composed of six consecutive leucines (L), a linker (6W) composed of six consecutive tryptophans (W), a linker (8Y) composed of eight consecutive tyrosines (Y), a linker (8L) composed of six consecutive leucines (L), and a linker (8W) composed of eight consecutive tryptophans (W), preferably 6Y, 6L or 6W.

[0123] The cancer antigen may be a tumor-associated antigen, a cancer testis antigen, a viral antigen, or a tumor-specific antigen (including a neoplastic tumor antigen). A single cancer antigen may be used, or two or more cancer antigens may be used in combination.

[0124] (Antigens derived from infectious diseases) As antigens derived from infectious diseases, there is no particular limitation as long as they are antigens derived from infectious pathogens and infectious pathogens. Examples of infectious pathogens include viruses, bacteria, fungi, nematodes, etc. Antigens derived from infectious disease pathogens may be antigenic proteins or antigenic peptides.

[0125] The diseases caused by the infectious pathogens are not particularly limited, and examples thereof include adenoviruses, herpes viruses (e.g., HSV-I, HSV-II, CMV, VZV), poxviruses (e.g., orthopoxviruses such as acne or vaccinia, molluscum contagiosum), picornaviruses (e.g., rhinoviruses, enteroviruses), orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)), coronaviruses (e.g., SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS-CoV-2), papovaviruses (e.g., papillomaviruses that cause condyloma acuminatum, common cystitis, plantar warts, etc.), hepadnaviruses (e.g., hepatitis B virus), flaviviruses (e.g., hepatitis C virus), and SARS-CoV-2. Viral diseases such as diseases caused by viral infections such as hepatitis virus, dengue virus), retroviruses (such as HIV and other slow viruses); bacterial diseases such as diseases caused by bacterial infections such as Escherichia coli, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, Bordetella; fungal diseases such as chlamydiosis, candidal disease, aspergillosis, histoplasmosis, cryptococcal meningitis; malaria, Pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, trypanosome infection, etc.

[0126] As the structure of the antigen that can be used in the pharmaceutical composition of the present embodiment, as long as it is at least a part of the various components constituting the pathogen, there is no particular limitation, and for example, it can include: live vaccines, inactive whole particles, their parts, protein subunits, proteins, peptides, etc. Among them, from the viewpoint of the complexation with the hyaluronic acid derivative, preferably protein subunits, proteins or peptides.

[0127] The above-mentioned influenza virus is an RNA enveloped virus with a particle size of about 100nm in diameter belonging to the family Orthomyxoviridae, and is divided into A, B and C types based on the antigenicity of the internal protein. The above-mentioned influenza virus is composed of the core and external glycoprotein of the ribonucleic acid (RNA) formed by the association of the internal nucleocapsid (Nucleocapsid) or nucleoprotein surrounded by the viral envelope with a lipid bilayer structure. The inner layer of the above-mentioned viral envelope is mainly composed of matrix protein, and the outer layer is mostly composed of lipid substances from the host. In addition, the RNA of the above-mentioned influenza virus is a segmented structure. In addition, the influenza that is prevalent in the world is influenza caused by type A influenza virus, which has the envelope glycoproteins of hemagglutinin and 2 kinds of neuraminidase, which are different due to different antigenicity, and hemagglutinin is divided into 16 subtypes, and neuraminidase is divided into 9 subtypes.

[0128] As the infectious disease-derived antigen, antigens derived from influenza virus type A and type B can be appropriately used. In addition, the subtypes of the influenza virus type A and type B are not particularly limited, and may be subtypes isolated so far or may be subtypes to be isolated in the future.

[0129] As the antigen derived from influenza virus, as long as it is at least a part of the various components constituting the above-mentioned influenza virus, there is no particular limitation, for example, it can be cited: the whole virus particle inactivated by organic solvent / surfactant or other reagent, or the virus subunit made by refining hemagglutinin and / or neuraminidase after removing impurities from the whole virus particle, etc. From the viewpoint of immunogenicity, it is preferably a hemagglutinin subunit or a whole virus particle. The above-mentioned whole virus particle is more preferably a whole virus particle inactivated by formalin, etc. In addition, it is particularly effective for the hemagglutinin subunit (cleavage) that has fewer impurities and requires adjuvants such as immunostimulants.

[0130] The method for preparing the influenza virus antigen is not particularly limited, and a known method can be used without limitation. For example, a method can be mentioned in which a virus strain isolated from an influenza-infected animal or influenza patient is infecting eggs, etc., and then cultured according to a conventional method, and the antigen is prepared from the purified virus stock solution. In addition, an antigen derived from a virus prepared in a cultured cell by a genetic engineering method can also be used.

[0131] (Antigens in immune diseases) As antigens in immune diseases, there is no particular limitation as long as they are antigens containing epitopes of target proteins of immune diseases. There is no particular limitation on immune diseases, and examples thereof include: psoriasis vulgaris, ankylosing spondylitis, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, bronchial asthma, chronic urticaria, hay fever, atopic dermatitis, etc. The target protein is not particularly limited, and examples thereof include IL-17A, DPP4, S100A9, PCSK9, IL-23, IgE, TNFα, IL-12 / 23p40, IL-6, α4β7 integrin, IL-4 / 13, IL-5, BLyS, IL-13, etc. Peptides derived from IL-17A are described in Reference 2 (International Publication No. 2017 / 164409). The peptide may contain not only the epitope of the target protein but also epitopes other than the target protein, such as B cell epitopes or T cell epitopes. The peptide is a sequence having an epitope and may contain a cyclic structure. The early peptide may have multiple cyclic structures in the molecule. Furthermore, the above peptides can be coupled or conjugated to proteins. By producing antibodies against the administered protein or peptide in the body, a therapeutic effect can be expected.

[0132] [Pharmacologically active peptides or proteins] A pharmaceutically active peptide or protein refers to a substance that has a positive or beneficial effect on the state or symptoms of a subject when a therapeutically effective amount is administered to the subject. A preferred pharmaceutically active peptide or protein is a substance that has a radical or symptomatic nature and can be administered to improve, relieve, alleviate, restore one or more symptoms of a disease or disorder, delay the onset of symptoms, or reduce the severity of symptoms. Pharmaceutically active peptides or proteins also have preventive properties and can be used to delay the onset of a disease or reduce the severity of such a disease or symptom. The term "pharmaceutically active peptide or protein" contains the meaning of a full-length protein or peptide, and also refers to a pharmacologically active fragment thereof. The term also includes pharmacologically active analogs of peptides or proteins.

[0133] Examples of pharmaceutically active proteins are not limited to the following, and include: cytokines such as immunologically active compounds and immune system proteins (e.g., interleukins, colony stimulating factors (CSFs), granulocyte colony stimulating factors (G-CSFs), granulocyte macrophage colony stimulating factors (GM-CSFs), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, immunoglobulins, antibodies, hormones (insulin, thyroid hormones, catecholamines, gonadotropins, troponins, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic enzymes or degrading enzymes, steroidogenic (sterio

[0013] The present invention relates to a group of proteins including glycosylated oligonucleotides, oligonucleotides, oligodeoxycholate ...

[0134] In one embodiment, the pharmaceutically active protein is a cytokine involved in the control of lymphocyte homeostasis, preferably a cytokine that is involved in one or more of the groups consisting of the generation, initial stimulation, proliferation, differentiation and survival of T cells and induces or enhances these cytokines. 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 also have a cyclic structure. The above peptide may also have multiple cyclic structures in the molecule.

[0135] [Nucleic Acids] As nucleic acid, for example, DNA, RNA, antisense nucleic acid, decoy nucleic acid, ribozyme, low molecular weight interfering RNA, aptamer, etc. In addition, when the above-mentioned antigen is a peptide or protein, it is also preferred to use nucleic acids (DNA or mRNA, etc.) encoding these antigen peptides or proteins.

[0136] [Low molecular weight compounds] Examples of low molecular weight compounds include carcinogens (e.g., alkylating agents, metabolic antagonists, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroids, non-steroidal anti-inflammatory agents, etc.), anti-rheumatic agents, antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfonamides, etc.), etc.

[0137] In addition, as the medicinal ingredient, from the viewpoint of being able to give full play to the interaction with the steryl group of the above-mentioned hyaluronic acid derivative, it is also preferred to use a medicinal ingredient with high hydrophobicity, i.e., poor water solubility. In addition, "poor water solubility" means that the amount of water required to dissolve 1g of solute in the 17th revised edition of the Japanese Pharmacopoeia needs to be more than 30mL.

[0138] Examples of the poorly water-soluble and solid active ingredients include active ingredients of medicines listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia Extra-Pharmacopoeia Standards, the USP (United States Pharmacopoeia), the NF (National Pharmaceutical Collection), and the EP (European Pharmacopoeia), such as acetaminophen, ibuprofen, benzoic acid, ethsalamide, caffeine, camphor, quinine, calcium gluconate, dimercaprol, sulfamide, theophylline, theobromine, riboflavin, toluenepropanol, phenobarbital, aminophylline, thiosemicarbazide, quercetin, rutin, salicylic acid, theophylline sodium salt, pyrabital, quinine hydrochloride, irgapyridine, in), antipyretic analgesics such as digoxin, griseofulvin, and phenacetin, nervous system drugs, sedatives and hypnotics, muscle relaxants, hypotensive agents, antihistamines, etc.; antibiotics such as acetylspiramycin, ampicillin, erythromycin, kitasamycin, chloramphenicol, triacetyloleandomycin, nystatin, and colistin sulfate; steroid hormones such as methyltestosterone, methylandrostenediol, progesterone, estradiol benzoate, ethinylestradiol, deoxycorticosterone acetate, cortisone acetate, hydrocortisone, hydrocortisone acetate, and prednisolone; non-steroidal orgestrel hormones such as dienestrol, hexestrol, diethylstilbestrol, diethylstilbestrol dipropionate, and chlorethoxyquin; other fat-soluble vitamins, etc. One selected from these active ingredients can be used, or two or more can be used in combination.

[0139] The active ingredient can be an oily or liquid component that is insoluble in water. As an oily or liquid active ingredient that is insoluble in water, the active ingredients of medicines recorded in "Japanese Pharmacopoeia", "Japanese Pharmacopoeia External Drug Standards", "USP", "NF", "EP" etc. can be cited, such as: vitamins such as teprenone, indomethacin farnesate, tetraene methyl quinone, phytomenadione, vitamin A oil, phenylpentyl alcohol, vitamin D, vitamin E, DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), liver oil and other high-level unsaturated fatty acids, coenzyme Q, orange oil, lemon oil, peppermint oil and other oil-soluble flavorings etc. There are various homologues and derivatives in vitamin E, but as long as it is liquid at room temperature, it is not particularly limited, for example, dl-α-tocopherol, acetic acid dl-α-tocopherol, d-α-tocopherol, acetic acid d-α-tocopherol etc. can be cited. One selected from these active ingredients can be used, and two or more can also be used in combination.

[0140] The active ingredient can also be a semi-solid substance that is poorly soluble in water. As the semi-solid active ingredient that is poorly soluble in water, for example, it can be enumerated: earthworm, licorice, cinnamon bark, peony root, peony bark, spider seven, pepper, ginger, tangerine peel, ephedra, Nandina domestica, cherry bark, Polygala, Platycodon, Plantago, Plantago, Lycoris, Polygala, Fritillaria, fennel, Phellodendron, Coptis, Curcuma, chamomile, gentian, bezoar, animal gall, Adenophora, ginger, Atractylodes, cloves, tangerine peel, Atractylodes, bamboo ginseng, carrot, Pueraria Decoction, Guizhi Decoction, Xiangsusan, Zihu Guizhi Decoction, Xiaozihu Decoction, Xiaoqinglong Decoction, Maimendong Decoction, Banxia Houpo Decoction, Mahuang Decoction and other Chinese herbal medicines or crude drug extracts; oyster meat extract, propolis and propolis extracts, coenzyme Q, etc. One selected from these active ingredients can be used, and two or more can be used in combination.

[0141] In the pharmaceutical composition of the present embodiment, the content of the active ingredient varies depending on the structure of the active ingredient, but can be set to 0.001 mass% to 10,000 mass%, preferably 0.1 mass% to 1000 mass%, more preferably 1.0 mass% to 100.0 mass%, further preferably 1.5 mass% to 50.0 mass%, particularly preferably 3.0 mass% to 30.0 mass%, and most preferably 5.0 mass% to 20.0 mass%, relative to the mass of the hyaluronic acid derivative. Alternatively, in the pharmaceutical composition of the present embodiment, the content of the active ingredient, relative to the mass of the hyaluronic acid derivative, is more preferably 0.1 mass % to 100.0 mass %, further preferably 0.1 mass % to 50.0 mass %, further preferably 0.1 mass % to 30.0 mass %, further preferably 0.5 mass % to 30.0 mass %, further preferably 1.0 mass % to 30.0 mass %, further preferably 1.0 mass % to 25.0 mass %, further preferably 1.0 mass % to 20.0 mass %, further preferably 1.0 mass % to 15.0 mass %, particularly preferably 1.0 mass % to 10.0 mass %.

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

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

[0144] When the content of the medicinal ingredient is equal to or greater than the lower limit, immune cells can be activated more efficiently, while when the content is equal to or less than the upper limit, the medicinal ingredient can be enclosed 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-mentioned active ingredients and the above-mentioned hyaluronic acid derivatives, an adjuvant may be further included. Thus, immunity can be induced more effectively. Among them, the induced immunity can be humoral immunity or cellular immunity. That is, the pharmaceutical composition of the present embodiment is preferably a pharmaceutical composition comprising the above-mentioned hyaluronic acid derivative and an antigen (preferably a cancer antigen or an antigen derived from an infectious disease) as the above-mentioned active ingredient and further comprising an adjuvant.

[0146] In addition, humoral immunity generally refers to an immune mechanism centered on B cells and antibodies. B cells are stimulated by cytokines produced by helper T cells (Th2 cells), and B cells differentiate into plasma cells, producing a large amount of antibodies, which circulate in body fluids and spread throughout the body. In addition, some stimulated B cells become memory B cells that remember antigen information. When infected again, they react faster than the initial reaction and can produce a larger amount of antibodies with high affinity for the antigen. On the other hand, cellular immunity refers to an immune mechanism that uses cells as the main effector to eliminate foreign substances such as pathogens themselves, virus-infected cells, cancer cells, etc. It is an elimination mechanism that uses immune active cells such as macrophages, cytotoxic T cells (CTL, cytotoxic T cells), and natural killer cells (NK cells).

[0147] The adjuvant is not particularly limited as long as it is a substance generally used for vaccines, and examples thereof include aluminum salts, squalene, and ligands for natural immune receptors.

[0148] The "ligand" referred to herein refers to a substance that specifically binds to a receptor, and in particular, substances that specifically bind to a receptor and exhibit various physiological effects can be used. Such substances are also called "agonists."

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

[0150] As the TLR ligand, for example, a ligand that interacts 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 may be appropriately selected.

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

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

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

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

[0155] Examples of 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, and loxoribine.

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

[0158] Among them, as the adjuvant, anionic compounds are preferred, and CpG oligodeoxynucleotide is more preferred, from the viewpoint of being able to further enhance the antigen presentation function.

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

[0160] <Other additives> The pharmaceutical composition of the present embodiment can also be administered separately, or can also be administered according to conventional means with a pharmacologically acceptable carrier. When used in combination with a pharmacologically acceptable carrier, for example, it can be mixed with the above-mentioned hyaluronic acid derivative and the above-mentioned active ingredient and the adjuvant added as required and water or its physiologically acceptable liquid (such as normal saline, aqueous ethanol, phosphate buffered saline (PBS)) etc., and can also contain physiologically acceptable buffer, excipient, vehicle, preservative, stabilizer, bonding agent, freeze-dried supplement etc.

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

[0162] Examples of the preservative include benzalkonium chloride, methyl paraben, propyl paraben, chlorobutanol, sorbic acid, and alkyldiaminoethylglycine.

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

[0164] As the pharmaceutical composition of this embodiment, a pharmaceutical composition in the form of a preparation can be used. The preparation can be in the form of a solid, semisolid or liquid. In the case of a solid, the form may include powder, granules, pills, granules, tablets, capsules, etc. Among them, freeze-dried powder is preferred as a solid. In the case of a semisolid, the form of a gel or the like is exemplified. In the case of a liquid, the form may be a suspension obtained by diluting or suspending a powder in water or a buffer solution such as phosphate buffered saline (PBS).

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

[0166] The method for producing a pharmaceutical composition according to the present embodiment is a method for producing a pharmaceutical composition containing a hyaluronic acid derivative and a medicinal ingredient, and includes the following steps. A preparation step of dissolving the above-mentioned medicinal ingredient in an organic solvent or water containing an organic solvent to prepare an oil phase containing the above-mentioned 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 of the oil phase and the aqueous phase containing the hyaluronic acid derivative becomes 20:100 to 0.01:100 in terms of volume ratio.

[0167] All along, due to the ratio of the oil phase to the aqueous phase, the interaction between the steryl groups in the hyaluronic acid derivative is hindered by the oil phase, and the particles are dissociated or partially condensed, etc., making it difficult to maintain the particle size of the hyaluronic acid derivative. Therefore, the pharmaceutical composition only contains a complex of a hyaluronic acid derivative of a smaller size from which particles have dissociated or a hyaluronic acid derivative containing partially condensed particles and an active ingredient. In addition, since the pharmaceutical composition is manufactured using a dialysis method, there is a trend that it is difficult to control the weight ratio of the hyaluronic acid derivative and the active ingredient. Further, in order to reach the active ingredient concentration of the target, a concentration step is also required.

[0168] In this regard, for the manufacturing method of the pharmaceutical composition of the present embodiment, dialysis is not used, and the mixing ratio of the above-mentioned composition, especially the oil phase and the aqueous phase, is made to be within the above-mentioned numerical range by mixing, so that the particle size distribution of the hyaluronic acid derivative before the manufacture of the pharmaceutical composition can be maintained, and a complex with the pharmaceutical ingredient can be formed, and the weight ratio of the hyaluronic acid derivative and the pharmaceutical ingredient can also be controlled to a certain extent. Thus, a pharmaceutical ingredient-hyaluronic acid derivative complex with stable structure can be obtained. As a result, a pharmaceutical composition with better delivery to immune cells in lymph nodes and the activation ability of the immune cells can be obtained.

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

[0170] [Preparation process] 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] From the viewpoint of being soluble in an organic solvent or water containing an organic solvent, a poorly water-soluble component can be preferably used as the medicinal component. Specifically, the components exemplified in the above-mentioned "medical component" can be used as the medicinal component.

[0172] Examples of the organic solvent for dissolving the medicinal ingredient include known organic solvents commonly used in the preparation of pharmaceutical compositions, and specific examples thereof include 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, and dimethylformamide.

[0173] [Mixing process] In the mixing step, the oil phase obtained in the preparation step and the aqueous phase containing the hyaluronic acid derivative are mixed at a volume ratio of 20:100 to 0.01:100.

[0174] Hyaluronic acid derivatives are used in advance by being dissolved or dispersed in water etc. with the concentration becoming expectation.At this moment, the pH of the aqueous phase containing hyaluronic acid derivatives is preferably more than 6.00, preferably more than 6.00 and below 11.00.By making pH be more than the above-mentioned lower limit, can more effectively suppress the coagulation of hyaluronic acid derivatives, form a complex with the medicinal ingredient, thus, can obtain the medicinal ingredient-hyaluronic acid derivative complex with more stable structure.By making pH be below the above-mentioned upper limit, can maintain the dispersion stability of hyaluronic acid derivatives, by drug compounding.In addition, because the electric charge of medicine tends to negative electricity, therefore also have the hydrophobic part of hyaluronic acid derivatives and the one side that medicine is more easily interacted.On the other hand, by making pH be below the above-mentioned upper limit, the main chain decomposition of hyaluronic acid derivatives can be suppressed more.

[0175] In the mixing step, due to the hydrophobic interaction between the sterol group of the hyaluronic acid derivative and the medicinal ingredient, a medicinal ingredient-hyaluronic acid derivative complex is formed, which has a structure in which the sterol group and the medicinal ingredient are present inside and the hydrophilic part derived from hyaluronic acid is present on the outer edge. Depending on the structure of the medicinal ingredient, the complex can be formed not only by the above-mentioned hydrophobic interaction but also by electrostatic interaction, hydrogen bonding, etc.

[0176] In the mixing step, the mixing ratio of the oil phase to the aqueous phase is 20:100 to 0.01:100, preferably 10:100 to 0.05:100, more preferably 5:100 to 0.1:100, and further preferably 2.5:100 to 0.5:100 in terms of volume ratio. By making the mixing ratio of the oil phase to the aqueous phase 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 interaction between the steryl groups of the particles of the hyaluronic acid derivative can be effectively inhibited from being hindered by the oil phase and causing association and dissociation. However, it is not limited to hydrophobic interactions, and the collapse of interactions that can contribute to structural stability, such as electrostatic interactions and hydrogen bonds, can also be effectively inhibited.

[0177] In the mixing step, the concentration of the medicinal ingredient dissolved in the oil phase is not particularly limited, but is preferably 0.1 parts by mass or more and 50 parts by mass or less, more preferably 0.2 parts by mass or more and 25 parts by mass or less, and further preferably 0.5 parts by mass or more and 10 parts by mass or less, relative 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. The concentration of the hyaluronic acid derivative is preferably 0.01 parts by mass to 10 parts by mass, more preferably 0.1 parts by mass to 10 parts by mass, and further preferably 0.5 parts by mass to 5 parts by mass, relative to 100 parts by mass of the aqueous phase.

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

[0180] In the mixing step, the method is not particularly limited. For example, an oil phase containing a medicinal ingredient is added to an aqueous phase containing the above-mentioned hyaluronic acid derivative in any proportion, or an aqueous phase containing a medicinal ingredient is added to an oil phase containing the above-mentioned hyaluronic acid derivative in any proportion, and a mixing step is performed in which the final mixing ratio of the oil phase to the aqueous phase becomes 20:100 to 0.01:100 in terms of volume ratio, thereby maintaining the particle size of the hyaluronic acid derivative.

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

[0182] The material and shape of the reactor in the flow method are not particularly limited as long as they are suitable for the production method of the present invention. For example, the inner diameters of the pipes through which the oil phase and the water phase pass may be different.

[0183] The material and shape of the pipe in the flow method are not particularly limited as long as they are applicable to the method for producing the pharmaceutical composition of this embodiment. Examples thereof include tubes made of Teflon (registered trademark), stainless steel tubes, glass tubes, and plastic tubes.

[0184] In the mixing step, the time is not particularly limited, and may be, for example, 30 minutes to 90 hours, or 1 hour to 80 hours.

[0185] The mixing step may further include a step of removing the organic solvent that has dissolved the medicinal ingredient by tangential flow filtration (TFF), centrifugal filtration, multiple TFFs in an ultrafiltration / diafiltration (UFDF) membrane, etc. after the medicinal ingredient is complexed with the hyaluronic acid derivative.

[0186] [Sterilization process] After the mixing step, a sterilization step can be performed to sterilize the obtained solution containing the medicinal ingredient-hyaluronic acid derivative complex to obtain a sterilized preparation. As the sterilization method of the preparation, filtration sterilization using a sterilizing filter, gas sterilization, gamma ray sterilization or electron beam sterilization can be mentioned. Filtration sterilization using a sterilizing filter is most preferred.

[0187] [Sterilization process] After the mixing step, a sterilization step may be performed to sterilize the solution containing the medicinal ingredient-hyaluronic acid derivative complex to obtain a sterilized product. The sterilization method may be the same as the method exemplified in the above-mentioned "method for producing hyaluronic acid derivatives".

[0188] <Administration Method> Examples of subjects to whom the pharmaceutical composition of the present embodiment can be administered include animals classified as mammals including humans (monkeys, marmosets, mice, rats, cows, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.).

[0189] The administration route may be, for example, intramedullary injection, intraarterial injection, intravenous injection, subcutaneous injection, etc., and may be intranasal, transbronchial, transpulmonary, intramuscular, transdermal or oral, as known to those skilled in the art. Wherein, when the pharmaceutical composition of the present embodiment is a vaccine composition, subcutaneous injection or intramuscular injection is preferred.

[0190] In the pharmaceutical composition of the present embodiment, in the case of non-oral administration, the dosage can be appropriately selected in consideration of the type of the subject of administration (including age, gender, etc.). For example, in humans (weight 60 kg), the amount of the active ingredient (preferably an antigen) per dose can be set to more than 0.01 μg and less than 5 mg, can be set to more than 0.1 μg and less than 500 μg, and can be set to more than 1 μg and less than 100 μg.

[0191] The administration frequency may be a single administration of the above dosage, or multiple administrations of the above dosage may be administered once every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 1 month, every 2 months, every 3 months or every 6 months, or more than 2 times. Alternatively, administration may be performed at 2 or more sites in 1 administration.

[0192] <Other Implementation Methods> 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, comprising the step of administering an effective amount of the above-mentioned pharmaceutical composition to a patient or animal. Infectious diseases include those listed as examples of the "antigen derived from an infectious disease" in the above-mentioned "antigen". In addition, the "effective amount" referred to herein refers to an amount effective for prevention or treatment, that is, it includes an amount suitable for the prevention or treatment of the onset of the above-mentioned diseases.

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

[0194] In one embodiment, the present invention provides a use of the above-mentioned medicinal ingredient-hyaluronic acid derivative complex for preparing 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 in that it comprises a composition containing a hyaluronic acid derivative and an antigen and lymphocytes expressing immune receptors for the above antigen. By combining the composition containing a hyaluronic acid derivative and an antigen with antigen-specific lymphocytes, the anti-tumor effect can be exerted more strongly, more effectively, more continuously and / or in a wider range.

[0196] The present application provides a pharmaceutical composition for combined use with a composition containing the above-mentioned hyaluronic acid derivative and an antigen, wherein the pharmaceutical composition contains lymphocytes expressing immune receptors against the antigen.

[0197] The pharmaceutical composition characterized by being composed of 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 hyaluronic acid derivatives and antigen is preferably administered before administration of antigen-specific lymphocytes. In a preferred embodiment of the present invention, when the composition containing hyaluronic acid derivatives and antigen is administered once and injected with 1 antigen-specific lymphocyte as the unit of administration number of times, preferably after administration of 1 unit or 2 units, the composition containing hyaluronic acid derivatives and antigen is administered at least once.

[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, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days or more than 14 days. In addition, the interval can be, for example, less than 28 days, less than 24 days, less than 21 days, less than 17 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days or less than 2 days.

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

[0201] In the case of combined administration, 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 0.1 to 20 mg / time, 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.

[0202] The antibody can be administered at the same time as the composition containing the hyaluronic acid derivative and the antigen (including the case where the antibody is contained in the preparation), or at a different time. When administered at different times, it is preferred to administer one, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours, after administering one.

[0203] The composition containing hyaluronic acid derivatives and antigens can also be administered in combination with the above-mentioned adjuvant and antibody. The dosage of the composition containing hyaluronic acid derivatives and antigens at this time is, for example, 0.01 to 100 mg / time, preferably 0.1 to 50 mg / time, and 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, and more preferably 0.1 to 10 mg / kg body weight; 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, and more preferably 1 to 40 mg / kg body weight. The adjuvant and the antibody can be administered at the same time as the composition containing the hyaluronic acid derivative and the antigen (including the case where the adjuvant is contained in the preparation), or at different times. In the case of administration at different times, it is preferred that all of the vaccine preparation, adjuvant and antibody are administered within, for example, 1 minute to 24 hours, preferably within 1 minute to 5 hours.

[0204] In one embodiment, the present invention provides a composition for enhancing, promoting or strengthening the incorporation of a medicinal ingredient into immune cells, which contains the medicinal ingredient-hyaluronic acid derivative complex.

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

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

[0207] In one embodiment, the present invention provides a method for enhancing, promoting or strengthening the incorporation of a pharmaceutical ingredient into immune cells in vivo or in vitro, comprising the step of administering a composition containing the pharmaceutical ingredient-hyaluronic acid derivative complex.

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

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

[0210] Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not intended to be limited to the examples.

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

[0212] 1. Process 1 (Synthesis of cholesteryl 6-aminohexyl carbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following step 1-1 and then according to the step 1-2.

[0213] (1) Process 1-1 Under an argon atmosphere, triethylamine (TEA, 1.05 mL) was added to a solution of cholesterol acetyl chloride (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL) and stirred. After stirring for 30 minutes under ice cooling, the mixture was warmed to room temperature (about 25°C) and stirred overnight, except that 6-(tert-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise under ice cooling. The reaction mixture was washed with ultrapure water and saturated brine, sterilized with anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified using silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:4), combined with the fraction of the target product, and the solvent was distilled off under reduced pressure.

[0214] (2) Process 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 generated precipitate was recovered by centrifugation. The obtained solid was washed 4 times with ethyl acetate and dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexyl carbamate hydrochloride (Chol hydrochloride).

[0215] 2. Process 2 (Preparation of Tetrabutylammonium (TBA) Salt of Hyaluronic Acid) According to the following step 2-1, and then according to the step 2-2, TBA salt of hyaluronic acid (HA) (HA-TBA) was prepared.

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

[0217] (2) Process 2-2 The raw material sodium hyaluronic acid 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 a cation exchange resin chlorinated with TBA in "(1) Step 2-1" was added in a 5-fold molar equivalent relative to the molar number of HA units (unit molecular weight 401.3) according to the ion exchange capacity of the resin. After stirring at room temperature (about 25°C) for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain TBA salt of hyaluronic acid (HA-TBA) as a white solid.

[0218] 3. Process 3 Prepare an anhydrous DMSO solution (10 mg / mL) of the HA-TBA prepared in "2. (2) Step 2-2". Then, add Chol hydrochloride in an amount such that the molar ratio is 44 / 100 relative to the disaccharide repeating unit (HA unit) present in the HA-TBA synthesized in "1. Step 1". Next, add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMT-MM) in an amount such that the molar ratio is 48 / 100 relative to the HA unit, and stir at room temperature (about 25°C) overnight. The reaction solution is dialyzed using a Spectra / Por 7 dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cutoff (MWCO): 3,500) in the order of 0.3M ammonium acetate / DMSO solution, 0.15M NaCl aqueous solution, and ultrapure water. The obtained dialysate was freeze-dried to obtain the target product (HA-C6-Chol) as a white solid.

[0219] The cholesterol incorporation rate in the obtained white solid was calculated as follows. A 0.02N DCl DMSO-d6 / D2O mixed solution (2N DCl D2O:DMSO-d6=1:99) was used as the measurement solvent, and a JNM-ECS400 (manufactured by JEOL Ltd.) was used as the NMR apparatus. The measurement was performed at 85°C to obtain a white solid. 1 H-NMR spectrum. 1 In the H-NMR spectrum, peaks derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm or more, 2.0 ppm or less, 3H) and peaks derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%.

[0220] A freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at a concentration of 1 mg / mL, and the results were measured by gel permeation chromatography under the following conditions: Figure 3A The obtained chromatogram.

[0221] (Measurement conditions) Device: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8 μm, inner diameter 7.8 mm, length 30 cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0222] [Synthesis example 1-2] According to the same procedure as in Synthesis Example 1-1, a hyaluronic acid derivative was synthesized. In the generated 1 In the H-NMR spectrum, the peaks of the acetyl group derived from N-acetyl-D-glucosamine (COCH3, 1.6ppm or more, 2.0ppm or less, 3H) and the peaks of the methyl group derived from the cholesteryl group (CH3, 0.7ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. The measurement based on gel permeation chromatography was carried out under the conditions shown in the above-mentioned synthesis example 1-1, and the obtained chromatogram is as follows: Figure 3B shown.

[0223] [Synthesis Example 1-3] According to the same procedure as in Synthesis Example 1-1, a hyaluronic acid derivative was synthesized. In the generated 1In the H-NMR spectrum, the peaks of the acetyl group derived from N-acetyl-D-glucosamine (COCH3, 1.6ppm or more, 2.0ppm or less, 3H) and the peaks of the methyl group derived from the cholesteryl group (CH3, 0.7ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. The measurement based on gel permeation chromatography was carried out under the conditions shown in the above-mentioned synthesis example 1-1, and the obtained chromatogram is as follows: Figure 3C shown.

[0224] [Example 1-1] The preparation and separation of the hyaluronic acid derivative obtained in Synthesis Example 1-1 were performed as follows. The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis box (Float-A-Lyzer G2, MWCO: 300,000, Ieda Trading Co., Ltd.), and dialyzed 9 times against 10 mM phosphate buffer pH 7.4. The hyaluronic acid derivative in the obtained dialysate was measured by gel permeation chromatography. Figure 4A The obtained chromatogram. Fig.21 and Fig. 22 This is a diagram showing superimposed chromatograms of the hyaluronic acid derivative prepared in Example 1-1 and polyacrylic acid as a standard substance in order to explain the calculation method of the area ratio A1 / A2 and the distance ratio Da / Db described later. The filtrate was concentrated to a desired concentration using an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius).

[0225] [Example 1-2] The hyaluronic acid derivative obtained in Synthesis Example 1-2 was prepared and separated in the same manner as in Example 1-1. The hyaluronic acid derivative in the obtained dialysate was measured by gel permeation chromatography. Figure 4B The obtained chromatogram.

[0226] [Examples 1-3] The hyaluronic acid derivative obtained in Synthesis Example 1-3 was prepared and separated in the same manner as in Example 1-1. The hyaluronic acid derivative in the obtained dialysate was measured by gel permeation chromatography. Figure 4C The obtained chromatogram.

[0227] [Comparative Example 1-1] The preparation and separation of the hyaluronic acid derivative obtained in Synthesis Example 1-1 were performed as follows. The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at 10 mg / mL and diluted to 10 mM phosphate buffer (pH 7.4) using 100 mM phosphate buffer. Afterwards, an ultrafilter (Vivaspin (registered trademark) 20, MWCO: 300,000, Sartorius) was used for filtration, and the hyaluronic acid derivative in the obtained filtrate was determined based on gel permeation chromatography. Figure 5A The filtrate was concentrated to a desired concentration using an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius).

[0228] [Comparative Example 1-2] The hyaluronic acid derivative prepared in Synthesis Example 1-2 was prepared and separated in the same steps as in Comparative Example 1-1. The hyaluronic acid derivative in the obtained filtrate was concentrated to the desired concentration using a superconcentrator (Vivaspin20, MWCO: 10,000, Sartorius). After that, it was diluted to 1 mg / mL using water for injection and measured by gel permeation chromatography. Figure 5B The obtained chromatogram.

[0229] [Comparative Examples 1-3] The hyaluronic acid derivative prepared in Synthesis Example 1-3 was prepared and separated in the same steps as Comparative Example 1-2. The hyaluronic acid derivative in the obtained filtrate was concentrated to the desired concentration using a superconcentrator (Vivaspin20, MWCO: 10,000, Sartorius). After that, it was diluted to 1 mg / mL using water for injection and measured by gel permeation chromatography. Figure 5C The obtained chromatogram.

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

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

[0232] According to the following method, from Fig.21 In the gel permeation chromatogram shown, areas A1 and A2 were calculated, and the area ratio A1 / A2 was calculated.

[0233] (i) First, the intersection of a vertical line drawn from the maximum refractive index intensity point Kb on the chromatogram of the standard substance 50 kDa polyacrylic acid to the baseline B and the chromatogram of the above hyaluronic acid derivative is set as Ub; the intersection of a vertical line drawn from the maximum refractive index intensity point Kb to the baseline B and the baseline is set as Bb. At this time, the dissolution time at the maximum refractive index intensity point Kb is 7.41 minutes. (ii) Next, on the chromatogram of the hyaluronic acid derivative, the area value enclosed by the curve from Ub to the end point, the vertical line drawn from the point of maximum refractive index intensity Kb to the baseline B, and the baseline is set to A2; on the chromatogram of the above hyaluronic acid derivative, the area value enclosed by the curve from the starting point to the above Ub, the vertical line drawn from the above point of maximum refractive index intensity Kb to the above baseline B, and the baseline is set to A1.

[0234] According to the following method, from Fig. 22 The distances Da and Db were calculated from the gel permeation chromatogram shown, and the distance ratio Da / Db was calculated.

[0235] (i) First, a vertical line was drawn from the maximum refractive index intensity point Ka on the chromatogram of the standard substance 150 kDa polyacrylic acid to the baseline B, and the intersection of the vertical line and the baseline was set as Ba, and the length between the maximum refractive index intensity point Ka and Ba was set as La. The dissolution time at the maximum refractive index intensity point Kb was 6.46 minutes. (ii) Next, of the two points on the chromatogram with a refractive index intensity of La / 20, the point with an earlier dissolution time is defined as point R1, and the point with a later dissolution time is defined as point S1. The dissolution time at point R1 is 5.74 minutes, and the dissolution time at point S1 is 7.68 minutes. (iii) Next, the intersection of a vertical line drawn from the maximum refractive index intensity point Kb on the chromatogram of the standard substance 50 kDa polyacrylic acid to the baseline B and the baseline is defined as Bb. At this time, 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 above-mentioned point R1 and the above-mentioned point S1 and the vertical line drawn from the above-mentioned point Ka with the maximum refractive index intensity to the baseline B is set as Ta, and the intersection point of the vertical line drawn from the above-mentioned point Kb with the above-mentioned maximum refractive index intensity to the baseline B and the above-mentioned straight line D1 is set as Tb. (v) Finally, let the distance between the point R1 and Ta be Da, and let the distance between Ta and Tb be Db.

[0236] The area ratio A1 / A2 and the distance ratio Da / Db were calculated from the gel permeation chromatograms of the hyaluronic acid derivatives prepared 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 in the same manner as for the hyaluronic acid derivative prepared in Example 1-1.

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

[0238] [Table 1] A1 / A2 Da / Db Synthesis example 1-1 0.51 0.68 Synthesis example 1-2 0.89 0.69 Synthesis Example 1-3 0.60 0.71 Example 1-1 1.79 0.76 Example 1-2 4.24 0.91 Examples 1-3 3.87 0.92 Comparative Example 1-1 0.31 0.22 Comparative Example 1-2 0.245 0.021 Comparative Examples 1-3 0.350 0.28 Comparative Examples 1-4 0.343 -

[0239] As shown in Table 1, the hyaluronic acid derivatives of Examples 1-1 to 1-3 having higher area ratios A1 / A2 than the hyaluronic acid derivatives of Synthesis Examples 1-1 to 1-3 before purification were obtained.

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

[0241] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(serial number 1)

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

[0243] Afterwards, solid refined sucrose (manufactured by Fujifilm and Light Company, special for manufacturing, product number 198-18385) is added so that sucrose is 10% by mass relative to the total mass of the composition and sucrose containing fluorescently labeled peptide-hyaluronic acid derivative complex, and stirred for more than 1 hour at room temperature (about 25 ° C). Confirm that sucrose is fully dissolved, and the concentration of peptide is diluted to about 0.3mg / mL with 10mM phosphate buffer pH7.4 containing 10% by mass sucrose, and 0.2μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25mmΦ) is used for sterilization filtration, and the pharmaceutical composition containing fluorescently labeled peptide-hyaluronic acid derivative complex is obtained. Relative to the pharmaceutical composition of 100 mass parts, the content of fluorescently labeled peptide is 0.0317 mass parts.

[0244] The hyaluronic acid derivative was diluted to 1 mg / mL by the method described in Synthesis Example 1-1, and the measurement was performed based on gel permeation chromatography. As a result, no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after the peptide was encapsulated ( Fig. 6A ). At this time, the peptide concentration was 0.1 mg / mL, and it was also confirmed that the introduced peptide was 100% enclosed.

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

[0246] [Example 2-3] Using the hyaluronic acid derivative prepared in Example 1-3, a pharmaceutical composition containing a fluorescent labeled peptide-hyaluronic acid derivative complex was prepared by the same procedure as in Example 2-1. The content of the fluorescent labeled peptide was 0.0303 parts by mass relative to 100 parts by mass of the pharmaceutical composition. The measurement based on gel permeation chromatography was carried out by the method described in Synthesis Example 1-1. As a result, no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after the peptide was encapsulated ( 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 another container, the fluorescent labeled peptide was dissolved in DMSO so that the concentration was 50 mg / mL.

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

[0249] Afterwards, add solid refined sucrose (manufactured by Fujifilm and Light Company, special for manufacturing, product number 198-18385) so that sucrose is 10% by mass relative to the total mass of the composition and sucrose containing fluorescently labeled peptide-hyaluronic acid derivative complex, and stir for more than 1 hour at room temperature (about 25 ° C). Confirm that sucrose is fully dissolved, and the concentration of peptide is diluted to 0.3mg / mL with 10mM phosphate buffer pH7.4 containing 10% by mass sucrose, and sterile filtration is performed with 0.2μm PES (polyethersulfone) (manufactured by Pall Company, Acrodisc syringe filter, 25mmΦ), and a pharmaceutical composition containing fluorescently labeled peptide-hyaluronic acid derivative complex is obtained. Relative to the pharmaceutical composition of 100 mass parts, the content of fluorescently labeled peptide is 0.0308 mass parts.

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

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

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

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

[0254] Afterwards, transfer to a dialysis cassette (manufactured by Thermo Company, Slide-A-Lyzer G2 Dialysis Cassettes, 3.5KMWCO, 15mL, No.87724), and dialyze using 0.6M phosphate buffered saline pH7.4 containing urea. Next, dialyze in the order of 0.06M phosphate buffered saline pH7.4 containing urea and phosphate buffered saline pH7.4. Further, use an ultraconcentrator (Vivaspin20, MWCO: 10,000, Sartorius) to concentrate it to the desired concentration.

[0255] Finally, sterile filtration was performed using 0.2 μm PES (polyethersulfone) (manufactured by Pall Corporation, Acrodisc syringe filter, 25 mmφ) to prepare a pharmaceutical composition containing a fluorescently labeled peptide-CHP complex. The content of the fluorescently labeled peptide was 0.0420 parts by mass per 100 parts by mass of the pharmaceutical composition.

[0256] [Comparative Example 4-1] The freeze-dried product of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added to a concentration of 5 mg / mL, followed by stirring overnight to allow the mixture to fully dissolve. On the other hand, in another container, a fluorescent labeled peptide was dissolved in DMSO to a concentration of 50 mg / mL.

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

[0258] Afterwards, solid refined sucrose (manufactured by Fujifilm and Light Company, special for manufacturing, product number 198-18385) is added so that sucrose is 10% by mass relative to the total mass of the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose, and stirred for more than 1 hour at room temperature (about 25 ° C). Confirm the full dissolution of sucrose, and the concentration of peptide is diluted to 0.3mg / mL with 25mM phosphate buffer pH7.4 containing sucrose by 10 mass%, and 0.2μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25mmΦ) is used for sterilization filtration, and the pharmaceutical composition containing fluorescently labeled peptide-hyaluronic acid derivative complex is obtained. Relative to the pharmaceutical composition of 100 mass parts, the content of fluorescently labeled peptide is 0.0316 mass parts.

[0259] [Comparative Example 4-2] Using the freeze-dried product of the hyaluronic acid derivative obtained in Synthesis Example 1-2, a composition containing a fluorescent labeled peptide-hyaluronic acid derivative complex was prepared in the same manner as in Comparative Example 4-1.

[0260] Afterwards, solid refined sucrose (manufactured by Fujifilm and Light Company, special for manufacturing, product number 198-18385) is added so that sucrose is 10% by mass relative to the total mass of the composition and sucrose containing fluorescently labeled peptide-hyaluronic acid derivative complex, and stirred for more than 1 hour at room temperature (about 25 ° C). Confirm the full dissolution of sucrose, the concentration of peptide is diluted to 0.33mg / mL with 30mM phosphate buffer pH7.4 containing 10% by mass sucrose, and 0.2μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25mmΦ) is used for sterilization filtration, and the pharmaceutical composition containing fluorescently labeled peptide-hyaluronic acid derivative complex is obtained. Relative to the pharmaceutical composition of 100 mass parts, the content of fluorescently labeled peptide is 0.0358 mass parts.

[0261] [Confirmation of fluorescent labeled peptide concentration] By high performance liquid chromatography under the following conditions, it was confirmed that the fluorescently labeled peptide 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 was complexed and solubilized with the hyaluronic acid derivative. In addition, the concentration of the fluorescently labeled peptide in each pharmaceutical composition was provided.

[0262] (Measurement conditions) HPLC device: Japan Spectroscopic HPLC-EXTREMA Chromatographic column: PLRP-S1000A 8μm, length 50mm×inner diameter 4.6mm (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: 2mL / min Injection volume: 30μL Detector: UV (215nm) Gradient program: as shown in the following Table 2. In Table 2, "%" means "v / v%".

[0263] [Table 2] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0→0.5 5 95 0.5→3.4 5→100 95→0 3.4→3.45 100→5 0→95 3.45→13.0 100→5 0→95

[0264] [Test Example 1-1] (Analysis of peptide incorporation into myeloid cells in lymph nodes and activation) 1. Materials Adjuvant: CpG oligo DNA1668 (purchased from Ajinomoto Bio-Pharma GeneDesign) Compositions for administration: pharmaceutical compositions of Example 2-1, Comparative Example 2-1, Comparative Example 3 and Comparative Example 4-1.

[0265] The fluorescent labeled antibodies used for evaluation are as follows and 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] In addition, the combination of antibodies used in the FACS analysis described below is as follows.

[0267] [Table 3]

[0268] 2. Analytical methods The pharmaceutical compositions of Example 2-1, Comparative Example 2-1 and Comparative Example 4-1 were diluted with 10 mass% sucrose 10mM phosphate buffer pH 7.4 to a fluorescent (fluorescein) labeled peptide concentration of about 0.3mg / mL. In addition, the pharmaceutical composition of Comparative Example 3 was diluted with phosphate buffered saline pH 7.4, and then the dilutions of each pharmaceutical composition were subcutaneously administered to the right back of BALB / c mice at 60μg of peptide. At the same time, 50μg of CpG oligoDNA1668 (Ajinomoto Bio-Pharma Gene Design) dissolved in PBS was administered as an adjuvant.

[0269] 20 hours after the start of administration, the lymph nodes at the administration site (right inguinal lymph nodes) were extracted, the lymph nodes were ground using a slide, and the cells were suspended in RPMI1640 medium. At this time, the cells of 2 mice in 1 group were combined. After centrifugation (400×g, 5 minutes, 4°C), the supernatant was removed, and the cells were washed twice with RPMI1640 medium and suspended in RPMI1640 medium containing 10v / v% FBS. The number of cells was measured using a hemocytometer, and the cell concentration was prepared to be 1.2×10 7 Pieces / mL.

[0270] Add 50 μL of cell suspension to each well of a 96-well V-bottom microplate (Nunc, Thermo Fisher Scientific) to 6 × 10 5 . The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C), and after removing the supernatant, it was washed twice with 200 μL of staining buffer (PBS containing 0.5 v / v% FBS) and then suspended. After centrifugation to remove the supernatant, 50 μL of a solution containing 50-fold diluted anti-mouse CD16 / CD32 antibodies was added, and the cells were allowed to stand in a dark room at 4°C for 10 minutes. 150 μL of staining buffer was added, the supernatant was removed by centrifugation, and the cells were further washed twice with 200 μL of staining buffer. According to the concentration recommended by the manufacturer of each antibody, 50 μL of a solution containing the first, second and third antibodies was added and mixed to form a combination described in Table 3 above, and then allowed to stand in a dark room at 4°C for 15 minutes. 150 μL of staining buffer was added, the supernatant was removed by centrifugation, and the cells were further washed twice with 200 μL of staining buffer. After centrifugation and removal of the supernatant, the cells were resuspended in 200 μL of staining buffer and transferred to a round-bottom polystyrene tube (BD Bioscience). The cells were analyzed using a flow cytometer FACSCanto II (BD Bioscience) and the accompanying analysis software (FACSDiva).

[0271] Dendritic cells (DC) were detected as a CD11c-positive group, macrophages (Mph) were detected as a F4 / 80-positive group, and type 1 conventional DC (cDC1) was detected as a CD11c-positive and XCR1-positive group. For each cell type, FITC (fluorescein) positive cells were detected as peptide-incorporated cells. The expression of costimulatory molecules CD80 and CD86 was analyzed for FITC-positive cells of each cell type. FACS analysis methods such as Figure 7A to Figure 7F shown. In addition, the analysis results are shown in Tables 4-1 to 4-3. For example, in Table 4-1, "peptide + CD80 + DC" means "peptide-incorporated dendritic cells (DC) expressing CD80", "peptide + CD86 + DC" means "peptide-incorporated dendritic cells (DC) expressing CD86", and "peptide + DC" means "peptide-incorporated 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, it was confirmed that in the mouse group administered with the pharmaceutical composition of Example 2-1, the co-stimulatory molecules (CD80 and CD86) in macrophages and DC (especially cDC1) were expressed more highly than in the mouse group administered with the pharmaceutical composition of Comparative Example 2-1 and Comparative Example 4-1 (hyaluronic acid derivatives before entrapment). In addition, it was confirmed in DC (especially cDC1) that in the mouse group administered with the pharmaceutical composition of Example 2-1, the co-stimulatory molecules (CD80 and CD86) in macrophages and DC (especially cDC1) were expressed more highly than in the mouse group administered with the pharmaceutical composition of Comparative Example 3 as other polysaccharide (pullulan) derivatives.

[0276] [Test Example 1-2] The same method as in Test Example 1-1 was followed except that the pharmaceutical compositions of Example 2-2, Comparative Example 2-2, and Comparative Example 4-2 were diluted with 10 mass% sucrose 10 mM phosphate buffer pH 7.4 to a fluorescent (fluorescein) labeled peptide concentration of about 0.2 mg / mL and 40 μg of each peptide was subcutaneously administered. 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] As in Test Example 1-1, in the mouse group administered with the pharmaceutical composition of Example 2-2, it was also confirmed that the co-stimulatory molecules (CD80 and CD86) in macrophages and DC (especially cDC1) were expressed more highly than those in the mouse group administered with the pharmaceutical composition of Comparative Example 2-2 and Comparative Example 4-2 (pre-entrapment hyaluronic acid derivative). Although the expression of CD80 and CD86 relative to cDC1 was higher in Comparative Example 4-2, the expression of CD80 and CD86 in macrophages was lower, which was worse than the pharmaceutical composition of Example 2-2.

[0281] [Test Examples 1-3] The same method as in Test Example 1-1 was followed except that the pharmaceutical composition of Example 2-3 was used and 40 μg of each peptide was subcutaneously administered. 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] As in Test Examples 1-1 and 1-2, in the mouse group administered with the pharmaceutical composition of Example 2-3, high expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (particularly cDC1) was confirmed.

[0286] It is reported that when the expression of CD86 on macrophages decreases, it will exert or is exerting an immunosuppressive effect (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.). Based on the content of the report in the above reference 3 and the results of the above test examples 1-1 to 1-3, it is expected that the pharmaceutical compositions of Examples 2-1, 2-2 and 2-3, which highly express CD86 not only in DCs but also in macrophages, will induce stronger immunity.

[0287] Based on these results, it is speculated that by using the pharmaceutical composition of this embodiment, the active ingredients such as peptides can be stably and effectively delivered to immune cells (especially cDC1, macrophages), and further, while improving, promoting or enhancing the incorporation of the active ingredients into immune cells, the activity of immune cells (especially the expression of co-stimulatory molecules CD80 and CD86 in cDC1) can be improved, promoted, maintained or enhanced.

[0288] <Study on 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 a peptide, gp100RP2RP1_6Y peptide manufactured by Biologica and having the following amino acid sequence was used.

[0290] SVYDFFVWLYYYYYYTWHRYHLLYYYYYYEGSRNQDWL (serial number 2)

[0291] Specifically, first, the freeze-dried product of the hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added to the mixture so as to have the concentration shown in Table 7 below, and the mixture was stirred overnight to be fully dissolved. On the other hand, in another container, the peptide was dissolved in DMSO so as to have a concentration of 50 mg / mL. After confirming the dissolution, 1 mol / L of sodium hydroxide aqueous solution (manufactured by Fujifilm Wako Co., Ltd.) was added to the above hyaluronic acid derivative aqueous solution according to the following Table 7.

[0292] The composition containing the peptide-hyaluronic acid derivative complex was prepared by mixing the hyaluronic acid derivative aqueous solution and the peptide dimethyl sulfoxide solution at room temperature (about 25° C.) at the volume ratio described in the following Table 7 and stirring at room temperature (about 25° C.) for 24 hours. 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 was visually determined 24 hours later to check whether white turbidity had occurred. The results are shown in Table 7 below.

[0294] [Table 7]

[0295] Afterwards, add solid refined sucrose (manufactured by Fujifilm and Light Company, special for manufacture, product number 198-18385) so that sucrose relative to the total mass of the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose reaches 10 mass %, and stir for more than 1 hour at room temperature (about 25 ° C). Then, the theoretical concentration of peptide is diluted to 0.3 mg / mL with 25mM phosphate buffer pH7.4 containing 10 mass % sucrose, and 0.2 μm PES (polyether sulfone) (manufactured by Pall Company, Acrodisc syringe filter, 25mmΦ) is used for sterilization filtration, and the pharmaceutical composition containing peptide-hyaluronic acid derivative complex is obtained. According to the same method as in Example 2-1 above, the peptide concentration in the pharmaceutical composition containing peptide-hyaluronic acid derivative complex at this time is quantified, and the results are as shown in Table 7 above.

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

[0297] (Measurement conditions) DLS device: Otsuka Electronics, ELSZ2000 Sample pool: micro-particle size sample pool Temperature: 25℃ Concentration of peptide-hyaluronic acid derivative complex: 4.95 mg / mL

[0298] As shown in Table 7 above, in the manufacture of the pharmaceutical composition, by adjusting the pH of the mixed solution to a specific range, it is apparent that the particle size of the complex of the active ingredient such as a peptide and the hyaluronic acid derivative can be maintained within a certain range, thereby making the stability of the complex in the solution 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 a peptide-hyaluronic acid derivative complex was prepared.

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

[0301] GSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYP (serial number 3)

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

[0303] The composition containing the peptide-hyaluronic acid derivative complex was prepared by mixing the hyaluronic acid derivative aqueous solution and the peptide DMSO solution at a volume ratio according to the following Table 8 at room temperature (about 25°C), and stirring at room temperature (about 25°C) for 24 hours. The pH of the solution at this time was also measured. Afterwards, solid refined sucrose (manufactured by Fujifilm and Light, dedicated for manufacture, product number 198-18385) is added so that the total mass of sucrose relative to the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose reaches 10% by mass, and stirred for more than 1 hour at room temperature (about 25 ° C). Then, 0.2 μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25 mm Φ) is used for sterile filtration to obtain a pharmaceutical composition containing a peptide-hyaluronic acid derivative complex. According to the same method as in Example 2-1 above, the peptide concentration in the pharmaceutical composition containing a peptide-hyaluronic acid derivative complex at this time is quantified, and according to the same method as in Reference Example 1 above, the average particle size of the pharmaceutical composition containing a peptide-hyaluronic acid derivative complex is determined. The results are shown in Table 8 below.

[0304] [Table 8]

[0305] As shown in Table 8 above, it is obvious that in the manufacture of a pharmaceutical composition using a peptide different from that in Reference Example 1, by adjusting the pH of the mixed solution to a specific range, the particle size of the complex of the active ingredient such as the peptide and the hyaluronic acid derivative can be maintained within a certain range, so that the stability of the complex in the solution is good, and the peptide concentration in the pharmaceutical composition can be increased.

[0306] <Manufacturing Conditions for Maintaining Appropriate Particle Size Distribution> [Test Examples 2-1 to 2-8] The hyaluronic acid derivative prepared and separated in the same manner as in Example 1-1 was subjected to detailed examination on the mixing ratio of the aqueous phase and the oil phase containing the hyaluronic acid derivative, by preparing an aqueous solution having a concentration of 5 mg / mL. According to Table 9 below, DMSO as the oil phase was mixed with 400 μL of hyaluronic acid derivative of 5 mg / mL. After sufficient mixing, water for injection was added according to Table 9 below until the final hyaluronic acid derivative concentration became a fixed concentration. Further, after culturing at room temperature (25°C) for 24 hours, the determination using gel permeation chromatography was performed according to the conditions shown below, and the area ratio A1 / A2 (Table 9) was calculated according to the same method as in Example 1-1. The resulting gel permeation chromatogram is shown in FIG. Figure 8 shown.

[0307] (Measurement conditions) Device: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0308] [Table 9]

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

[0310] [Table 10]

[0311] The results of Test Examples 2-1 to 2-8 and 3-1 to 3-6 show that the particle size of the hyaluronic acid derivative having excellent medicinal effects can be maintained by a mixing step in which the mixing ratio of the oil phase to the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100 by volume.

[0312] [Comparative Example 5-1] A powder of hyaluronic acid (manufactured by KIKKOMAN) having 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 the mixture was measured by gel permeation chromatography under the same conditions as those in Synthesis Example 1-1. The obtained chromatogram is as follows: Fig.10 shown.

[0313] Furthermore, hyaluronic acid powder having 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 prepare a hyaluronic acid aqueous solution. On the other hand, in another container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) at 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 Biologica was used.

[0314] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(serial number 1)

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

[0316] Afterwards, solid refined sucrose (manufactured by Fujifilm and Light Co., Ltd., dedicated for manufacture, product number 198-18385) is added so that the total mass of sucrose relative to the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose reaches 10 mass %, and is stirred at room temperature for more than 1 hour. Confirm that sucrose is fully dissolved, and the concentration of peptide is diluted to about 0.30mg / mL with 10mM phosphate buffer pH7.4 containing 10 mass % sucrose, and 0.2μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25mmΦ) is used for sterilization filtration, and the pharmaceutical composition containing peptide-hyaluronic acid derivative complex is obtained. According to the same method as Example 2-1, the peptide content in the pharmaceutical composition is quantified by the determination of high performance liquid chromatography. As a result, the peptide content in the pharmaceutical composition is lower than the detection limit.

[0317] [Comparative Example 5-2] Hyaluronic acid powder (manufactured by Kewpie Co., Ltd.) with a weight average molecular weight Mw (absolute molecular weight) of 650 kDa was dissolved in water for injection to a concentration of 1 mg / mL, and measured by gel permeation chromatography under the same conditions as those shown in Synthesis Example 1-1. The obtained gel permeation chromatogram is as follows: Fig.12 shown.

[0318] Hyaluronic acid powder having a weight average molecular weight Mw (absolute molecular weight) of 650 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 a hyaluronic acid aqueous 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 Biologica was used.

[0319] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(serial number 1)

[0320] The above-mentioned hyaluronic acid derivative aqueous solution and the DMSO solution of the antigen peptide were mixed at a volume ratio of 100:1 at room temperature (about 25°C), and stirred at room temperature (about 25°C) for 24 hours to try to prepare the antigen peptide-hyaluronic acid complex. Fig.11 As confirmed by the photograph described as "hyaluronic acid (640 kDa)", white turbidity was observed and the antigen peptide was not dissolved, indicating that the peptide was not enclosed.

[0321] Afterwards, add the refined sucrose (manufactured by Fujifilm and Light Co., Ltd., special for manufacturing, product number 198-18385) of solid, so that sucrose relative to the total mass of the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose reaches 10 mass %, and stir at room temperature for more than 1 hour.Confirm that sucrose is fully dissolved, and the concentration of peptide is diluted to about 0.30mg / mL with 10mM phosphate buffer pH7.4 containing 10 mass % sucrose, and 0.2μm PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe filter, 25mmΦ) is used for sterile filtration, and the pharmaceutical composition containing peptide-hyaluronic acid complex is obtained.According to the same method as Example 2-1, high performance liquid chromatography is used to measure, and the peptide content in the pharmaceutical composition is quantitatively carried out.As a result, the peptide content in the pharmaceutical composition is lower than the detection limit.

[0322] Judging from the results of Comparative Examples 5-1 and 5-2, it is difficult to encapsulate peptides in high molecular weight hyaluronic acid with an area ratio A1 / A2 of 0.9 or more, making it difficult to stably and effectively deliver pharmaceutical ingredients such as peptides to immune cells (especially cDC1 and macrophages). Furthermore, it is difficult to enhance, promote or strengthen the incorporation of the pharmaceutical ingredients into immune cells.

[0323] <Functional evaluation as an infectious disease vaccine (evaluation of antibody production against foreign antigens) and functional evaluation of antibody production in vivo for immune disease treatment> [Test Example 4]

[0324] [Comparative Example 6-1] The hyaluronic acid derivative was synthesized by the same method as in Synthesis Example 1-1. It was not intercepted and used as it was. The freeze-dried body of the hyaluronic acid derivative obtained was weighed, and water for injection was added to a concentration of 5 mg / mL, and stirred overnight at room temperature to fully dissolve it. For the above-mentioned hyaluronic acid derivative aqueous solution, it was diluted 5 times with water for injection to 1 mg / mL, and measured by gel permeation chromatography. Fig.13 The obtained gel permeation chromatogram is shown in Table 11. In addition, the particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1. The results are shown in Table 11 below.

[0325] [Example 6-1] The hyaluronic acid derivative synthesized by the same method as in Synthesis Example 1-1 was trapped in the same method as in Example 1-1 to obtain the hyaluronic acid derivative. The hyaluronic acid derivative in the obtained dialysate was measured by gel permeation chromatography. Fig.14 The obtained gel permeation chromatogram is shown in Table 11. In addition, the particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1. The results are shown in Table 11 below.

[0326] [Table 11] A1 / A2 Da / Db Comparative Example 6-1 0.81 0.62 Example 6-1 2.03 0.79

[0327] <Manufacturing of Pharmaceutical Composition> [Example 6-1-1] 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 and was found to be 4.28 mg / mL. In another container, ovalbumin (OVA, a low endotoxin reagent manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved in PBS (pH 7.4) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., product number: 166-23555) at a concentration of 5 mg / mL to obtain an OVA / PBS solution. In the order of water for injection, aqueous solution of hyaluronic acid derivative, and OVA / PBS solution, solid sucrose was added to form a 10% sucrose isotonic solution, and the mixture was stirred at room temperature until a clear solution was obtained. The concentration of OVA in the preparation was 0.15 mg / mL, and the concentration of hyaluronic acid derivative was 3.0 mg / mL, and the mixture was a 10% sucrose solution containing 10 mM PBS (pH 7.4). The mixture was cultured at 75°C and heated for 1 hour to obtain a composition containing an OVA-hyaluronic acid derivative complex.

[0328] [Comparative Example 6-1-1] Using the aqueous hyaluronic acid derivative solution 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 prepare a composition containing an OVA-hyaluronic acid derivative complex.

[0329] [Antibody production test] The composition containing the OVA-hyaluronic acid derivative complex prepared in Example 6-1-1 and Comparative Example 6-1-1 was administered subcutaneously to BALB / c mice with OVA at 20 μg on the 0th and 7th days, and serum was collected on the 10th day. In addition, when administering the OVA-hyaluronic acid derivative complex, 50 μg of CpG1826 (manufacturer: INVIVOGEN, product number: tlrl-1826-1) as an adjuvant was administered subcutaneously. Using an ELISA kit (manufactured by Chondrex, mouse anti-OVA IgG antibody detection kit, mouse anti-OVA IgG1 antibody detection kit, mouse anti-OVA IgG2a antibody detection kit), the antibody titer against OVA was determined according to the attached test protocol. The dilution ratio of serum was set to 10, 50, 250, 1250, 6250, 31250.

[0330] The IgG antibody titer (OD) was calculated using the mouse anti-OVA IgG antibody detection kit. Fig.15 As shown, the IgG1 antibody titer (OD) calculated using the mouse anti-OVA IgG1 antibody detection kit is as follows Fig.16 As shown, the IgG2a antibody titer (OD) calculated using the mouse anti-OVA IgG2a antibody detection kit is as follows Fig.17 The titers of the antibodies at a dilution ratio of 10 are shown in Fig.18A (IgG antibody titer), Fig.18B (IgG1 antibody titer) and Fig.18C (IgG2a antibody titer) shown.

[0331] It was confirmed that: when the serum was diluted 1250 times or more, there was no difference in antibody titer, but when the dilution ratio was lower than this ratio, the composition containing the OVA-hyaluronic acid derivative complex of Example 6-1-1 obtained a higher antibody titer than the composition containing the OVA-hyaluronic acid derivative complex of Comparative Example 6-1-1. Thus, the hyaluronic acid derivative of Example 6-1 (A1 / A2=2.03) obviously has a higher antibody production ability against foreign antigens than the hyaluronic acid derivative of Comparative Example 6-1 (A1 / A2=0.81), and also shows that the in vivo production function of therapeutic antibodies is high.

[0332] It is reported that the mature dendritic cells to which the antigen is added, through the induction of type I helper T cells that produce IFN-γ, promote the production of antigen-specific IgG2a (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 content of the report of the above reference 5 and the results of the above test example 4, it is speculated that the pharmaceutical composition containing the OVA-hyaluronic acid derivative of Example 6-1-1 activates the costimulatory molecules of DC, CD80 and CD86 are strongly expressed, and the subsequent induction of antigen-specific T cells and IFN-γ production ability are improved, and the production of antigen-specific IgG2a is promoted as a result. Therefore, the pharmaceutical composition of Example 6-1-1 not only induces humoral immunity, but also induces cellular immunity, and it is expected to induce a stronger immune response as an infectious disease vaccine.

[0333] It is speculated that by using the pharmaceutical composition of this embodiment, not only peptides but also active ingredients of protein antigens can be protected under heating conditions so as to maintain an appropriate three-dimensional structure and stably and effectively delivered to immune cells (especially cDC1, macrophages). Furthermore, while enhancing, promoting or strengthening the incorporation of the active ingredient into immune cells, the activity of immune cells (especially the expression of co-stimulatory molecules CD80 and CD86 in cDC1) is enhanced, promoted, maintained or strengthened. It is speculated that by promoting the activation of helper T cells, the activation and growth of B cells are further promoted, resulting in the production of antigens targeting the epitope.

[0334] It is believed that by using the hyaluronic acid derivatives of the first to third embodiments, a vaccine (mainly an infectious disease vaccine) against foreign antigens that is superior to that using conventional hyaluronic acid derivatives can be provided. It is believed that at the same time, an excellent drug containing a therapeutic antibody-inducing antigen can be provided.

[0335] <Functional Evaluation as Cancer Vaccine (Cancer Vaccine Antitumor Test Using an Administration Composition Containing a Hyaluronic Acid Derivative and an Antigen)> [Test Example 5] In order to clarify the anti-tumor effect of cancer vaccines of hyaluronic acid derivatives with different area ratios A1 / A2, tumor-bearing mice of the immune checkpoint inhibitor (ICI)-resistant fibrosarcoma cell line CMS5a were used to prepare hyaluronic acid derivative cancer vaccines carrying long-chain peptide antigens containing the CD8 epitope of the mutant ERK2 antigen (mERK2) expressed in CMS5a and various hyaluronic acid derivatives, and the therapeutic effects were studied.

[0336] Materials and methods are shown below. (1) Cells RPMI1640 medium (supplemented with 2-mercaptoethanol) was purchased from the Institute of Cell Science. Fetal bovine serum (FBS) was purchased from Gibco. Mouse fibrosarcoma CMS5a cell line distributed by the Memorial Sloan-Kettering Cancer Institute and propagated at Mie University was used. Mouse fibrosarcoma CMS5a cell line expresses mutant ERK2 protein. A peptide (QYIHSANVL (SEQ ID NO. 4)) containing the mutant portion of the mutant ERK2 protein is recognized by CD8 positive cell killer T cells of BALB / c mice. The T cell receptor (TCR) that recognizes the peptide is isolated, and a transgenic mouse (DUC18 mouse) into which the TCR gene is introduced is prepared.

[0337] (2) Experimental animals Female BALB / c mice (CD90.2 positive) aged 6 to 8 weeks were purchased from Japan SLC. The mice were raised in the animal experiment facility of the Center for Advanced Scientific Research, Mie University. The animal experiment protocol was approved by the Ethics Committee of the School of Medicine, Mie University.

[0338] (3) Long-chain peptide antigens Synthetic long-chain peptide antigens were purchased from Sigma-Genosys. The sequences are shown below. NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(sequence number 1). The sequence contains a 9-amino acid sequence from Q at position 16 to L at position 24 (QYIHSANVL (sequence number 4)) which is a CD8-positive cell-blocking T cell recognition epitope sequence of the above-mentioned mutant ERK2, and a 17-amino acid sequence from R at position 13 to K at position 29 (RGLQYIHSANVLHRDLK (sequence number 5)) which is a CD4-positive helper T cell recognition epitope sequence. The hyaluronic acid derivatives carrying the long-chain peptide antigen, which are complexed with the long-chain peptide antigen and various hyaluronic acid derivatives, may be expressed as "HA nanogel cancer vaccine carrying the long-chain peptide antigen" or "HA nanogel cancer vaccine".

[0339] [Example 7-1] The hyaluronic acid derivative prepared in Comparative Example 6-1 was retained as follows. The freeze-dried hyaluronic acid derivative (10k HA-C6-Chol-4 1.3%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Float-A-Lyzer G2, MWCO: 300,000, Ieda Trading Co., Ltd.), and dialyzed against 10 mM phosphate buffer pH 7.4. The hyaluronic acid derivative in the resulting dialysate was measured by gel permeation chromatography. Fig.19 The obtained gel permeation chromatogram is shown in Table 12. The particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1.

[0340] [Table 12] A1 / A2 Da / Db Example 7-1 6.62 0.80

[0341] <Manufacturing of Pharmaceutical Composition> [Example 7-1-1] The concentration of the hyaluronic acid derivative in the aqueous solution of the hyaluronic acid derivative prepared in Example 7-1 was calculated from the area value of the hyaluronic acid derivative with known concentration and was 3.8 mg / mL. On the other hand, in another container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg / mL to obtain a DMSO solution of the antigen peptide. The antigen peptide used was a peptide having the following amino acid sequence manufactured by Biologica.

[0342] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(serial number 1)

[0343] At room temperature (about 25°C), the above-mentioned hyaluronic acid derivative aqueous solution 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 try to prepare an antigen peptide-hyaluronic acid derivative complex. The state of the solution at this time was visually confirmed, and the result was as shown by Fig.11 As confirmed by the photograph described as "hyaluronic acid derivative" in the figure, a clear solution was obtained. This confirmed that the peptide could be encapsulated.

[0344] Afterwards, solid refined sucrose (manufactured by Fujifilm and Light Company, dedicated for manufacture, product number 198-18385) is added so that the total mass of sucrose relative to the composition containing fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose reaches 10% by mass, and stirred for more than 1 hour at room temperature (about 25 ° C). Confirm that sucrose is fully dissolved, and the concentration of peptide is diluted to about 0.30 mg / mL with 10mM phosphate buffer pH7.4 containing 10% by mass sucrose, and 0.2 μm PES (polyether sulfone) (manufactured by Pall Company, Acrodisc syringe filter, 25mmΦ) is sterilized and filtered to obtain a pharmaceutical composition containing peptide-hyaluronic acid derivative complex. Using the same method as Example 2-1, the peptide content in the pharmaceutical composition is quantified by the determination of high performance liquid chromatography. As a result, relative to 100 parts by mass of the pharmaceutical composition, the content of peptide is 0.383 parts by mass. Further, the pharmaceutical composition containing the above-mentioned peptide-hyaluronic acid derivative complex was diluted to a peptide concentration of 250 μg / mL using a 10 mM phosphate buffer pH 7.4 containing 10% by mass sucrose to obtain a pharmaceutical composition for administration. The pharmaceutical composition containing the above-mentioned peptide-hyaluronic acid derivative complex was measured using gel permeation chromatography under the following measurement conditions using the method described in Example 1-1. Fig. 20 The obtained gel permeation chromatogram is shown in Figure 2.

[0345] (Measurement conditions) Device: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

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

[0347] [Test Example 6] Tumor proliferation assay Mouse fibrosarcoma CMS5a cell line was cultured in RPMI1640 medium containing 10% FBS using T75 culture flasks (clone). The cultured cells were detached using PBS containing 0.5% trypsin and suspended in RPMI1640 medium containing 10% FBS. The suspension was centrifuged (400×g, 5 minutes, 4°C) and the supernatant was removed. After that, the suspension was washed twice with RPMI1640 medium and 1×10 6 The cells were suspended in RPMI1640 medium at a concentration of 100 μL / cell and subcutaneously transplanted into the right anterior back of BALB / c mice (5 mice per group) at a dosage of 100 μL / cell.

[0348] When administering the HA nanogel cancer vaccine carrying a long-chain peptide antigen, 50 μg of the HA nanogel cancer vaccine carrying a long-chain peptide antigen and 50 μg of CpG oligo DNA (Ajinomoto Bio-Pharma GeneDesign) dissolved in PBS were subcutaneously administered to the right back of the mouse 7 days, 10 days, 13 days, and 16 days after tumor transplantation. Afterwards, the tumor area was measured over time. When the long-chain peptide antigen of Comparative Example 7-1-1 was administered, 50 μg of the long-chain peptide was subcutaneously administered to the right back of the mouse together with 50 μg of CpG oligo DNA (Ajinomoto Bio-PharmaGene Design) dissolved in PBS 7, 10, 13, and 16 days after tumor transplantation. Afterwards, the tumor area was measured over time.

[0349] The tumor area values ​​(average values) over time are as follows Fig.23 shown. The pharmaceutical composition obtained by combining the HA nanogel cancer vaccine carrying the long-chain peptide antigen obtained by compounding the hyaluronic acid derivative (A1 / A2=6.62) of Example 7-1 with the above-mentioned long-chain peptide antigen and CpG oligo DNA significantly inhibited the proliferation of CMS5a tumors compared with the pharmaceutical composition obtained by combining the above-mentioned long-chain peptide antigen and CpG oligo DNA of Comparative Example 7-1. It is therefore believed that by using the hyaluronic acid derivative with A1 / A2=6.62, an excellent cancer vaccine can be provided.

[0350] <Effect of retention time at the peak top (maximum refractive index intensity point) obtained by size exclusion chromatography (SEC) on immune cell activation and vaccine function> [Test Example 7] [Retention time ratio of the peak top in the particle size distribution of the hyaluronic acid derivative] Regarding 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 standard substance polyacrylic acid (50 kDa), samples were prepared by the method described in Example 1-1 and measured by gel permeation chromatography under the conditions shown below.

[0351] (Measurement conditions) Device: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0352] The peak top retention time Pt ​​of the hyaluronic acid derivatives prepared 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 and 7-2 and the peak top retention time Pr of the standard substance polyacrylic acid (50 kDa) are shown in the following Table 13. In addition, the ratio of the peak top retention time Pt ​​of the hyaluronic acid derivative to the peak top retention time Pr of the standard substance polyacrylic acid (50 kDa), i.e., Pt / Pr, was calculated and recorded in the following Table 13.

[0353] [Table 13]

[0354] It is speculated that when the peak retention time Pt of the hyaluronic acid derivative is shorter than the peak retention time Pr of the reference substance polyacrylic acid (50 kDa) and the associated particle size is small, more aggregates with excellent activation of co-stimulatory molecules exist. By using the pharmaceutical composition of the present embodiment, the active pharmaceutical ingredients of peptides and proteins can be stably and effectively delivered to immune cells (particularly cDC1 and macrophages). Further, while enhancing, promoting, or augmenting the incorporation of the active pharmaceutical ingredient into immune cells, the activity of immune cells (particularly the expression of co-stimulatory molecules CD80 and CD86 in cDC1) can be enhanced, promoted, maintained, or augmented.

[0355] <Effect of the average molecular weight (converted to polyacrylic acid) of GPC-SEC on immune cell activation and vaccine function> [Test Example 8] The weight-average molecular weight (Mw) and number-average molecular weight (Mn) (converted to polyacrylic acid) of the hyaluronic acid derivatives prepared 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. A calibration curve was prepared using polyacrylic acid as a reference substance, and the molecular weights based on the polyacrylic acid of the hyaluronic acid derivatives prepared 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. As the above polyacrylic acid reference 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 prepared in the synthesis examples, examples, and comparative examples were 1 mg / ml, and the polyacrylic acid reference substance was 2 mg / ml Calibration curve conditions: As an approximate formula, it was used three times: At3+Bt2+Ct+D.

[0357] [Table 14] M Mw / Mn Synthesis example 1-1 69201 2.784 Synthesis example 1-2 86393 2.293 Example 1-1 133892 2.565 Example 1-2 152674 2.073 Comparative Example 1-1 45799 2.209 Comparative Example 1-2 43722 1.826 Example 6-1 137659 2.201 Comparative Example 6-1 74840 1.825 Example 7-1 204104 2.056

[0358] It is speculated that: when the weight average molecular weight Mw (converted into polyacrylic acid) of the hyaluronic acid derivative calculated with polyacrylic acid as the standard substance meets the condition of 110,000 or more, there are more complexes with excellent activation of co-stimulatory molecules, and by using the pharmaceutical composition of this embodiment, it is possible to stably and effectively deliver pharmaceutical ingredients such as peptides to immune cells (especially cDC1, macrophages), and further, while enhancing, promoting or strengthening the incorporation of the pharmaceutical ingredients into immune cells, it is possible to enhance, promote, maintain or strengthen the activity of immune cells (especially the expression of co-stimulatory molecules CD80 and CD86 in cDC1).

[0359] <Preparative separation method using SEC (size exclusion chromatography)> [Example 8]

[0360] Preparative separation methods are not limited to interception methods using dialysis membranes. The hyaluronic acid derivative obtained in Synthesis Example 1-1 was dissolved in water for injection at 20 mg / mL, and preparative separation was performed by gel permeation chromatography under the following conditions: For example, preparative separation was performed every 0.1 minutes.

[0361] (Measurement conditions) Installation: HITACHI, CHROMASTER Chromatographic 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 (6.0mm IDX 4CM) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 100mL Detector: RI

[0362] After the preparative separation, GPC analysis was performed according to the method described in Synthesis Example 1-1, and a fraction having an area ratio A1 / A2 of 0.90 or more was identified to obtain the target hyaluronic acid derivative.

[0363] <Production Conditions for Pharmaceutical Compositions Maintaining Appropriate Particle Size Distribution> [Examples 9-1 to 9-4, Comparative Examples 9-1 to 9-4] The hyaluronic acid derivative separated by the same method as in Example 1-1 was prepared, and an aqueous solution with a concentration of 5 mg / mL was prepared, and the mixing ratio of the aqueous phase containing the hyaluronic acid derivative and the oil phase was studied in detail. Cyclosporin (Tokyo Chemical Industry Co., Ltd., product number: C2408) as a peptide drug was weighed in a 6 mL clean vial and dissolved in DMSO (Fuji Film Wako Co., Ltd., product number: 045-24511) at a concentration of 50 mg / mL. Next, 400 μL of 5 mg / mL hyaluronic acid derivatives were added as 4 μL of 50 mg / mL cyclosporin as the oil phase containing the drug. After that, the oil phase DMSO was mixed according to Table 15 below. After sufficient mixing, water for injection was added according to Table 15 below until the final hyaluronic acid derivative concentration became a certain concentration. Further, after culturing at room temperature (25° C.) for 24 hours, the gel permeation chromatography was used for measurement according to the conditions shown below, and the area ratio A1 / A2 (Table 15) was calculated by the same method as in Example 1-1.

[0364] (Measurement conditions) Device: HLC8420-GPC (manufactured by Tosoh Corporation) Chromatographic column: G4000SWXL (manufactured by Tosoh Corporation, particle size 8μm, inner diameter 7.8mm, length 30cm, product number: 8542) Elution buffer: 10 mM phosphate buffer (pH 7.4) Flow rate: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0365] [Table 15]

[0366] The results of Examples 9-1 to 9-4 and Comparative Examples 9-1 to 9-4 show that the particle size of the hyaluronic acid derivative having excellent medicinal effects can be maintained by performing a mixing step in which the mixing ratio of the oil phase to the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100 in terms of volume ratio. Industrial Applicability

[0367] According to the hyaluronic acid derivative of the present embodiment, a hyaluronic acid derivative having excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells can be provided when formulated with a pharmaceutical ingredient. The pharmaceutical composition of the present embodiment contains the above-mentioned hyaluronic acid derivative, which has excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells. The manufacturing method of the pharmaceutical composition of the present embodiment uses the above-mentioned hyaluronic acid derivative to prepare a pharmaceutical composition having excellent delivery to immune cells in lymph nodes and the ability to activate the immune cells.

Claims

1. A hyaluronic acid derivative, which is a hyaluronic acid derivative into which a steryl group is introduced, From the chromatogram obtained by gel permeation chromatography, the ratio of area A1 to A2 calculated by the following method is 0.90 or more. (i) the intersection point of a vertical line drawn from the maximum refractive index intensity point Kb on the chromatogram of the standard substance 50 kDa polyacrylic acid to the baseline B and the chromatogram of the hyaluronic acid derivative is set as Ub; (ii) On the chromatogram of the hyaluronic acid derivative, let the curve from the Ub to the end point, the vertical line drawn from the point of maximum refractive index intensity Kb to the baseline B and the area enclosed by the baseline be A2; on the chromatogram of the hyaluronic acid derivative, let the curve from the starting point to the Ub, the vertical line drawn from the point of maximum refractive index intensity Kb to the baseline B and the area enclosed by the baseline be A1.

2. The hyaluronic acid derivative according to claim 1, wherein the ratio Da / Db of the distance Da to Db calculated by the following method is greater than 0.00 and less than 1.20 from the chromatogram obtained by gel permeation chromatography, (i) draw a vertical line from the maximum refractive index intensity point Ka on the chromatogram of the standard substance 150 kDa polyacrylic acid to the baseline B, set the intersection of the vertical line with the baseline as Ba, and set the length between the maximum refractive index intensity point Ka and Ba as La; (ii) Of the two points on the chromatogram with a refractive index intensity of La / 20, the point with an earlier dissolution time is point R1, and the point with a later dissolution time is point S1; (iv) The intersection point of the straight line D1 connecting the point R1 and the point S1 and the vertical line drawn from the maximum refractive index intensity point Ka to the baseline B is set as Ta, and the intersection point of the vertical line drawn from the maximum refractive index intensity point Kb to the baseline B and the straight line D1 is set as Tb; (v) Let the distance between the point R1 and Ta be Da, and let the distance between Ta and Tb be Db. 3 . The hyaluronic acid derivative according to claim 1 , wherein the ratio A1 / A2 of the areas A1 and A2 is 1.70 or more.

4. A hyaluronic acid derivative, which is a hyaluronic acid derivative introduced with a steryl group, wherein the ratio Pt / Pr of the retention time Pt ​​of the hyaluronic acid derivative when the refractive index intensity reaches a maximum point calculated from a chromatogram obtained by gel permeation chromatography to the retention time Pr of the refractive index intensity of a standard substance 50 kDa polyacrylic acid when the refractive index intensity reaches a maximum point is greater than 0.5 and less than 1.

0.

5. A hyaluronic acid derivative, which is a hyaluronic acid derivative introduced with a steryl group, wherein, according to a chromatogram obtained by gel permeation chromatography, the weight average molecular weight of the hyaluronic acid derivative calculated based on a calibration curve prepared using polyacrylic acid with molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa and 150 kDa as standard substances is 110,000 or more and less than 500,000.

6. The hyaluronic acid derivative according to claim 1, 4 or 5, wherein the hyaluronic acid derivative has one or more repeating units represented by the following general formula (I): In the formula, R 1 , R 2 , R 3 and R 4 Each independently from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 Select from the group consisting of 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 A group selected from the group consisting of groups represented by the following chemical formulae: -NR b -R、 -NR b -COO-R、 -NR b -CO-R、 -NR b -CO-NR c -R、 -COO-R, -O-COO-R, -SR, -WHAT-Y a -SR、 -O-CO-Y b -SR、 -NR b -CO-Y b -SR and -SSR; R a , R b and R c Each independently from the hydrogen atom, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group is selected from the group consisting of alkyl groups, in which case the alkyl group is inserted or not inserted with -O- and -NR f - A group selected from the group consisting of; R f From hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 The alkyl group is selected from the group consisting of alkyl, and the alkyl part of the group may be inserted with or without a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, in which case the alkylene is inserted or not inserted by -O-, -NR g - and -SS- group selected from the group consisting of; R g From hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 The alkyl group is selected from the group consisting of alkyl, and the alkyl part of the group may be inserted with or without a group selected from the group consisting of -O- and -NH-; Y a C 1-5 Alkylene; Y b C 2-8 Alkylene or C 2-8 alkenylene; m is an integer of 1 to 100.

7. The hyaluronic acid derivative according to claim 1, 4 or 5, wherein the steryl group is a cholesteryl group. 8 . The hyaluronic acid derivative according to claim 1 , 4 or 5 , wherein the introduction rate of the steryl group is 30% or more and 60% or less relative to the repeating unit of the disaccharide constituting the hyaluronic acid derivative. 9 . The hyaluronic acid derivative according to claim 1 , 4 or 5 , wherein the weight average molecular weight, that is, the 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 claim 9 , wherein the weight average molecular weight, that is, the absolute molecular weight, of the hyaluronic acid derivative is 5000 or more and 25,000 or less.

11. A pharmaceutical composition comprising the hyaluronic acid derivative according to claim 1, 4 or 5 and a medicinal ingredient. 12 . The pharmaceutical composition according to claim 11 , which is used 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 claim 11 , comprising, as the medicinal ingredient, at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases, and further comprising an adjuvant. 14 . The pharmaceutical composition according to claim 13 , comprising a cancer antigen or an antigen derived from an infectious disease as the medicinal ingredient.

15. A method for producing a pharmaceutical composition, The invention relates to a method for producing a pharmaceutical composition comprising the hyaluronic acid derivative according to claim 1, 4 or 5 and a medicinal ingredient. The method comprises the steps of dissolving the medicinal ingredient in an organic solvent or water containing an organic solvent, preparing 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 by volume ratio is 20:100 to 0.01:

100. 16 . The method for producing a pharmaceutical composition according to claim 15 , 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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