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

Abstract

To provide a hyaluronic acid derivative which exhibits excellent delivery properties to immune cells in lymph nodes and activation ability of 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.SOLUTION: A hyaluronic acid derivative in which multiple molecules are aggregated, the hyaluronic acid derivative having a weight-average molecular weight of 110,000 or more and less than 500,000, the weight-average molecular weight being calculated from a chromatogram obtained by a gel permeation chromatography measurement based on a calibration curve prepared using polyacrylic acids having molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa, and 150 kDa as standard substances.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a hyaluronic acid derivative, a pharmaceutical composition, and a method for producing the pharmaceutical composition. [Background technology]

[0002] In recent years, biopharmaceuticals, which are pharmaceuticals containing proteins, peptides, or nucleic acids as active ingredients, have been put into practical use, and their number continues to increase year by year. Biopharmaceuticals can fulfill unmet medical needs that could not be met by conventional small molecule drugs. However, they have issues such as poor absorption from the digestive tract or mucous membranes, instability in the body, and a short half-life in the blood. As a result, biopharmaceuticals require frequent injections, which places a significant burden on both patients and medical professionals. Therefore, there is a need for drug carriers (sustained-release drug delivery system carriers) that can encapsulate biopharmaceuticals without compromising their pharmacological activity and gradually release the active ingredient in the body.

[0003] In light of this background, Patent Document 1 proposes a sustained-release drug delivery system substrate made of a hyaluronic acid derivative with excellent safety. This hyaluronic acid derivative spontaneously aggregates in an aqueous solution, can efficiently encapsulate drugs, particularly biopharmaceuticals, while maintaining their biological activity, aggregates under physiological salt concentrations (or disperses under physiological salt concentrations), and has good blood retention. This hyaluronic acid derivative can be used as a carrier that can efficiently encapsulate many drugs while maintaining their pharmacological activity, particularly when biopharmaceuticals are used as active ingredients, and as a blood sustained-release carrier and targeting carrier with excellent blood retention, and can also be used as a local (e.g., subcutaneous) sustained-release carrier that can sustainably release drugs.

[0004] Cancer vaccines using the above-mentioned hyaluronic acid derivatives as carriers have also been reported (see, for example, Patent Document 2, etc.). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2010 / 053140 [Patent Document 2] International Publication No. 2020 / 158771 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the hyaluronic acid derivatives used in Patent Document 2 and the like have a broad and non-uniform particle size distribution, and the relationship between the particle size distribution of the hyaluronic acid derivatives and their delivery to immune cells in lymph nodes or their ability to activate these immune cells has not been specifically examined, leaving room for improvement.

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

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

[0010] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b -SR, and -SSR, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f - optionally inserted with a group selected from the group consisting of: R f is a hydrogen atom, C 1-12 Alkyl, Amino C 2-12 Alkyl and hydroxy C 2-12 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g optionally containing an inserted group selected from the group consisting of - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

[0011] (7) The hyaluronic acid derivative according to any one of (1) to (6), wherein the steryl group is a cholesteryl group. (8) A hyaluronic acid derivative according to any one of (1) to (7), wherein the introduction rate of steryl groups relative to the repeating units of the disaccharide constituting the hyaluronic acid derivative is 30% or more and 60% or less. (9) The hyaluronic acid derivative according to any one of (1) to (8), wherein the weight-average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 4,000 or more and 1,000,000 or less. (10) The hyaluronic acid derivative according to (9), wherein the weight-average molecular weight (absolute molecular weight) of the hyaluronic acid derivative is 5,000 or more and 25,000 or less. (11) A pharmaceutical composition comprising the hyaluronic acid derivative according to any one of (1) to (10) and a medicinal ingredient. (12) The pharmaceutical composition according to (11), which is for the prevention or treatment of 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), comprising, as the medicinal ingredient, at least one selected from the group consisting of a cancer antigen, an antigen derived from an infectious disease, and an autoantigen in an immune disease, and further comprising an adjuvant. (14) The pharmaceutical composition according to any one of (11) to (13), which contains a cancer antigen or an antigen derived from an infectious disease as the medicinal ingredient and further contains an adjuvant. (15) A method for producing a pharmaceutical composition containing the hyaluronic acid derivative according to any one of (1) to (10) and a medicinal ingredient, a preparation step of dissolving the medicinal ingredient in an organic solvent or water containing an organic solvent to prepare an oil phase containing the medicinal ingredient; a mixing step of mixing the oil phase with the aqueous phase containing the hyaluronic acid derivative so that the volume ratio of the oil phase to the aqueous phase is 20:100 to 0.01:100; A method for producing a pharmaceutical composition, comprising: (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 aspect, it is possible to provide a hyaluronic acid derivative that, when formulated with a medicinal ingredient, has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells.The pharmaceutical composition of the above-mentioned aspect comprises the hyaluronic acid derivative, and has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells.The manufacturing method of the pharmaceutical composition of the above-mentioned aspect uses the hyaluronic acid derivative, and can obtain a pharmaceutical composition that has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is an example of a gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment. [Figure 2] 1 is an example of a gel permeation chromatogram of the hyaluronic acid derivative of the present embodiment. [Figure 3A] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-1. [Figure 3B] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-2. [Figure 3C] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Synthesis Example 1-3. [Figure 4A] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Example 1-1. [Figure 4B] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Example 1-2. [Figure 4C] 1 shows a gel permeation chromatogram of the hyaluronic acid derivative in Example 1-3. [Figure 5A] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-1. [Figure 5B] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-2. [Figure 5C] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 1-3. [Figure 5D]1 is a gel permeation chromatogram of cholesteryl-modified pullulan (CHP) in Comparative Example 1-4. [Figure 6A] 1 shows gel permeation chromatograms of the hyaluronic acid derivative in Example 1-1 and the hyaluronic acid derivative in the pharmaceutical composition in Example 2-1. [Figure 6B] 1 shows gel permeation chromatograms of the hyaluronic acid derivative in Example 1-2 and the hyaluronic acid derivative in the pharmaceutical composition in Example 2-2. [Figure 6C] 1 shows gel permeation chromatograms of the hyaluronic acid derivatives in Examples 1-3 and the hyaluronic acid derivatives in the pharmaceutical compositions in Examples 2-3. [Figure 7A] 1 is a graph showing an example of FACS analysis in which the ratio (%) of peptide-uptaken dendritic cells (DCs) expressing CD80 to the total peptide-uptaken DCs was calculated in Test Example 1-1. [Figure 7B] 1 is a graph showing an example of FACS analysis in which the ratio (%) of peptide-uptaken DCs expressing CD86 to peptide-uptaken DCs was calculated in Test Example 1-1. [Figure 7C] 1 is a graph showing an example of FACS analysis in which the ratio (%) of peptide-uptaken standard type 1 dendritic cells (cDC1) expressing CD80 was calculated relative to peptide-uptaken standard type 1 dendritic cells (cDC1) in Test Example 1-1. [Figure 7D] 1 is a graph showing an example of FACS analysis in which the ratio (%) of peptide-uptaken cDC1 expressing CD86 to the peptide-uptaken cDC1 was calculated in Test Example 1-1. [Figure 7E] 1 is a graph showing an example of FACS analysis in which the ratio (%) of CD80-expressing macrophage cells (Mph) that have taken up the peptide relative to the total peptide-taken Mph cells in Test Example 1-1 was calculated. [Figure 7F] 1 is a graph showing an example of FACS analysis in which the ratio (%) of peptide-uptaken Mphs expressing CD86 to peptide-uptaken Mphs was calculated in Test Example 1-1. [Figure 8]1 shows gel permeation chromatograms of hyaluronic acid derivatives in Test Examples 2-1 to 2-8. [Figure 9] 1 shows gel permeation chromatograms of hyaluronic acid derivatives in Test Examples 3-1 to 3-6. [Figure 10] 1 is a gel permeation chromatogram of hyaluronic acid in Comparative Example 5-1. [Figure 11] 1 shows photographs of the mixtures used when attempting to prepare antigen peptide-hyaluronic acid complexes in Comparative Examples 5-1 and 5-2, and photographs of the solutions used when preparing antigen peptide-hyaluronic acid derivative complexes in Example 7-1-1. [Figure 12] 1 is a gel permeation chromatogram of hyaluronic acid in Comparative Example 5-2. [Figure 13] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Comparative Example 6-1. [Figure 14] 1 is a gel permeation chromatogram of the hyaluronic acid derivative in Example 6-1. [Figure 15] 1 is a graph showing IgG antibody titers (OD values) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 16] 6 is a graph showing the IgG1 antibody titer (OD value) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 17] 1 is a graph showing IgG2a antibody titers (OD values) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 18A] 1 is a graph showing the IgG antibody titer (OD value) at a dilution rate of 10 (titer 10-1) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 18B] 1 is a graph showing the IgG1 antibody titer (OD value) at a dilution rate of 10 (titer 10-1) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 18C] 1 is a graph showing the IgG2a antibody titer (OD value) at a dilution rate of 10 (titer 10-1) in Example 6-1-1 and Comparative Example 6-1-1. [Figure 19]1 is a gel permeation chromatogram of the hyaluronic acid derivative in Example 7-1. [Figure 20] 1 is a gel permeation chromatogram of a pharmaceutical composition containing an antigen peptide-hyaluronic acid derivative complex in Example 7-1-1. [Figure 21] 1 is a gel permeation chromatogram of the hyaluronic acid derivative obtained in Example 1-1. [Figure 22] 1 is a gel permeation chromatogram of the hyaluronic acid derivative obtained in Example 1-1. [Figure 23] 1 is a graph showing tumor area values ​​(average values) over time in Test Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0014] Below, we will explain in detail the embodiment of the present invention (hereinafter referred to as the ``present embodiment''), but the present invention is not limited to this and various modifications are possible within the scope of the gist of the present invention.

[0015] The terms used in this specification will be explained below.

[0016] As used herein, "C 1-20 The term "alkyl" means a straight or branched chain alkyl group having from 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, etc. 1-4 alkyl," and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, and the like. 1-20 Alkyl has 1 to 12 carbon atoms. 1-12 Alkyl, carbon number 1 to 6, C 1-6 Alkyl groups are also included.

[0017] As used herein, "C1-6 The term "alkylcarbonyl" refers to the alkyl moiety of the already mentioned C 1-6 It means an alkylcarbonyl group, for example, acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc. 1-4 alkylcarbonyl".

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

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

[0020] As used herein, "C 2-30 The term "alkylene" means a linear or branched divalent saturated hydrocarbon group having from 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc., and has from 2 to 20 carbon atoms. 2-20 Alkylene, C2-8 alkylene having 2 to 8 carbon atoms, the group "-(CH2) n -" (where n is 2 or more and 30 or less, preferably 2 or more and 20 or less, and more preferably 2 or more and 15 or less).

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

[0022] The term "C" referred to herein 2-8 The term "alkenylene" refers to a linear or branched divalent saturated hydrocarbon group having from 2 to 8 carbon atoms and containing one or more double bonds, and includes, for example, -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0023] <Hyaluronic acid derivatives> The hyaluronic acid derivative of the first embodiment of the present invention is a hyaluronic acid derivative into which a steryl group has been introduced, From the chromatogram shown in FIG. 1 obtained by gel permeation chromatography, the ratio A1 / A2 of the areas A1 and A2 calculated by the method described below is 0.90 or more. (i) The intersection point of a perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B on the chromatogram of a 50 kDa polyacrylic acid standard substance and the chromatogram of the hyaluronic acid derivative is designated as Ub, and the intersection point of a perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B and the baseline is designated as Bb; (ii) In the chromatogram of the hyaluronic acid derivative, the area enclosed by the curve from Ub to the end point, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A2, and in the chromatogram of the hyaluronic acid derivative, the area enclosed by the curve from the start point to Ub, the perpendicular line drawn from the refractive index intensity maximum point Kb to the baseline B, and the baseline is defined as A1.

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

[0025] The hyaluronic acid derivative of this embodiment is different from the conventional hyaluronic acid derivative, and in particle size distribution, the hyaluronic acid derivative of specific particle size is relatively large, and particle size distribution is sharply controlled.The inventors have found that by formulating this hyaluronic acid derivative of controlled particle size distribution with active ingredient, as shown in the example below, compared with the hyaluronic acid derivative of uncontrolled particle size distribution and the cholesterolized pullulan (CHP) that has been used as carrier in the past, the delivery ability to the immune cells in lymph node and the activation ability of this immune cell are significantly superior, and have completed the present invention.

[0026] The immune cells referred to here are preferably myeloid cells, more preferably macrophages or dendritic cells (DCs), even more preferably DCs, and particularly preferably standard type 1 dendritic cells (cDC1s).

[0027] Both macrophages and dendritic cells have antigen-presenting capabilities. Dendritic cells are the most potent antigen-presenting cells and are responsible for regulating the proliferation and function of T cells and natural killer cells in lymphoid and non-lymphoid tissues. Dendritic cells have several subtypes, including cDC1s, which specifically express the chemokine receptor XCR1 and the C-type lectin endocytosis receptor CLEC9A. These subtypes possess high cross-presentation capabilities and efficiently load antigens onto MHC class I molecules (Reference 1: Noubade R et al., “Beyond cDC1: Emerging Roles of DC Crosstalk in Cancer Immunity”, Front Immunol., Vol. 10, Article 1014, pp. 1-13, 2019). By simultaneously expressing costimulatory molecules, cDC1s can activate T cells (cytotoxic T cells [CTLs]) that attack virus- and bacteria-infected cells and cancer cells. That is, increasing the antigen delivery to macrophages or DCs (preferably cDC1s) improves the efficacy of preventing or treating cancer and infectious diseases.

[0028] Macrophages and DCs can also load antigens onto MHC class II molecules, which activate helper T cells and promote antibody production by B cells. That is, increasing the antigen delivery to macrophages or DCs (preferably cDC1s) improves the efficacy of preventing or treating infectious diseases and treating immune diseases.

[0029] Furthermore, the immune cell activation ability refers to the property of improving, promoting, or enhancing the activity of the cells as described above, and in particular, the property of improving, promoting, or maintaining the expression of costimulatory molecules in DCs (preferably cDC1). Examples of costimulatory molecules include CD80 and CD86. By improving, promoting, or maintaining the expression of these costimulatory molecules, T cells (cytotoxic T cells (CTLs)) can be significantly induced. Therefore, it is preferable that the hyaluronic acid derivative of this embodiment, when formulated with a medicinal ingredient, can improve, promote, or maintain the expression of both CD80 and CD86 in DCs (preferably cDC1).

[0030] When the hyaluronic acid derivative of this embodiment is formulated with a medicinal component, it can form a stable association state with the medicinal component, and can stably and efficiently deliver the medicinal component to immune cells (preferably DC, more preferably cDC1), and can improve, promote or enhance the uptake of the medicinal component into immune cells.It is presumed that this improves, promotes or enhances the activity of immune cells.In addition, even if the desired effect is obtained by a mechanism different from the above-mentioned mechanism, it is still included in the technical scope.

[0031] That is, the hyaluronic acid derivative of the present embodiment can be said to be a delivery enhancer, delivery promoter, or delivery enhancer of a medicinal component to immune cells, or an enhancer, promoter, or enhancer of uptake of a medicinal component into immune cells. Furthermore, the pharmaceutical composition described below, which contains the hyaluronic acid derivative of this embodiment and a medicinal ingredient, can also be referred to as a composition for activating immune cells, a composition for enhancing the expression of costimulatory molecules in DCs (preferably cDC1), a composition for promoting expression, or a composition for maintaining expression.

[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 shown by the ratio A1 / A2 of the area A1 and A2 calculated from the gel permeation chromatogram by the method shown below. 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 gel permeation chromatography in the following manner. Prepare a 1 mg / mL aqueous solution of hyaluronic acid derivative and a 2 mg / mL aqueous solution of polyacrylic acid standard, and perform measurements by gel permeation chromatography under the conditions shown below. (Measurement conditions) Equipment: High-speed GPC (gel permeation chromatography) equipment Column: G4000SWXL (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: 1mL / min Injection volume: 50μL Detector: RI (differential refractive index detector) Temperature: 30℃

[0034] Fig. 1 shows an example of a gel permeation chromatogram of the hyaluronic acid derivative of this embodiment. With reference to Fig. 1, the method for calculating the areas A1 and A2 will be described in detail below.

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

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

[0037] The ratio A1 / A2 of the area A1 to A2 shown in Figure 1 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, more preferably 1.40 or more, more preferably 1.50 or more, even more preferably 1.60 or more, and particularly preferably 1.70 or more. When the ratio A1 / A2 of the area A1 to A2 is above the lower limit, it is possible to contain a relatively large amount of hyaluronic acid derivative with a large particle size, and when formulated with a medicinal ingredient, it is possible to form a stable association state with the medicinal ingredient. This allows the medicinal ingredient to be delivered stably and efficiently to immune cells (preferably DC, more preferably cDC1), and furthermore, it is possible to improve, promote, or enhance the uptake of the medicinal ingredient into immune cells.

[0038] On the other hand, the upper limit of A1 / A2 is preferably the higher the ratio of area A1 to area A2. That is, in Figure 1, the particle size increases toward the left side of the baseline (shorter dissolution time) and decreases toward the right side (longer dissolution time), so the higher the ratio of large particle sizes relative to small particle sizes, the more preferable. 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 this embodiment is not particularly limited, but can have two or more maximum points (peak tops) of the refractive index intensity of the hyaluronic acid derivative.Not particularly limited, but can have one or more maximum points (peak tops) of the refractive index intensity of the hyaluronic acid derivative on each side (left and right) of the perpendicular line drawn from the refractive index intensity maximum point Kb to baseline B on the chromatogram of the 50 kDa polyacrylic acid of standard substance.At this time, the refractive index intensity at the maximum point (peak top) of the refractive index intensity of the hyaluronic acid derivative on the left side of the perpendicular line drawn from the refractive index intensity maximum point Kb to baseline B can be greater than the refractive index intensity at the maximum point (peak top) of the refractive index intensity of the hyaluronic acid derivative on the right side of the perpendicular line drawn from the refractive index intensity maximum point Kb to baseline B. Furthermore, the refractive index intensity at the maximum point (peak top) on the left side relative to the refractive index intensity at the maximum point (peak top) on the right side is not particularly limited, and may be 1.0 times or more, 2.0 times or more, 3.0 times or more, 5.0 times or more, or 10.0 times or more, and the upper limit may be larger, for example, 1000 times or less, 100 times or less, 50.0 times or less, 20.0 times or less, 10.0 times or less, 2.0 times or less, or 1.5 times or less.

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

[0041] Figure 2 is an example of the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment, and the gel permeation chromatogram of the hyaluronic acid derivative shown in Figure 2 and the gel permeation chromatogram of the standard substance 50 kDa polyacrylic acid are the same as Figure 1. However, Figure 2 differs from the above-mentioned Figure 1 in that the gel permeation chromatogram of the standard substance 150 kDa polyacrylic acid is added, and various intersections etc. are defined. With reference to Figure 2, the calculation method of distances Da and Db will be described in detail below.

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

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

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

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

[0046] (v) Finally, the distance between the point R1 and the point Ta is set to Da, and the distance between the point Ta and the point Tb is set to Db.

[0047] The ratio Da / Db of the distances Da to Db shown in Figure 2 is preferably greater than 0.00 and less than 1.20, more preferably 0.10 to 1.00, more preferably 0.20 to 0.8, even more preferably 0.30 to 0.95, even more preferably 0.40 to 0.94, and particularly preferably 0.50 to 0.93. By having the ratio Da / Db of the distances Da to Db within the above range, it is possible to contain a relatively large amount of hyaluronic acid derivative with a particle size larger than that of the standard substance 150 kDa polyacrylic acid, and when formulated with a medicinal component, it is possible to form a stable association state with the medicinal component. This allows the medicinal component to be delivered stably and efficiently to immune cells (preferably DCs, more preferably cDC1), and furthermore, it is possible to improve, promote, or enhance the uptake of the medicinal component into immune cells.

[0048] In the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment, the horizontal axis indicates elution time, and the vertical axis indicates the refractive index intensity obtained by using a differential refractometer, but there can be any number of refractive index maximum points.The gel permeation chromatogram of the hyaluronic acid derivative of this embodiment preferably has 1 to 5 maximum points in particle size distribution, more preferably has 1 to 3 maximum points in particle size distribution.In addition, it can be with or without minimum points. Furthermore, in the gel permeation chromatogram of the hyaluronic acid derivative of this embodiment, the chromatogram represented by the refractive index intensity and elution time obtained using a differential refractometer may be asymmetric or symmetric.

[0049] In the hyaluronic acid derivative of this embodiment, the steryl groups in the hyaluronic acid derivative self-associate in water, and a single molecule or multiple molecules associate to form a nano-sized hydrogel.

[0050] In the hyaluronic acid derivative, the steryl group may be directly bound to the hyaluronic acid or may be bound via a linker.

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

[0052] [Steryl group] The term "steryl group" used herein is not particularly limited as long as it is a group having a steroid skeleton. Specific examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simiarenolol, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycortisterone. Examples of steryl groups include cholesteryl, stigmasteryl, lanosteryl, and ergosteryl groups, with cholesteryl groups (particularly cholest-5-en-3β-yl groups) being preferred.

[0053] The weight-average molecular weight (absolute molecular weight) of hyaluronic acid derivative is not particularly limited, but from the viewpoint of increasing the number of steryl groups introduced per molecule of hyaluronic acid derivative, forming a complex with medicinal ingredients, and also from the viewpoint of increasing molecular entanglement and increasing retention in blood, the hyaluronic acid derivative with relatively large molecular weight is preferred.The weight-average molecular weight (absolute molecular weight) of such hyaluronic acid derivative is preferably 4000 (4k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 500k or less, more preferably 6k or more and 500k or less, more preferably 7k or more and 300k or less, more preferably 7k or more and 100k or less, more preferably 7k or more and 50k or less, more preferably 7k or more and 25k or less, and particularly preferably 8k or more and 15k or less.Or, the weight-average molecular weight (absolute molecular weight) of hyaluronic acid derivative is more preferably 5k or more and 25k or less. The weight-average molecular weight (absolute molecular weight) of hyaluronic acid derivative is above the lower limit, so that molecular entanglement can be further enhanced, and the retention time in blood can be further improved.On the other hand, the weight-average molecular weight (absolute molecular weight) of hyaluronic acid derivative is below the upper limit, so that the increase in viscosity can be suppressed, and the hyaluronic acid derivative can be dissolved in pharmaceutical composition at a higher concentration.The weight-average molecular weight (absolute molecular weight) of hyaluronic acid derivative can generally be adjusted by using the raw material with corresponding molecular weight.

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

[0055] In the chromatogram obtained by gel permeation chromatography measurement, the ratio Pt / Pr of the retention time Pt ​​of the refractive index intensity maximum point (peak top) of hyaluronic acid derivative to the retention time Pr (elution time Bb) of the refractive index intensity maximum point (peak top) of 50 kDa polyacrylic acid as standard substance is not particularly limited, but is preferably 0.5 or more and less than 1.0, more preferably 0.7 or more and less than 0.95, and even more preferably 0.75 or more and less than 0.92.When the ratio Pt / Pr is within the above range, it can contain a relatively large amount of hyaluronic acid derivative with large particle size, and when formulated with medicinal ingredients, it can form a stable association state with medicinal ingredients.Therefore, it can stably and efficiently deliver medicinal ingredients to immune cells (preferably DC, more preferably cDC1), and further improve, promote or enhance the uptake of medicinal ingredients into immune cells. In addition, in the chromatogram obtained by the gel permeation chromatography measurement, when there are multiple refractive index intensity maximum points (peak tops) of the hyaluronic acid derivative, the elution time at the refractive index intensity maximum point (peak top) where the refractive index intensity is the largest is adopted as the retention time Pt ​​of the refractive index intensity maximum point (peak top) of the 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, it is more preferably Order No. PSS-Paa series (sodium polyacrylate) manufactured by Polymer Standards Service-USA.

[0056] The weight-average molecular weight (polyacrylic acid equivalent) of hyaluronic acid derivative is not particularly limited, but the weight-average molecular weight (polyacrylic acid equivalent) of hyaluronic acid derivative is preferably 110,000 or more, more preferably 110,000 or more and less than 500,000, more preferably 130,000 or more and less than 300,000, and most preferably 140,000 or more and less than 250,000.When the weight-average molecular weight (polyacrylic acid equivalent) of hyaluronic acid derivative is within the above-mentioned range, it can contain a relatively large amount of hyaluronic acid derivative with large particle size, and when formulated with medicinal components, it can form a stable association state with medicinal components.Therefore, it can deliver medicinal components to immune cells (preferably DC, more preferably cDC1) stably and efficiently, and can improve, promote or enhance the uptake of medicinal components into immune cells. On the other hand, if the weight-average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative exceeds the upper limit, the viscosity increases, making it difficult to use as a formulation.

[0057] The weight-average molecular weight (polyacrylic acid equivalent) of the hyaluronic acid derivative is calculated from the chromatogram obtained by the gel permeation chromatography measurement, based on a calibration curve prepared using polyacrylic acid (PSS-Paa2k (2kDa), 4k (4kDa), 8k (8kDa), 18k (18kDa), 40k (40kDa), 150k (150kDa) (PSS Polymer Standard Service GmbH, sodium polyacrylate) with molecular weights of 2kDa, 4kDa, 8kDa, 18kDa, 40kDa, and 150kDa as a standard substance, 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 unit (I)").

[0059] [ka]

[0060] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b -SR, and -SSR, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f - optionally inserted with a group selected from the group consisting of: R f is a hydrogen atom, C 1-12 Alkyl, Amino C 2-12 Alkyl and hydroxy C 2-12alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g optionally containing an inserted group selected from the group consisting of - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

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

[0062] [ka]

[0063] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X is -NR a -Y-NR b-COO-R is a hydrophobic group; R a and R b are each independently selected from the group consisting of a hydrogen atom and a C alkyl; R is a steryl group; Y is C 2-30 alkylene, or —(CH2CH2O)m-CH2CH2—; m is an integer between 1 and 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 hydroxy group may be modified by -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 The carboxy group may be converted to an amide or ester, or may form a salt.

[0066] Repeating Unit The group "-ZN(R a )YX 1 " is the 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 )-CH 2 -SR; -NH-(CH 2 ) mz -SSR; and -Z-NR a -Y-NR b -COO-R (where mz is an integer between 2 and 30, and R 8 is a hydrogen atom or a methyl group, and R and m are as defined above in this specification. Preferably, the group consisting of the groups represented by -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -SSR (wherein mz, ​​R, and m are as previously defined herein.) It is more preferred that the hydroxyl group is selected from the group consisting of:

[0067] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 Ha-NR b Preferably, it is —COO—R. Furthermore, in another embodiment, Z is —NH—[CH(—Z a )-CONH] n-1 -CH(-Z a )-CO-, where n is an integer of 2 or more and 30 or less, and Z a are each independently H2N-CH(-Z a The peptide linker is attached to the carboxy group of the glucuronic acid moiety at the N-terminus and to the group -N(-R)-COOH at the C-terminus. a )-YX 1 Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural (L-form) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as D-forms thereof, and all α-amino acids, including synthetic amino acids, can be used. That is, Z a Examples of such linkers include -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, and the like. Furthermore, n Zs may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, and more preferably 2 to 4. Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, and the like.

[0068] (Y) In general formula (I), Y is —(CH2) n1 -and-(CH2CH2O) m1A group selected from the group consisting of -CH2CH2- (where n1 is an integer of 2 or more and 20 or less, preferably an integer of 2 or more and 15 or less, more preferably an integer of 2 or more and 12 or less, and even more preferably an integer of 2 or more and 6 or less; m1 is an integer of 1 or more and 4 or less) is preferred. Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12 -, or -(CH2CH2O)2-CH2CH2- is preferred. From the viewpoint of realizing high solubility in pure water or at low salt concentrations and exhibiting high precipitate-forming ability at physiological salt concentrations, Y is preferably -(CH2)2-, -(CH2)6-, -(CH2)8-, or -(CH2) 12 A group selected from the group consisting of - is preferred, with -(CH2)6- being more preferred.

[0069] Y may 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-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, and the like.

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

[0071] (Y b ) Y b As the alkyl group, -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, or octa-2,4,6-triene-1,8-diyl is preferred, and -CH2-CH2- or -CH(CH3)CH2- is more preferred.

[0072] Group "-ZN(R a )YX 1" is exemplified by -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C -NH-(CH)-NH-(CH)-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH-(CH)-NH)-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-CO-NH-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-CO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-cholesteryl, and the like are preferred. a )YX 1 " as R a , R b and R c is a hydrogen atom, and Y is a linear C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, and Y a However, linear C 1-5 alkylene, or Y b However, linear C 2-8 Alkylene or linear C 2-8 It is alkenylene.

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

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

[0075] [ka]

[0076] (In the formula, R 1a , R 2a , R 3a , and R 4a are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X a is hydroxy and -OQ + wherein Q is selected from the group consisting of + is the countercation.)

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

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

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

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

[0081] In the hyaluronic acid derivative of this embodiment, the introduction rate of steryl groups relative to the repeating units of the disaccharide constituting the hyaluronic acid derivative (hereinafter sometimes simply 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, more preferably 30% or more and 55% or less, even more preferably 35% or more and 50% or less, and particularly preferably 35% or more and 45% or less. By ensuring that the steryl group introduction rate is equal to or greater than the lower limit, the hydrogel can be maintained stable without precipitating in vivo, while by ensuring that the steryl group introduction rate is equal to or less than the upper limit, the average particle size of the hydrogel can be kept within the above range.

[0082] The steryl group introduction rate is 1 It can be measured by H-NMR measurement. 1 It can be calculated based on the following formula using the integral value of the peak derived from the steryl group of the hyaluronic acid derivative in the H-NMR spectrum and the integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH3, 1.6 ppm to 2.0 ppm, 3H). H represents the number of hydrogen atoms corresponding to the peak. The aforementioned 1H-NMR can be performed, for example, using a 0.02N DCl DMSO-d6 / D2O mixture (2N DCl D2O:DMSO-d6 = 1:99) as a measurement solvent at a measurement temperature of 85°C. Furthermore, since the peak (5H) derived from cholesteryl groups overlaps with the peak near 1.6 to 2.0 ppm, which includes the peak derived from the acetyl group of glucosamine, the value calculated by subtracting 5 / 3 of the integral value of the peak derived from cholesteryl group methyl (0.7 ppm) from the integral value of the peak near 1.6 to 2.0 ppm (i.e., integral value (1.6 to 2.0 ppm) - integral value (0.7 ppm) × 5 / 3) can be used to calculate the introduction rate as the integral value of the acetyl group derived from hyaluronic acid.

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

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

[0085] As a method for converting the carboxy group of glucuronic acid into an amide and introducing a steryl group, specifically, for example, starting material hyaluronic acid or a derivative thereof, preferably hyaluronic acid or a derivative thereof composed only of repeating unit (II), is ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then, in the presence of a suitable condensing agent, the hyaluronic acid salt and a compound of the formula: "HNR a -Y-NR b -R, NHR a -Y-NR b -COO-R, HNR a -Y-NR b -COO-R, HNRa -Y-NR b -CO-R, HNR a -Y-NR b -CO-NR c -R, HNR a -Y-COO-R, HNR a -YO-COO-R, HNR a -YSR, HNR a -Y-CO-Y a -SR, HNR a -YO-CO-Y b -SR, HNR a -Y-NR b -CO-Y b -SR, HNR a -YSSR or -Z-NR a -Y-NR b -COO-R(wherein, R a , R b , R c , Y, Y a , Y b , Z and R are as defined above) with an amine having a steryl group (particularly, a cholesteryl group) introduced therein.

[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-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), and benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium. Examples include hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0087] In particular, but not limited to, DMT-MM is preferred because the reaction proceeds highly efficiently even in a mixed solvent of water and organic solvent.In addition, by using DMT-MM as a condensing agent, in a system where a large number of hydroxyl groups coexist, it is possible to suppress the formation of ester bonds and highly selectively form amide bonds between amino groups and carboxyl groups.The use of this condensing agent can, for example, prevent the reaction of the alcohol solvent with the carboxyl group of hyaluronic acid moiety, or prevent the intramolecular or intermolecular bonding of the carboxyl group and hydroxyl group simultaneously present in hyaluronic acid moiety, resulting in the formation of undesired crosslinks.

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

[0089] Alternatively, the starting hyaluronic acid or its derivative can be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then react this hyaluronic acid salt with a spacer moiety in a solvent in the presence of a suitable condensing agent (at this time, protection and deprotection reactions can be carried out as necessary), converting the carboxyl group (-COOH) of the starting hyaluronic acid or its derivative, and then reacting it with a suitable reagent. Examples of the combination of groups derived from carboxyl groups and reaction reagents are shown below. -CONRa -Y-NR b H + Hal-R; -CONR a -Y-NR b H + Hal-COOR; -CONR a -Y-NR b H + HOCO-R; -CONR a -Y-NR b H + Hal-CO-R; -CONR a -Y-NR b -COOH + HNR c -R; -CONR a -Y-NR b -CO-NR c H + Hal-R; -CONR a -Y-NR b H + HOCO-NR c -R; -CONR a -Y-NR b H + Hal-CO-NR c -R; -CONR a -Y-COOH + HO-R; -CONR a -Y-OH + Hal-COO-R; -CONR a -Y-OCOOH + HO-R; -CONR a -Y-OCOOH + Hal-R; -CONR a -Y-OCO-Hal + HO-R; -CONR a -Y-SH + Hal-R; -CONR a -Y-Hal + HS-R; -CONR a -Y-CO-Y a -Hal + HS-R; -CONR a -Y-CO-Y a -SH + Hal-R; -CONR a -YO-CO-CH=CH2+ HS-R; -CONR a -Y-NR b -CO-CH(CH3)=CH2+ HS-R; -CONR a -Y-SH + HS-R; -COZ-OH + HNR a -Y-NR b -COO-R; -COZ-NR a -Y-NR b H + Hal-COO-R (In the formula, R a , R b , R c , Y, Y a , Y b and Z are as previously defined herein, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0090] Examples of reaction modes include dehydrohalogenation reactions, condensation reactions, dehydration reactions, nucleophilic addition reactions such as Michael addition, and oxidative disulfide formation reactions. These are well-known reactions, and can be appropriately selected by those skilled in the art and carried out under preferred reaction conditions. When the conversion product or reaction product has a carboxy group, it may be converted into an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester and then reacted.

[0091] Another method is to react the carboxyl group of the raw material hyaluronic acid or its derivative with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative having a spacer with a mercapto group modified with a leaving group at the end, and then subject this to a nucleophilic substitution reaction with thiocholesterol to form a disulfide bond.

[0092] Furthermore, the method of preparing the carboxyl group of hyaluronic acid or its derivatives that introduces a part of spacer, and the method of introducing a part of spacer into steryl group, and then reacting them.Some specific examples have been mentioned above, but when -SS- is inserted into Y, the carboxyl group of hyaluronic acid can also be prepared by introducing the spacer that has mercapto group at the end of the hyaluronic acid derivative, and the steryl group that has mercapto group at the end of the spacer, and then oxidatively react them to form disulfide bond.At this time, one mercapto group can be reacted with 2-mercaptopyridine to form disulfide, and then be replaced with the other mercapto group.

[0093] After preparing the hyaluronic acid derivative, other substituents may be further introduced. For example, 0.1 mol % to 99.5 mol % of the carboxyl groups in the hyaluronic acid derivative essentially consisting of the repeating unit (I) and the repeating unit (II) may be replaced by -CO-X z , where X z is the following group: -NH-(CH2) p1 -O-CO-C(R 17 )=CH2; -NH-(CH2) p1 -O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -SH; -NH-(CH2) p1 -NH-CO-C(R 17 )=CH2; -NH-(CH2) p1 -NH-C(=NH)-(CH2)3-SH; -NH-(CH2) p1 -NH-CO-(CH2) r -SH; -NH-(CH2) p1 -NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-(CH2)2-SH; -NH-NH-CO-(CH2)4-CO-NH-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-(CH2) r -SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-(CH2)2-SH; -NH-CH(CO2H)-(CH2)-SH; -NH-CH(CO2H)-(CH2)2-SH; and -NH-CH(CO2H)-(CH2)2-CONH-CH(CONH-CH2-CO2H)-CH2-SH (where R 17 is a hydrogen atom or C 1-6 p1 is an alkyl group, p2 is an integer of 2 or more and 10 or less, q is an integer of 1 or more and 100 or less, and r is an integer of 1 or more and 3 or less. By converting the compound into the above, it is possible to chemically crosslink the compound within a molecule or between molecules including other molecules to form a gel.

[0094] When controlling particle size within a specific range by dialysis, dialysis membrane with MWCO (molecular weight cutoff) 300kDa is used for 1 to 9 times, preferably 3 to 9 times, more preferably 6 to 9 times, so that hyaluronic acid derivative with particle size distribution that satisfies area ratio A1 / A2 of 0.9 or more can be obtained.The number of dialysis times can be selected by those skilled in the art as long as area ratio A1 / A2 satisfies 0.9 or more.In some cases, dialysis can be performed for 9 times or more.

[0095] At this time, the method for obtaining the hyaluronic acid derivative with particle size distribution that satisfies area ratio A1 / A2 of 0.9 or more is not limited to dialysis.For example, by using ultrafiltration membrane or microfiltration membrane centrifugal filtration separation, preparative purification HPLC, preparative purification GPC, ultracentrifugal separation, separation by ion exchange resin, separation by membrane distillation, membrane separation by organic or inorganic membrane, separation by adsorption and desorption method using activated carbon, zeolite, MOF (Metal Organic Framework), separation by TFF (Tangential Flow Filtration), using filter liquid transfer, pressure or vacuum filtration separation, precipitation separation method using salting out etc., separation by using hyaluronic acid receptor, etc., similarly, by removing the hyaluronic acid derivative below the particle size distribution of standard polyacrylic acid (50kDa, Polymer Standards Service-USA, ORDER No.PSS-Paa50k), can obtain the hyaluronic acid derivative with particle size distribution that satisfies area ratio A1 / A2 of 0.9 or more.

[0096] The hyaluronic acid derivative obtained can be dried.Drying method can be for example ventilation drying, drying in thermostatic oven, drying under reduced pressure, hot air circulation drying, freeze-drying etc.Among them, freeze-drying is preferred.When freeze-drying is carried out, it is preferred that the hyaluronic acid derivative further comprises cryoprotectant, from the viewpoint of more effectively suppressing the particle size increase of the microparticles formed by this hyaluronic acid derivative.

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

[0098] Disaccharide is not particularly limited, and for example, can be sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, scillabiose, neohesperidose, rutinose, rutinulose, vicianose, xylobiose, primeverose etc.Among them, sucrose, trehalose, maltose or lactose are preferred, because they are widely used as cryoprotective agent.In addition, sucrose is more preferred, because of its use as pharmaceutical additive and from the viewpoint of more effectively suppressing the increase in particle size of the microparticles formed by hyaluronic acid derivative during freeze-drying.

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

[0100] The amount of cryoprotectant to be added is not particularly limited, but it is preferably more than 20 parts by weight for 100 parts by weight of hyaluronic acid derivative.By adding the amount of cryoprotectant to be more than the lower limit value, it can obtain more sufficient particle size increase suppression effect.On the other hand, the upper limit of the amount of cryoprotectant to be added is not particularly limited, but it can be, for example, 100,000 parts by weight.

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

[0102] The hyaluronic acid derivative of the second embodiment of the present invention is a hyaluronic acid derivative that has been introduced with steryl groups, and in the chromatogram obtained by gel permeation chromatography, the ratio Pt / Pr of the retention time Pt ​​at the refractive index intensity maximum point (peak top) of the hyaluronic acid derivative to the retention time Pr at the refractive index intensity maximum point (peak top) of the 50 kDa polyacrylic acid as a standard substance is 0.5 or more and less than 1.0, more preferably 0.7 or more and less than 0.95, and most preferably 0.75 or more and less than 0.92.By making the ratio Pt / Pr within the above range, it is possible to contain a relatively large amount of hyaluronic acid derivative with large particle size, and when formulated with medicinal ingredients, it can form a stable association state with medicinal ingredients.Therefore, it is possible to stably and efficiently deliver medicinal ingredients to immune cells (preferably DC, more preferably cDC1), and furthermore, it is possible to improve, promote or enhance the uptake of the medicinal ingredients 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 preferable. The measurement by gel permeation chromatography can be carried out, for example, by the following method. Prepare a 1 mg / mL aqueous solution of hyaluronic acid derivative and a 2 mg / mL aqueous solution of polyacrylic acid standard, and perform measurements by gel permeation chromatography under the conditions shown below. (Measurement conditions) Equipment: High-speed GPC (gel permeation chromatography) equipment Column: G4000SWXL (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: 1mL / min Injection volume: 50μL Detector: RI (differential refractive index detector) Temperature: 30℃ Other than that, the description of the same configuration as the hyaluronic acid derivative of the first embodiment will be omitted.

[0104] The hyaluronic acid derivative of the third embodiment of the present invention is the hyaluronic acid derivative that steryl group is introduced, and the weight-average molecular weight (polyacrylic acid equivalent) of the hyaluronic acid derivative is 110,000 or more and less than 500,000, preferably 130,000 or more and less than 300,000, more preferably 140,000 or more and less than 250,000.When the weight-average molecular weight (polyacrylic acid equivalent) of the hyaluronic acid derivative satisfies the above range, it can contain a relatively large amount of hyaluronic acid derivative with large particle size, and when formulated with medicinal components, it can form a stable association state with medicinal components.Therefore, it can deliver medicinal components to immune cells (preferably DC, more preferably cDC1) stably and efficiently, and furthermore, it can improve, promote or enhance the uptake of the medicinal components into immune cells. On the other hand, if the weight-average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative exceeds the upper limit, the viscosity increases and it may be difficult to use the hyaluronic acid derivative as a formulation.

[0105] The weight-average molecular weight (in terms of polyacrylic acid) of the hyaluronic acid derivative is calculated from the chromatogram obtained by gel permeation chromatography measurement, based on a calibration curve prepared using polyacrylic acid having 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 carried out by the method described in the first or second embodiment. Other than that, the description of the same configuration as the hyaluronic acid derivative of the first embodiment and / or the second embodiment will be omitted.

[0107] Pharmaceutical Composition The hyaluronic acid derivatives of the first to third embodiments can be formulated with medicinal ingredients and used as pharmaceutical compositions. That is, the pharmaceutical composition of this embodiment contains the above-mentioned hyaluronic acid derivative and a medicinal ingredient.

[0108] In the pharmaceutical composition of this embodiment, the above-mentioned hyaluronic acid derivative forms a complex with a medicinal ingredient (hereinafter, may be referred to as "medicinal ingredient-hyaluronic acid derivative complex").Specifically, the steryl group in the hyaluronic acid derivative and the hydrophobic part of the medicinal ingredient form a complex by hydrophobic interaction, and it is presumed that the medicinal ingredient and the hydrophobic part such as the steryl group are present in the center, while the hydrophilic part such as the part derived from hyaluronic acid in the hyaluronic acid derivative are present in the outer periphery, thus presenting a core-shell-like spherical structure.In other words, it is presumed that the medicinal ingredient is encapsulated or encapsulated in the hyaluronic acid derivative.

[0109] In the pharmaceutical composition of this embodiment, the average particle size of the spherical structures containing the medicinal ingredient-hyaluronic acid derivative complex can be 20 nm to 100 nm, 20 nm to 95 nm, or 20 nm to 90 nm. The average particle size here refers to a value expressed as the z-average. Having an average particle size within the above range allows the structure to remain stable in vivo and pass through lymph nodes more easily. The average particle size can be measured, for example, by DLS (Dynamic Light Scattering), a nanotracking particle analyzer, size exclusion chromatography, high-performance liquid chromatography, electron microscopy, or the like. More specifically, the hyaluronic acid derivative is diluted with 10 mM phosphate buffer containing 10 w / v% sucrose to a concentration of 1 mg / mL and then measured using a DLS device.

[0110] In the pharmaceutical composition of this embodiment, the weight-average molecular weight Mw (in terms of polyacrylic acid) of the hyaluronic acid derivative or the medicinal ingredient-hyaluronic acid derivative complex is preferably 110,000 or more.The weight-average molecular weight Mw (in terms of polyacrylic acid) can be calculated by creating a calibration curve using multiple polyacrylic acid standards with different molecular weights.The polyacrylic acid standard used is preferably sodium polyacrylate, and more preferably, it is manufactured by Polymer Standards Service-USA, Inc., Order No. PSS-Paa series (sodium polyacrylate). When Mw is within the above-mentioned range, there are more aggregate sizes that are excellent in activating costimulatory molecules, and it is believed that use of the pharmaceutical composition of this embodiment can stably and efficiently deliver medicinal ingredients such as peptides to immune cells (particularly, cDC1 and macrophages), and further improve, promote, or enhance the uptake of the medicinal ingredients into immune cells, while at the same time improving, promoting, maintaining, or enhancing the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1). Mw can be measured by, for example, 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 and measured using a size exclusion chromatography device.

[0111] In the pharmaceutical composition of this embodiment, the peak top time in the particle size distribution of the hyaluronic acid derivative or the medicinal ingredient-hyaluronic acid derivative complex by size exclusion chromatography is preferably shorter than the peak top time of the standard substance polyacrylic acid (50 kDa). A peak top time within the above range allows for a greater number of aggregate sizes that are excellent at activating costimulatory molecules. Use of the pharmaceutical composition of this embodiment is believed to enable stable and efficient delivery of medicinal ingredients such as peptides to immune cells (particularly, cDC1 and macrophages), and to improve, promote, or enhance the uptake of the medicinal ingredients into immune cells, while also improving, promoting, maintaining, or enhancing immune cell activity (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1). The peak top time can be measured, for example, by size exclusion chromatography. More specifically, the hyaluronic acid derivative is diluted to a concentration of 1 mg / mL and measured using a size exclusion chromatography device.

[0112] In the pharmaceutical composition of this embodiment, the content of the hyaluronic acid derivative is not particularly limited, but for example, it is preferably 0.01 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 even more preferably 0.20 parts by mass or more and 10.00 parts by mass or less, per 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 medicinal ingredient is not particularly limited, but 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 these, antigens are preferred, more preferably at least one selected from the group consisting of cancer antigens, antigens derived from infectious diseases, and autoantigens in immune diseases, and more preferably cancer antigens or antigens derived from infectious diseases.

[0115] That is, the pharmaceutical composition of this 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 even more preferably a pharmaceutical composition for preventing or treating cancer or infectious diseases. When the applicable disease is cancer, infectious diseases, or immune diseases, the pharmaceutical composition of this embodiment can also be referred to as a vaccine composition.

[0116] [antigen] (cancer antigens) Cancer antigens are antigens that are highly expressed on, and in some cases exclusively by, cancer cells. Cancer antigens can be expressed within or on the surface of cancer cells.

[0117] Antigen proteins that can be used in the pharmaceutical composition of this embodiment include, but are not limited to, ERK1, ERK2, WT1, MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, and prostate-specific membrane antigen (PSMA). ), T cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE- 1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, large These include adenomatous polyposis protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus proteins, the Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, and c-erbB-2.

[0118] The antigen protein may have its entire sequence or a sequence with a partial deletion.

[0119] The antigenic peptide that can be used in the pharmaceutical composition of this embodiment is an antigenic peptide that contains, in the sequence of an antigenic protein, one or more epitopes selected from the group consisting of epitopes recognized by CD8-positive cytotoxic T cells and epitopes recognized by CD4-positive helper T cells. In one embodiment, the antigenic peptide preferably contains two or more epitopes, from the viewpoint of being loaded onto an MHC class I molecule or an MHC class II molecule after degradation in antigen-presenting cells. Specifically, the antigenic peptide includes an antigenic peptide containing an epitope of an antigenic protein of a tumor cell.

[0120] In one embodiment, the antigen peptide has, for example, 8 to 120 amino acids, preferably 8 to 80 amino acids, more preferably 15 to 80 amino acids, even more preferably 16 to 80 amino acids, even more preferably 23 to 80 amino acids, still more 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 antigen peptide is an antigen peptide that contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0122] In one embodiment, when two or more epitopes are contained, an amino acid linker may be placed between the epitopes. The linker may have, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, and more preferably 4 to 8 amino acids. Examples of amino acids used in the linker include glycine (G), tyrosine (Y), leucine (L), tryptophan (W), etc. Preferred are tyrosine (Y), leucine (L), and tryptophan (W). Specific examples of the amino acid linker include a linker (4Y) consisting of four consecutive tyrosines (Y), a linker (4L) consisting of four consecutive leucines (L), a linker (4W) consisting of four consecutive tryptophans (W), a linker (6G) consisting of six consecutive glycines (G), a linker (6Y) consisting of six consecutive tyrosines (Y), a linker (6L) consisting of six consecutive leucines (L), a linker (6W) consisting of six consecutive tryptophans (W), a linker (8Y) consisting of eight consecutive tyrosines (Y), a linker (8L) consisting of six consecutive leucines (L), and a linker (8W) consisting of eight consecutive tryptophans (W), and 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 neoantigens). One cancer antigen may be used alone, or two or more cancer antigens may be used in combination.

[0124] (Antigens derived from infectious diseases) Antigens derived from infectious diseases are not particularly limited as long as they are infectious pathogens or antigens derived from infectious pathogens. Examples of infectious pathogens include viruses, bacteria, fungi, 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, herpesviruses (e.g., HSV-I, HSV-II, CMV, VZV), poxviruses (e.g., orthopoxviruses such as smallpox, vaccinia, and molluscum contagiosum), picornaviruses (e.g., rhinoviruses and enteroviruses), orthomyxoviruses (e.g., influenza viruses), paramyxoviruses (e.g., parainfluenza viruses, mumps viruses, measles viruses, and respiratory syncytial viruses (RSV)), coronaviruses (e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2), papovaviruses (e.g., papillomaviruses such as those causing genital warts, bladder warts, and plantar warts), hepadnaviruses (e.g., hepatitis B virus), and flaviviruses (e.g., hepatitis C virus). viral diseases such as those caused by infection with viruses such as Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium Examples of such diseases include bacterial diseases such as those caused by bacterial infections with bacteria such as Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, and Bordetella; fungal diseases such as chlamydia, candidiasis, aspergillosis, histoplasmosis, and cryptococcal meningitis; and malaria, Pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosoma infection.

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

[0127] Influenza viruses are RNA-enveloped viruses with a particle size of approximately 100 nm and belong to the Orthomyxoviridae family. They are classified into types A, B, and C based on the antigenicity of their internal proteins. Influenza viruses consist of an internal nucleocapsid or ribonucleic acid (RNA) core associated with nucleoprotein, surrounded by a viral envelope with a lipid bilayer structure, and an external glycoprotein. The inner layer of the viral envelope is primarily composed of matrix protein, while the outer layer is largely composed of host-derived lipid substances. Furthermore, the RNA of the influenza virus has a segmented structure. Globally widespread influenza is caused by influenza A viruses, which contain two envelope glycoproteins, hemagglutinin and neuraminidase, and are divided into 16 hemagglutinin subtypes and 9 neuraminidase subtypes based on antigenic differences.

[0128] Antigens derived from influenza viruses type A and type B are preferably used as the antigens derived from infectious diseases. The subtypes of influenza viruses type A and type B described above are not particularly limited, and may be subtypes that have been isolated so far or subtypes that will be isolated in the future.

[0129] The influenza virus-derived antigen is not particularly limited as long as it is at least a portion of the various components constituting the influenza virus. Examples include whole virus particles inactivated by an organic solvent, surfactant, or other reagent from purified virus particles, and virus subunits produced by removing impurities from the whole virus particles and purifying hemagglutinin and / or neuraminidase. From the viewpoint of immunogenicity, hemagglutinin subunits or whole virus particles are preferred. The whole virus particles are more preferably inactivated with formalin or the like. Furthermore, hemagglutinin subunits (split), which contain few impurities and require adjuvants such as immunostimulants, are particularly effective.

[0130] The method for preparing the influenza virus antigen is not particularly limited, and any known method can be used without limitation. For example, a method can be used in which a virus strain isolated from an influenza-infected animal or an influenza patient is infected into chicken eggs or the like, followed by culturing in a conventional manner, and an antigen is prepared from the purified virus stock solution. Alternatively, a virus-derived antigen prepared in cultured cells by genetic engineering may be used.

[0131] (Antigens in immune diseases) Antigens for immune diseases are not particularly limited as long as they contain an epitope of a target protein of the immune disease. Examples of immune diseases include, but are not limited to, 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. Reference 2 (WO 2017 / 164409) describes peptides derived from IL-17A. The peptide may have not only an epitope of the target protein but also an epitope of a protein other than the target protein, and may be a B cell epitope or a T cell epitope. A peptide is a sequence having an epitope, and may have a cyclic structure. The peptide may have multiple cyclic structures within the molecule. Additionally, the peptide may be conjugated to a protein. The therapeutic effect can be expected by producing antibodies against the administered protein or peptide in the body.

[0132] [Pharmaceutically active peptides or proteins] A pharmaceutically active peptide or protein refers to one that, when administered to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or pathology. Preferred pharmaceutically active peptides or proteins have curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may also have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathology. The term "pharmaceutically active peptide or protein" encompasses a full-length protein or polypeptide and may also refer to a pharmaceutically active fragment thereof. The term also encompasses pharmaceutically active analogs of peptides or proteins.

[0133] Examples of pharmaceutically active proteins include, but are not limited to, cytokines and immune system proteins such as immunoactive compounds (e.g., interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factors (G-CSF), granulocyte-macrophage colony-stimulating factors (GM-CSF), erythropoietin, tumor necrosis factors (TNF), interferons, integrins, addressins, seletins, homing receptors, T cell receptors, immunoglobulins, antibodies, hormones (insulin, thyroid hormones, catecholamines, gonadotropins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic enzymes or degradative enzymes, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate cyclase or guanylate cyclase, neuramidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone binding proteins or growth factor binding proteins, etc.), transcription factors and translation factors, tumor growth suppressor proteins (for example, proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin, etc.), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, modified factor VIII, anticoagulant factors), etc.

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

[0135] [Nucleic acid] Examples of nucleic acids include DNA, RNA, antisense nucleic acids, decoy nucleic acids, ribozymes, small interfering RNAs, nucleic acid aptamers, etc. When the antigen is a peptide or protein, nucleic acids (DNA, mRNA, etc.) encoding the antigen peptide or protein are also preferably used.

[0136] [Low molecular compounds] Examples of low molecular weight compounds include anticancer agents (e.g., alkylating agents, antimetabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory agents (steroids, non-steroidal anti-inflammatory agents, etc.), antirheumatic agents, antibacterial agents (β-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfa drugs, etc.).

[0137] In addition, as the medicinal ingredient, since it can fully exert the interaction with the steryl group of the above-mentioned hyaluronic acid derivative, it is also preferable to use highly hydrophobic, that is, poorly water-soluble.In addition, in the 17th edition of the Japanese Pharmacopoeia, "poorly water-soluble" means that the amount of water required to dissolve 1 g of solute is 30 mL or more.

[0138] Examples of poorly water-soluble solid medicinal ingredients include antipyretics and analgesics such as acetaminophen, ibuprofen, benzoic acid, ethenzamide, caffeine, camphor, quinine, calcium gluconate, dimethylcaprol, sulfamine, theophylline, theopromine, riboflavin, mephenesin, phenobarbital, aminophylline, thioacetazone, quercetin, rutin, salicylic acid, theophylline sodium salt, pyrapital, quinine hydrochloride, irgapyrin, digitoxin, griseofulvin, and phenacetin, nervous system drugs, sedatives and hypnotics, muscle relaxants, blood pressure sclerosing agents, and antihistamines; acetylspiramycin, ampicillin, erythromycin, xatamycin, chloramphenicol, and triacetyloleandomycin. Examples of effective ingredients include antibiotics such as methyltestosterone, methylandrosterone diol, progesterone, estradiol benzoate, ethinylestradiol, deoxycorticosterone acetate, cortisone acetate, hydrocortisone, hydrocortisone acetate, and prednisolone; nonsteroidal yolk hormones such as dienstrol, hexastrol, diethylstilbesterol, diethylstilbesterol dibromochloride, and chlorotrianisene; and other fat-soluble vitamins, as listed in the Japanese Pharmacopoeia, the United States Pharmacopoeia (USP), the National Formulary (NF), and the European Pharmacopoeia (EP). These active ingredients may be used singly or in combination.

[0139] The medicinal ingredient may be in a water-insoluble oily or liquid form. Examples of water-insoluble oily or liquid medicinal ingredients include teprenone, indomethacin farnesyl, menatetrenone, phytonadione, vitamin A oil, phenipentol, vitamin D, vitamin E, and other vitamins; higher unsaturated fatty acids such as DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), and liver oil; coenzyme Q; and oil-soluble flavorings such as orange oil, lemon oil, and peppermint oil, all of which are listed in the Japanese Pharmacopoeia, USP, NF, and EP. Vitamin E includes various homologs and derivatives, but is not limited thereto as long as they are liquid at room temperature. Examples include dl-α-tocopherol, dl-α-tocopherol acetate, d-α-tocopherol, and d-α-tocopherol acetate. One or more of these medicinal ingredients may be used alone or in combination.

[0140] The medicinal ingredient may be a semi-solid substance that is poorly water-soluble. Examples of poorly water-soluble semi-solid medicinal ingredients include Chinese herbal or crude drug extracts such as earth dragon root, licorice, cinnamon bark, peony root, moutan pea, valerian, Japanese pepper, ginger, tangerine peel, ephedra, nandina fruit, scutellaria, onion root, platycodon, rhododendron, rhododendron bark, garlic, seneca, fritillary root, fennel, Phellodendron bark, coptis, zedoary, chamomile, gentian, bezoar, animal gall, siberian rhizome, ginger, atractylodes rhizome, clove, tangerine peel, atractylodes rhizome, chikusetsuninjin, ginseng, kakkonto, keishito, kososan, shikokeishito, shosaikoto, shoseiryuto, bakumondoto, hangehoubokuto, and maoto; oyster meat extract, propolis and propolis extract, and coenzyme Q. One of these medicinal ingredients may be used alone, or two or more of them may be used in combination.

[0141] In the pharmaceutical composition of this embodiment, the content of the medicinal ingredient varies depending on the structure of the medicinal ingredient, but can be from 0.001% by mass to 10,000% by mass, preferably from 0.1% by mass to 1,000% by mass, more preferably from 1.0% by mass to 100.0% by mass, even more preferably from 1.5% by mass to 50.0% by mass, particularly preferably from 3.0% by mass to 30.0% by mass, and most preferably from 5.0% by mass to 20.0% by mass. Alternatively, in the pharmaceutical composition of this embodiment, the content of the active ingredient is more preferably 0.1% by mass or more and 100.0% by mass or less, even more preferably 0.1% by mass or more and 50.0% by mass or less, even more preferably 0.1% by mass or more and 30.0% by mass or less, even more preferably 0.5% by mass or more and 30.0% by mass or less, even more preferably 1.0% by mass or more and 30.0% by mass or less, even more preferably 1.0% by mass or more and 25.0% by mass or less, even more preferably 1.0% by mass or more and 20.0% by mass or less, even more preferably 1.0% by mass or more and 15.0% by mass or less, and particularly preferably 1.0% by mass or more and 10.0% by mass or less.

[0142] Alternatively, in the pharmaceutical composition of this embodiment, the content of the active ingredient is preferably 0.0001 to 1.00 parts by mass, more preferably 0.001 to 0.100 parts by mass, and even more preferably 0.002 to 0.500 parts by mass, per 100 parts by mass of the pharmaceutical composition.

[0143] Alternatively, in the pharmaceutical composition of this embodiment, the content of the active ingredient is preferably 0.001 to 1.00 parts by mass, more preferably 0.001 to 0.500 parts by mass, even more preferably 0.001 to 0.200 parts by mass, and even more preferably 0.005 to 0.100 parts by mass, per 100 parts by mass of the pharmaceutical composition.

[0144] When the content of the medicinal ingredient is equal to or greater than the above-mentioned lower limit, immune cells can be activated more effectively, while when the content is equal to or less than the above-mentioned upper limit, the medicinal ingredient can be encapsulated in the hyaluronic acid derivative component, resulting in a more stable structure.

[0145] <Adjuvant> When the pharmaceutical composition of this embodiment is a vaccine composition, it can further contain an adjuvant in addition to the medicinal ingredient and the hyaluronic acid derivative. This allows for more effective induction of immunity. Here, the induced immunity may be either humoral immunity or cellular immunity. That is, the pharmaceutical composition of this embodiment is preferably a pharmaceutical composition that contains the hyaluronic acid derivative and an antigen (preferably a cancer antigen or an infectious disease-derived antigen) as the medicinal ingredient, and further contains an adjuvant.

[0146] Generally, humoral immunity refers to an immune system centered on B cells and antibodies. When B cells are stimulated by cytokines produced by helper T cells (Th2 cells), they differentiate into plasma cells and produce large amounts of antibodies, which circulate in bodily fluids and spread throughout the body. In addition, some of the stimulated B cells become memory B cells that remember antigen information, and in the event of a second infection, they can produce large amounts of antibodies with higher affinity to the antigen more quickly than in the initial response. Cellular immunity, on the other hand, is an immune system in which cells act as the main effectors in eliminating foreign substances such as pathogens themselves, virus-infected cells, and cancer cells. This is an elimination mechanism using immune cells themselves, such as macrophages, cytotoxic T cells (CTLs, killer T cells), and natural killer cells (NK cells).

[0147] The adjuvant is not particularly limited as long as it is one that is commonly used in vaccines, and examples thereof include aluminum salts, squalene, and ligands for innate immune receptors.

[0148] The term "ligand" as used 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 actions can be used. Such substances are also called "agonists."

[0149] Examples of innate immune receptors include toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs).

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

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

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

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

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

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

[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.

[0158] Among these, anionic compounds are preferred as adjuvants, and CpG oligodeoxynucleotides are more preferred, as they can further improve the antigen-presenting function.

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

[0160] <Other additives> The pharmaceutical composition of this embodiment can be administered alone, or can be administered with pharmacologically acceptable carrier by conventional means.When used in combination with pharmacologically acceptable carrier, for example, above-mentioned hyaluronic acid derivative and above-mentioned medicinal component, and if necessary, adjuvant can be mixed with water or other physiologically acceptable liquid (for example, physiological saline, aqueous ethanol, phosphate buffered saline (PBS)) etc., and can also contain physiologically acceptable buffer solution, excipient, vehicle, preservative, stabilizer, binder, lyophilization aid etc.

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

[0162] Examples of preservatives include benzalkonium chloride, methyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, sorbic acid, and alkylpolyaminoethylglycine.

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

[0164] The pharmaceutical composition of this embodiment may be prepared as a formulation, which may be in the form of a solid, semi-solid, or liquid. In the case of a solid, examples of the solid include powder, granules, pills, pellets, tablets, capsules, etc. Among these, the solid is preferably a freeze-dried powder. In the case of a semi-solid, examples include a gel and the like. In the case of a liquid, examples include a form such as a suspension in which the powder is diluted or suspended in water or a buffer solution such as phosphate buffered saline (PBS).

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

[0166] The method for producing a pharmaceutical composition of this 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 medicinal ingredient in an organic solvent or in water containing an organic solvent to prepare an oil phase containing the medicinal ingredient; and a mixing step in which the oil phase and the aqueous phase containing the hyaluronic acid derivative are mixed so that the volume ratio of the oil phase to the aqueous phase containing the hyaluronic acid derivative is 20:100 to 0.01:100;

[0167] In the past, depending on the ratio of oil phase and water phase, the interaction between steryl groups of hyaluronic acid derivatives is hindered by oil phase, so that particles are disintegrated or partly aggregated, making it difficult to maintain the particle size of hyaluronic acid derivatives.Therefore, pharmaceutical composition only contains the hyaluronic acid derivatives of relatively small size that particles are disintegrated, or the complex of hyaluronic acid derivatives and medicinal ingredients that contains partly aggregated particles.In addition, because pharmaceutical composition is produced by dialysis method, the weight ratio of hyaluronic acid derivatives and medicinal ingredients is difficult to control, and it is just a matter of course.In addition, in order to achieve the target concentration of medicinal ingredients, it is also necessary to carry out a concentration process.

[0168] On the other hand, the method for producing pharmaceutical composition of this embodiment does not use dialysis, and by mixing the above-mentioned composition, especially the mixing ratio of oil phase and water phase, so as to be within the above-mentioned numerical range, can form a complex with medicinal ingredients while maintaining the particle size distribution of hyaluronic acid derivative before producing pharmaceutical composition, and can also control the weight ratio of hyaluronic acid derivative and medicinal ingredients to be constant.Therefore, the medicinal ingredient-hyaluronic acid derivative complex can be obtained with stable structure.As a result, the pharmaceutical composition can be obtained that is more excellent in the delivery to immune cells in lymph nodes and the activation ability of said immune cells.

[0169] Next, each step of the method for producing the pharmaceutical composition of this 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] Since the active ingredient is dissolved in an organic solvent or water containing an organic solvent, it is preferable to use an active ingredient that is poorly water-soluble. Specific examples of the active ingredient that can be used include those exemplified in the above "active ingredient."

[0172] Examples of organic solvents that dissolve medicinal ingredients include known organic solvents that are commonly used in the production of pharmaceutical compositions, such as 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 so that the volume ratio is 20:100 to 0.01:100.

[0174] The hyaluronic acid derivative is dissolved or dispersed in water or the like to a desired concentration before use. In this case, the pH of the aqueous phase containing the hyaluronic acid derivative is preferably 6.00 or higher, and more preferably 6.00 to 11.00. By having a pH above the lower limit, the aggregation of the hyaluronic acid derivative can be more effectively suppressed while forming a complex with the medicinal ingredient, thereby obtaining a medicinal ingredient-hyaluronic acid derivative complex with a more stable structure. By having a pH below the upper limit, the drug can be complexed while maintaining the dispersion stability of the hyaluronic acid derivative. Furthermore, since the charge of the drug becomes negative, there is also the aspect that the hydrophobic portion of the hyaluronic acid derivative and the drug can more easily interact with each other. On the other hand, by having a pH below the upper limit, the main chain decomposition of the hyaluronic acid derivative can be more effectively suppressed.

[0175] In the mixing step, the hydrophobic interaction between the steryl group of the hyaluronic acid derivative and the medicinal ingredient forms a medicinal ingredient-hyaluronic acid derivative complex, which has a structure in which the steryl group and the medicinal ingredient are present inside, while the hydrophilic part derived from hyaluronic acid is present on the outer periphery. Depending on the structure of the medicinal ingredient, the complex can be formed not only by the above-mentioned hydrophobic interaction but also by interactions such as electrostatic interaction and hydrogen bond.

[0176] In the mixing step, the volume 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 even more preferably 2.5:100 to 0.5:100. By keeping the oil phase to aqueous phase ratio within the above range, a sufficient amount of the medicinal ingredient can be encapsulated in the hyaluronic acid derivative to form a medicinal ingredient-hyaluronic acid derivative complex, and the oil phase can effectively prevent the hydrophobic interaction between steryl groups of the hyaluronic acid derivative particles from being inhibited by the hydrophobic interaction, which would lead to dissociation. However, this is not limited to hydrophobic interactions, and disruption of interactions that may contribute to structural stability, such as electrostatic interactions and hydrogen bonds, can also be effectively prevented.

[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 to 50 parts by mass, more preferably 0.2 to 25 parts by mass, and even more preferably 0.5 to 10 parts by mass, per 100 parts by mass of the oil phase.

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

[0179] In the mixing step, in order to expose the hydrophobic portion of the medicinal ingredient to some extent and facilitate its interaction with the hyaluronic acid derivative, the temperature is preferably set to 20° C. or higher and 65° C. or lower, more preferably 23° C. or higher and 55° C. or lower, and even more preferably 25° C. or higher and 40° C. Alternatively, the optimum temperature can be appropriately set depending on the structure and properties of the medicinal ingredient.

[0180] In the mixing step, the method is not particularly limited, but for example, the oil phase containing the medicinal ingredient is added to the aqueous phase containing the hyaluronic acid derivative in any ratio, or the aqueous phase containing the hyaluronic acid derivative is added to the oil phase containing the medicinal ingredient in any ratio, and the final mixing ratio of the oil phase and the aqueous phase is 20:100 to 0.01:100 by volume ratio, and the particle size of the hyaluronic acid derivative can be maintained by going through the mixing step.

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

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

[0183] The material and shape of the tube used 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, and examples thereof include Teflon (registered trademark) tubes, stainless steel tubes, glass tubes, plastic tubes, etc.

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

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

[0186] [Sterilization process] After mixing process, the solution that contains the obtained medicinal ingredient-hyaluronic acid derivative complex can be sterilized to obtain sterilized preparation.The sterilization method of preparation can include filtration sterilization with sterilizing filter, gas sterilization, γ-ray sterilization or electron beam sterilization etc.The filtration sterilization with sterilizing filter is the most preferred.

[0187] [Drying process] After mixing process, the solution that contains the obtained medicinal ingredient-hyaluronic acid derivative complex can be dried to obtain a dried product.As drying method, the same method as the method exemplified in the above-mentioned "method for producing hyaluronic acid derivative" can be used.

[0188] <Administration method> The pharmaceutical composition of this embodiment can be administered to animals classified as mammals, including humans (monkeys, marmosets, mice, rats, cows, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.).

[0189] The administration route can be any route known to those skilled in the art, such as intrathecal injection, intraarterial injection, intravenous injection, subcutaneous injection, as well as intranasal, transbronchial, transpulmonary, intramuscular, transdermal, or oral administration. Among these, when the pharmaceutical composition of this embodiment is a vaccine composition, subcutaneous injection or intramuscular injection is preferred.

[0190] When the pharmaceutical composition of this embodiment is administered parenterally, the dosage can be appropriately selected taking into consideration the type of recipient (including age, sex, etc.), but generally, for a human (body weight 60 kg), the amount of the active ingredient (preferably, an antigen) per dose can be from 0.01 μg to 5 mg, from 0.1 μg to 500 μg, or from 1 μg to 100 μg.

[0191] The administration frequency may be a single administration of the above-mentioned dose, or multiple administrations of the above-mentioned dose twice or more, such as once every week, every two weeks, three weeks, four weeks, one month, two months, three months, or six months. Alternatively, administration may be performed at two or more sites in a single administration.

[0192] Other Embodiments In one embodiment, the present invention provides a method for preventing or treating one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases, comprising administering an effective amount of the pharmaceutical composition to a patient or animal. In addition, examples of infectious diseases include those exemplified in the "antigens derived from infectious diseases" in the "antigens" section above. Furthermore, the term "effective amount" as used herein includes an amount effective for prevention or treatment, that is, an amount suitable for preventing the onset or treatment of the above-mentioned diseases.

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

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

[0195] In one embodiment, the present invention further provides a pharmaceutical composition comprising a composition containing a hyaluronic acid derivative and an antigen, and lymphocytes expressing an immune receptor for the antigen. The combination of the composition containing a hyaluronic acid derivative and an antigen with antigen-specific lymphocytes can produce stronger, more efficient, more sustained, and / or more extensive antitumor effects.

[0196] The present invention provides a pharmaceutical composition for use in combined administration with a composition containing the hyaluronic acid derivative and an antigen, the composition containing lymphocytes expressing an immune receptor for the antigen.

[0197] The pharmaceutical composition, which is characterized by a combination of a composition containing a hyaluronic acid derivative and an antigen with antigen-specific lymphocytes, can also be in the form of a kit, such as 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 comprising hyaluronic acid derivative and antigen is preferably administered before the administration of antigen-specific lymphocytes.In a preferred embodiment of the present invention, when the number of administrations is defined as one administration of the composition comprising hyaluronic acid derivative and antigen followed by one infusion of antigen-specific lymphocytes, it is preferred that the composition comprising hyaluronic acid derivative and antigen is administered at least once after one or two units are administered.

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

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

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

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

[0203] The composition containing a hyaluronic acid derivative and an antigen may be administered in combination with both the adjuvant and the antibody. In this case, the dosage of the composition containing a hyaluronic acid derivative and an antigen is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably, 0.1 to 20 mg / dose. 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 antibody may be administered at the same time as the composition containing a hyaluronic acid derivative and an antigen (including cases where an adjuvant is included in the formulation), or at different times. When administered at different times, it is preferable that the vaccine formulation, adjuvant, and antibody are all administered within, for example, 1 minute to 24 hours, preferably 1 minute to 5 hours.

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

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

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

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

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

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

[0210] The present invention will be described in detail below with reference to examples, but it is not intended that the scope of the present invention be limited to these examples.

[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-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1 and then 1-2.

[0213] (1) Process 1-1 To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred for 30 minutes under ice cooling. After that, the temperature was raised to room temperature (approximately 25 °C), and the mixture was stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:4). The fractions containing the target compound were combined, and the solvent was evaporated under reduced pressure.

[0214] (2) Process 1-2 The resulting residue was dissolved in ethyl acetate (40 mL), and a 4N hydrochloric acid / ethyl acetate solution (40 mL) was added. The mixture was stirred overnight at room temperature (approximately 25°C). The resulting precipitate was collected by centrifugation. The resulting solid was washed four times with ethyl acetate and then dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

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

[0216] (1) Process 2-1 DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water approximately three times by decantation. Approximately 1.5 molar equivalents of 40 wt% tetrabutylammonium hydroxide aqueous solution (TBA-OH) (manufactured by Aldrich) based on the cation exchange capacity of the resin was added, and the mixture was stirred for 30 minutes at room temperature (approximately 25°C). The excess TBA-OH solution was removed by decantation, and the resin was further washed with excess ultrapure water to obtain a TBA salt-formed cation exchange resin.

[0217] (2) Process 2-2 Hyaluronic acid sodium 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. The suspension of the cation exchange resin converted to TBA salt in "(1) Step 2-1" was added in an amount equivalent to 5 times the molar ion exchange capacity of the resin relative to the number of moles of HA units (unit molecular weight 401.3). After stirring for 15 minutes at room temperature (approximately 25°C), the mixture was filtered through a 0.45 μm filter, and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid.

[0218] 3.Process 3 A 10 mg / mL solution of HA-TBA (prepared in "2.(2) Step 2-2") in anhydrous DMSO was prepared. Chol hydrochloride was then added to the HA-TBA synthesized in "1. Step 1" at a molar ratio of 44 / 100 relative to the disaccharide repeating units (HA units). Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added at a molar ratio of 48 / 100 relative to the HA units, and the mixture was stirred overnight at room temperature (approximately 25°C). The reaction solution was dialyzed against 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water using a Spectrapore 7 dialysis membrane (Spectrum Laboratories, molecular weight cutoff (MWCO): 3,500). The resulting dialyzate was lyophilized to obtain the target product (HA-C6-Chol) as a white solid.

[0219] The cholesterol introduction rate in the obtained white solid was calculated by the following method. The measurement solvent was a 0.02N DCl DMSO-d6 / D2O mixture (2N DCl D2O:DMSO-d6 = 1:99), and the NMR apparatus was a JNM-ECS400 (manufactured by JEOL Ltd.). Measurements were carried out at 85°C. 1 H-NMR spectra were obtained. 1In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%.

[0220] The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at 1 mg / mL and subjected to gel permeation chromatography under the following conditions. Figure 3A shows the resulting chromatogram.

[0221] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

[0222] [Synthesis Example 1-2] A hyaluronic acid derivative was synthesized in the same manner as in Synthesis Example 1-1. Product 1 In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. Measurement was performed by gel permeation chromatography under the conditions shown in Synthesis Example 1-1 above, and the resulting chromatogram is shown in Figure 3B.

[0223] [Synthesis Example 1-3] A hyaluronic acid derivative was synthesized in the same manner as in Synthesis Example 1-1. Product 1In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 44%. Measurement was performed by gel permeation chromatography under the conditions shown in Synthesis Example 1-1 above, and the resulting chromatogram is shown in Figure 3C.

[0224] [Example 1-1] The hyaluronic acid derivative obtained in Synthesis Example 1-1 was separated as follows. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Floatalizer G2, MWCO: 300,000, Ieda Trading Co., Ltd.), and dialyzed nine times against 10 mM phosphate buffer, pH 7.4. The hyaluronic acid derivative in the resulting dialyzed solution was measured by gel permeation chromatography. Figure 4A shows the resulting chromatogram. Figures 21 and 22 show the chromatograms of the hyaluronic acid derivative obtained in Example 1-1 and the standard polyacrylic acid, superimposed on each other, to explain the calculation methods for the area ratio A1 / A2 and the distance ratio Da / Db, which will be described later. The filtrate was concentrated to the desired concentration using an ultraconcentrator (Vivaspin20, MWCO: 10,000, Sartorius).

[0225] [Example 1-2] The hyaluronic acid derivative obtained in Synthesis Example 1-2 was separated by the same procedure as in Example 1-1. The hyaluronic acid derivative in the obtained dialyzed solution was measured by gel permeation chromatography. Figure 4B shows the obtained chromatogram.

[0226] [Examples 1-3] The hyaluronic acid derivative obtained in Synthesis Example 1-3 was separated by the same procedure as in Example 1-1. The hyaluronic acid derivative in the obtained dialyzed solution was measured by gel permeation chromatography. Figure 4C shows the obtained chromatogram.

[0227] [Comparative Example 1-1] The hyaluronic acid derivative obtained in Synthesis Example 1-1 was separated as follows. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-44%) was dissolved in water for injection at 10 mg / mL and diluted with 100 mM phosphate buffer to 10 mM phosphate buffer (pH 7.4). Then, it was filtered through an ultrafilter (Vivaspin® 20, MWCO: 300,000, Sartorius), and the hyaluronic acid derivative in the obtained filtrate was measured by gel permeation chromatography. Figure 5A shows the obtained chromatogram. The filtrate was concentrated to the desired concentration using an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius).

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

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

[0230] [Comparative Example 1-4] Lyophilized cholesteryl-modified pullulan (CHP: NOF Corporation, product number CHP-80T) was dissolved in water for injection to a concentration of 1 mg / mL and subjected to gel permeation chromatography. Figure 5D shows the resulting chromatogram.

[0231] [Evaluation of particle size distribution of hyaluronic acid derivatives: area ratio A1 / A2 and distance ratio Da / Db] The area ratio A1 / A2 and the distance ratio Da / Db were calculated from the gel permeation chromatograms of the hyaluronic acid derivative obtained in Example 1-1 and the standard substance polyacrylic acid. The standard polyacrylic acid (50 kDa) used was Order No. PSS-Paa50k (sodium polyacrylate) manufactured by Polymer Standards Service-USA. The standard polyacrylic acid (150 kDa) used was Order No. PSS-Paa150k (sodium polyacrylate) manufactured by Polymer Standards Service-USA. The standard polyacrylic acid powder was dissolved in water for injection to a concentration of 2 mg / mL. All polyacrylic acid used as a standard substance hereafter was manufactured by Polymer Standards Service-USA (sodium polyacrylate).

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

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

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

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

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

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

[0238] [Table 1]

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

[0240] <Production of Pharmaceutical Composition> [Example 2-1] The hyaluronic acid derivative obtained in Example 1-1 was diluted with 10 mM phosphate buffer, pH 7.4, to a concentration of 5 mg / mL. Separately, in a separate container, the fluorescently labeled peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL. The fluorescently labeled peptide used was a Biologica fluorescently labeled peptide with the following amino acid sequence, in which fluorescein was bound to the N-terminus:

[0241] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

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

[0243] Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex so that the sucrose concentration was 10% by mass relative to the total mass of the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex and sucrose, and the mixture was stirred at room temperature (approximately 25°C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 10 mM phosphate buffer, pH 7.4, to a peptide concentration of approximately 0.3 mg / mL, and sterilized and filtered using 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide per 100 parts by mass of the pharmaceutical composition was 0.0317 parts by mass.

[0244] Using the method described in Synthesis Example 1-1, the hyaluronic acid derivative was diluted to 1 mg / mL and analyzed by gel permeation chromatography. No significant changes were observed in the chromatogram of the hyaluronic acid derivative before and after peptide encapsulation (Figure 6A). At this time, the peptide concentration was 0.1 mg / mL, confirming that 100% of the peptide was encapsulated.

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

[0246] [Example 2-3] Using the hyaluronic acid derivative obtained in Example 1-3, a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex was obtained by the same procedure as in Example 2-1. The content of the fluorescently labeled peptide relative to 100 parts by mass of the pharmaceutical composition was 0.0303 parts by mass. Measurement by gel permeation chromatography was carried out using the method described in Synthesis Example 1-1, and no significant change was observed in the chromatogram of the hyaluronic acid derivative before and after peptide encapsulation (FIG. 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. Meanwhile, in a separate container, the fluorescently labeled peptide was dissolved in DMSO to a concentration of 50 mg / mL.

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

[0249] Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex so that the sucrose concentration was 10% by mass relative to the total mass of the composition and sucrose, and the mixture was stirred at room temperature (approximately 25°C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 10 mM phosphate buffer, pH 7.4, to a peptide concentration of 0.3 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide per 100 parts by mass of the pharmaceutical composition was 0.0308 parts by mass.

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

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

[0252] Comparative Example 3 A freeze-dried form of CHP (NOF Corporation, product number CHP-80T) was dissolved in phosphate buffered saline containing 6 M urea, pH 7.4, to a concentration of 10 mg / mL. Meanwhile, in a separate container, the fluorescently labeled peptide was dissolved in dimethyl sulfoxide to a concentration of 50 mg / mL.

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

[0254] The mixture was then transferred to a dialysis cassette (Thermo, Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL, No. 87724) and dialyzed against 0.6 M urea-containing phosphate-buffered saline, pH 7.4. Subsequently, dialysis was performed against 0.06 M urea-containing phosphate-buffered saline, pH 7.4, and then against phosphate-buffered saline, pH 7.4. The mixture was then concentrated to the desired concentration using an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius).

[0255] Finally, the mixture was sterilized through a 0.2 μm PES (polyethersulfone) filter (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing the fluorescently labeled peptide-CHP conjugate. The content of the fluorescently labeled peptide per 100 parts by mass of the pharmaceutical composition was 0.0420 parts by mass.

[0256] [Comparative Example 4-1] The freeze-dried hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added to give a concentration of 5 mg / mL, followed by stirring overnight to dissolve thoroughly. Meanwhile, in a separate container, the fluorescently labeled peptide was dissolved in DMSO to a concentration of 50 mg / mL.

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

[0258] Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex so that the sucrose concentration was 10% by mass relative to the total mass of the composition and sucrose, and the mixture was stirred at room temperature (approximately 25 ° C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 25 mM phosphate buffer, pH 7.4, to a peptide concentration of 0.3 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide per 100 parts by mass of the pharmaceutical composition was 0.0316 parts by mass.

[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 fluorescently labeled peptide-hyaluronic acid derivative complex was obtained in the same manner as in Comparative Example 4-1.

[0260] Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition containing the fluorescently labeled peptide-hyaluronic acid derivative complex so that the sucrose concentration was 10% by mass relative to the total mass of the composition and sucrose, and the mixture was stirred at room temperature (approximately 25°C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 30 mM phosphate buffer, pH 7.4, to a peptide concentration of 0.33 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a fluorescently labeled peptide-hyaluronic acid derivative complex. The content of the fluorescently labeled peptide per 100 parts by mass of the pharmaceutical composition was 0.0358 parts by mass.

[0261] [Confirmation of fluorescently labeled peptide concentration] High performance liquid chromatography measurement under the following conditions confirmed that the fluorescently labeled peptides 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 were complexed with the hyaluronic acid derivative and solubilized, and the concentration of the fluorescently labeled peptide in each pharmaceutical composition was also provided.

[0262] (Measurement conditions) HPLC equipment: JASCO HPLC-EXTREMA Column: PLRP-S 1000A 8 μm, length 50 mm, inner diameter 4.6 mm (Agilent Technologies, product number: PL1512-1802) Column temperature: 40℃ 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 (215 nm) Gradient program: shown in Table 2 below. In Table 2, "%" means "v / v%".

[0263] [Table 2]

[0264] [Test Example 1-1] (Analysis of peptide uptake and activation in lymph node myeloid cells) 1.Material Adjuvant: CpG oligo DNA1668 (purchased from Ajinomoto Bio-Pharma Services Gene Design) Administered compositions: pharmaceutical compositions of Example 2-1, Comparative Example 2-1, Comparative Example 3, and Comparative Example 4-1.

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

[0266] The antibody combinations used in the FACS analysis described below are as follows:

[0267] [Table 3]

[0268] 2.Analysis method The pharmaceutical compositions of Example 2-1, Comparative Example 2-1, and Comparative Example 4-1 were diluted with 10% sucrose, 10 mM phosphate buffer, pH 7.4, to a fluorescent (fluorescein)-labeled peptide concentration of approximately 0.3 mg / mL, and the pharmaceutical composition of Comparative Example 3 was diluted with phosphate-buffered saline, pH 7.4, and then the diluted pharmaceutical compositions were subcutaneously administered to the right dorsal region of BALB / c mice at a dose of 60 μg of peptide each. Simultaneously, 50 μg of CpG oligoDNA1668 (Ajinomoto Bio-Pharma Services Gene Design) dissolved in PBS was administered as an adjuvant.

[0269] Twenty hours after administration, lymph nodes (right inguinal lymph nodes) were collected from the administration site, and after grinding the lymph nodes using a glass slide, the cells were suspended in RPMI1640 medium. Cells from two animals per group were pooled. After centrifugation (400 × g, 5 minutes, 4°C), the supernatant was removed, and the cells were washed twice with RPMI1640 medium and then suspended in RPMI1640 medium containing 10% FBS (v / v). The cell count was measured using a hemocytometer, and the cell concentration was 1.2 × 10 7 The concentration was adjusted to 1 / mL.

[0270] 6 × 10 cells per well in a 96-well V-bottom microplate (Nunc, Thermo Fisher Scientific). 550 μL of cell suspension was added to the cells so that the total number of cells was 1. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C), the supernatant was removed, and the cells were washed twice with 200 μL of staining buffer (PBS containing 0.5% v / v FBS) and then suspended. After centrifugation and removal of the supernatant, 50 μL of a solution containing a 50-fold diluted anti-mouse CD16 / CD32 antibody was added and the cells were left to stand in the dark at 4°C for 10 minutes. 150 μL of staining buffer was added, centrifuged, and the supernatant was removed. The cells were then washed twice with 200 μL of staining buffer. 50 μL of a solution containing the first, second, and third antibodies was added to the cells in the combinations listed in Table 3, according to the manufacturer's recommended concentrations for each antibody, mixed, and left to stand in the dark at 4°C for 15 minutes. 150 μL of staining buffer was added, centrifuged, and the supernatant was removed. The cells were then 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 Biosciences). The cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and the accompanying analysis software (FACSDiva).

[0271] Dendritic cells (DCs) were detected as CD11c-positive, macrophages (Mphs) as F4 / 80-positive, and canonical type 1 DCs (cDC1s) as CD11c-positive and XCR1-positive populations. For each cell type, FITC (fluorescein)-positive cells were detected as cells that had taken up the peptide. The expression of costimulatory molecules CD80 and CD86 was analyzed for FITC-positive cells in each cell type. The FACS analysis method is shown in Figures 7A to 7F. The analysis results are shown in Tables 4-1 to 4-3. For example, in Table 4-1, "peptide + CD80 + DC" means "dendritic cells (DCs) that express CD80 and have taken up the peptide," "peptide + CD86 + DC" means "dendritic cells (DCs) that express CD86," and "peptide + DC" means "dendritic cells (DCs) that have taken up the peptide."

[0272] [Table 4-1]

[0273] [Table 4-2]

[0274] [Table 4-3]

[0275] As shown in Tables 4-1 to 4-3, the mice administered with the pharmaceutical composition of Example 2-1 showed higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) than the mice administered with the pharmaceutical compositions of Comparative Examples 2-1 and 4-1 (pre-fractionation hyaluronic acid derivative). Furthermore, in DCs (especially cDC1), the mice administered with the pharmaceutical composition of Example 2-1 showed higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) than the mice administered with the pharmaceutical composition of Comparative Example 3, a derivative of another polysaccharide (pullulan).

[0276] [Test Example 1-2] The pharmaceutical compositions of Example 2-2, Comparative Example 2-2, and Comparative Example 4-2 were diluted with 10% by mass sucrose, 10 mM phosphate buffer, pH 7.4, to a fluorescent (fluorescein)-labeled peptide concentration of approximately 0.2 mg / mL, and administered subcutaneously at 40 μg of peptide, in the same manner as in Test Example 1-1. The analytical 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, the mice administered with the pharmaceutical composition of Example 2-2 showed higher expression of costimulatory molecules (CD80 and CD86) in macrophages and DCs (especially cDC1) than the mice administered with the pharmaceutical compositions of Comparative Example 2-2 and Comparative Example 4-2 (pre-fractionation hyaluronic acid derivative).Comparative Example 4-2 showed relatively high expression of CD80 and CD86 in cDC1, but low expression of CD80 and CD86 in macrophages, which was inferior to the pharmaceutical composition of Example 2-2.

[0281] [Test Example 1-3] The test was carried out in the same manner as in Test Example 1-1, except that the pharmaceutical composition of Example 2-3 was used and administered subcutaneously so that each peptide amounted to 40 μg. The analytical 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, high expression of costimulatory molecules (CD80 and CD86) was confirmed in macrophages and DCs (especially cDC1) in the mouse group administered with the pharmaceutical composition of Example 2-3.

[0286] It has been reported that reduced expression of CD86 on macrophages is or has been shown to be immunosuppressive (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 report in Reference 3 and the results of Test Examples 1-1 to 1-3, the pharmaceutical compositions of Examples 2-1, 2-2, and 2-3, in which CD86 is strongly expressed not only on DCs but also on macrophages, are expected to induce stronger immunity.

[0287] From these results, it is presumed that use of the pharmaceutical composition of this embodiment enables stable and efficient delivery of medicinal ingredients such as peptides to immune cells (particularly, cDC1 and macrophages), and further improves, promotes, or enhances the uptake of the medicinal ingredients into immune cells, while at the same time improving, promoting, maintaining, or enhancing the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1).

[0288] <Consideration of manufacturing conditions for pharmaceutical compositions> [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] The peptide used was gp100RP2RP1_6Y peptide manufactured by Biologica and having the following amino acid sequence:

[0290] SVYDFFVWLYYYYYYTWHRYHLLYYYYYYEGSRNQDWL (SEQ ID NO: 2)

[0291] Specifically, first, the freeze-dried hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added to the mixture so as to obtain the concentration shown in Table 7 below, followed by stirring overnight to dissolve the mixture thoroughly. Meanwhile, in a separate container, the peptide was dissolved in DMSO to a concentration of 50 mg / mL. After confirming dissolution, 1 mol / L of aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Co., Ltd.) was added to the aqueous hyaluronic acid derivative solution according to Table 7 below.

[0292] At room temperature (about 25 ℃), above-mentioned hyaluronic acid derivative aqueous solution and peptide dimethyl sulfoxide solution are mixed in the volume ratio shown in the following table 7, and stirred at room temperature (about 25 ℃) for 24 hours, thereby obtaining the composition that contains peptide-hyaluronic acid derivative complex.At this time, the pH of the solution is also measured.

[0293] (Stability of peptide-hyaluronic acid derivative complex: Appearance in aqueous solution) After 24 hours, the appearance of the composition (aqueous solution) containing the peptide-hyaluronic acid derivative complex was visually inspected to see if it was cloudy. The results are shown in Table 7 below.

[0294] [Table 7]

[0295] Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for exclusive use in manufacturing, product number 198-18385) is added to the composition containing peptide-hyaluronic acid derivative complex and sucrose in such a manner that the sucrose is 10% by mass, and stirred at room temperature (about 25 ° C) for more than 1 hour.Subsequently, dilute with 10% by mass sucrose-containing 25 mM phosphate buffer pH 7.4 to make the theoretical concentration of peptide 0.3 mg / mL, and sterilize and filter with 0.2 μm PES (polyethersulfone) (Pall Co., Ltd., Acrodisc syringe filter, 25 mm Φ), to obtain the pharmaceutical composition containing peptide-hyaluronic acid derivative complex.The peptide concentration in the pharmaceutical composition containing peptide-hyaluronic acid derivative complex at this time is quantified in the same manner as in Example 2-1 above, and the results are shown in Table 7 above.

[0296] (Average particle size) The composition containing the peptide-hyaluronic acid derivative complex produced by the method described 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 equipment: Otsuka Electronics, ELSZ2000 Cell: Micro particle size cell Temperature: 25℃ Concentration of peptide-hyaluronic acid derivative complex: 4.95 mg / mL

[0298] As shown in Table 7 above, it has been revealed that in the production of pharmaceutical compositions, by adjusting the pH of the mixed solution within a specific range, the particle size of the complex of the medicinal ingredient such as a peptide and a hyaluronic acid derivative can be maintained within a certain range, and the stability of the complex in the solution can be improved.

[0299] [Reference examples 2-1~2-3] 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] The peptide used was manufactured by Biologica and had the following amino acid sequence:

[0301] GSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 3)

[0302] Specifically, first, the freeze-dried hyaluronic acid derivative obtained in Synthesis Example 1-1 was weighed, and water for injection was added to the mixture so as to obtain the concentration shown in Table 8 below, followed by stirring overnight to dissolve the mixture thoroughly. Meanwhile, in a separate container, the peptide was dissolved in DMSO to a concentration of 50 mg / mL. After confirming dissolution, 1 mol / L of aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Co., Ltd.) was added to the aqueous hyaluronic acid derivative solution according to Table 8 below.

[0303] At room temperature (about 25°C), the above-mentioned hyaluronic acid derivative aqueous solution and peptide DMSO solution are mixed in the volume ratio shown in Table 8 below, and stirred at room temperature (about 25°C) for 24 hours to obtain the composition containing peptide-hyaluronic acid derivative complex.The pH of the solution at this time is also measured. Then, solid purified sucrose (Fujifilm Wako Co., Ltd., for exclusive use in manufacturing, product number 198-18385) was added to the composition containing the peptide-hyaluronic acid derivative complex so that the sucrose content was 10% by mass relative to the total mass of the composition containing the peptide-hyaluronic acid derivative complex and sucrose, and the mixture was stirred at room temperature (about 25 ° C) for more than 1 hour. Subsequently, the mixture was sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall Co., Ltd., Acrodisc syringe filter, 25 mm diameter), to obtain a pharmaceutical composition containing a peptide-hyaluronic acid derivative complex. The peptide concentration in the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex at this time was quantified in the same manner as in Example 2-1 above, and the average particle size of the pharmaceutical composition containing the peptide-hyaluronic acid derivative complex was measured in the same manner as in Reference Example 1 above. The results are shown in Table 8 below.

[0304] [Table 8]

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

[0306] <Manufacturing conditions to maintain appropriate particle size distribution> [Test Examples 2-1 to 2-8] A 5 mg / mL aqueous solution of the hyaluronic acid derivative separated using the same method as in Example 1-1 was prepared, and the mixing ratio of the aqueous phase containing the hyaluronic acid derivative to the oil phase was examined in detail. 400 μL of 5 mg / mL hyaluronic acid derivative was mixed with DMSO, which is an oil phase, according to Table 9 below. After thorough mixing, water for injection was added according to Table 9 below so that the final hyaluronic acid derivative concentration was constant. After incubation at room temperature (25 ° C) for 24 hours, measurement was carried out by gel permeation chromatography under the conditions shown below, and the area ratio A1 / A2 was calculated in the same manner as in Example 1-1 (Table 9). The obtained gel permeation chromatogram is shown in Figure 8.

[0307] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 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 Test Examples 2-1 to 2-8, except that ethanol was used as the oil phase instead of DMSO. Measurements were carried out by gel permeation chromatography, and the area ratio A1 / A2 was calculated in the same manner as in Example 1-1. The results are shown in Table 10 below. The obtained gel permeation chromatogram is shown in Figure 9.

[0310] [Table 10]

[0311] The results of Test Examples 2-1 to 2-8 and 3-1 to 3-6 showed that the particle size of the hyaluronic acid derivative, which has excellent medicinal properties, can be maintained by undergoing a mixing process 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] Hyaluronic acid powder (manufactured by Kikkoman Corporation) with a weight-average molecular weight Mw (absolute molecular weight) of 980 kDa was dissolved in water for injection to a concentration of 1 mg / mL, and measurement was performed by gel permeation chromatography under the same conditions as those shown in Synthesis Example 1-1. The resulting chromatogram is shown in Figure 10.

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

[0314] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0315] We attempted to prepare an antigen peptide-hyaluronic acid complex by mixing the above hyaluronic acid aqueous solution and the antigen peptide DMSO solution at a volume ratio of 100:1 at room temperature (approximately 25°C) and stirring for 24 hours at room temperature (approximately 25°C). As can be seen in the photograph labeled "Hyaluronic acid (980 kDa)" in Figure 11, the resulting mixture was cloudy, suggesting that the antigen peptide was not dissolved and that a large amount of peptide was not encapsulated.

[0316] Subsequently, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition so that the sucrose concentration was 10% by weight relative to the total weight of the composition containing the peptide-hyaluronic acid complex and sucrose, and the mixture was stirred at room temperature for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by weight sucrose-containing 10 mM phosphate buffer, pH 7.4, to a peptide concentration of approximately 0.30 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a peptide-hyaluronic acid complex. The peptide content in the pharmaceutical composition was quantified by high-performance liquid chromatography using the same method as in Example 2-1. As a result, the peptide content in the pharmaceutical composition was below the detection limit.

[0317] [Comparative Example 5-2] Hyaluronic acid powder (manufactured by Kewpie Corporation) 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 measurement was performed by gel permeation chromatography under the same conditions as those shown in Synthesis Example 1-1. The resulting gel permeation chromatogram is shown in Figure 12.

[0318] Hyaluronic acid powder with 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 an aqueous hyaluronic acid solution. Separately, in a separate container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL to obtain a DMSO solution of the antigen peptide. The antigen peptide used was a peptide manufactured by Biologica having the following amino acid sequence:

[0319] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0320] We attempted to prepare an antigen peptide-hyaluronic acid complex by mixing the above hyaluronic acid aqueous solution and the antigen peptide DMSO solution at a volume ratio of 100:1 at room temperature (approximately 25°C) and stirring for 24 hours at room temperature (approximately 25°C). The resulting mixture was cloudy, as can be seen in the photograph labeled "Hyaluronic acid (640 kDa)" in Figure 11, suggesting that the antigen peptide was not dissolved and not encapsulated.

[0321] Subsequently, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition so that the sucrose concentration was 10% by mass relative to the total mass of the composition containing the peptide-hyaluronic acid complex and sucrose, and the mixture was stirred at room temperature (approximately 25°C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 10 mM phosphate buffer, pH 7.4, to a peptide concentration of approximately 0.30 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a peptide-hyaluronic acid complex. The peptide content in the pharmaceutical composition was quantified by high-performance liquid chromatography using the same method as in Example 2-1. As a result, the peptide content in the pharmaceutical composition was below the detection limit.

[0322] The results of Comparative Examples 5-1 and 5-2 show that it is difficult to encapsulate peptides in high molecular weight hyaluronic acid with an area ratio A1 / A2 of 0.9 or more, and it is difficult to stably and efficiently deliver medicinal ingredients such as peptides to immune cells (especially cDC1 and macrophages), and further to improve, promote, or enhance the uptake of the medicinal 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 the treatment of immune diseases> [Test Example 4]

[0324] [Comparative Example 6-1] A hyaluronic acid derivative was synthesized in the same manner as in Synthesis Example 1-1. It was used as it was without fractionation. The obtained lyophilized hyaluronic acid derivative was weighed, and water for injection was added to the solution to a concentration of 5 mg / mL, and the solution was stirred overnight at room temperature to be fully dissolved. The aqueous solution of the hyaluronic acid derivative was diluted 5 times with water for injection to a concentration of 1 mg / mL, and then measured by gel permeation chromatography. Figure 13 shows the gel permeation chromatogram obtained. In addition, the particle size distribution of the hyaluronic acid derivative was evaluated in the same manner as in Example 1-1, and 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 is fractionated by the same method as in example 1-1, and obtains hyaluronic acid derivative.The hyaluronic acid derivative of the obtained dialysis fluid is measured by gel permeation chromatography.Figure 14 shows the obtained gel permeation chromatogram.In addition, the particle size distribution of hyaluronic acid derivative is evaluated by the same method as in example 1-1, and the result is shown in the following table 11.

[0326] [Table 11]

[0327] <Production 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 of known concentration to be 4.28 mg / mL. In a separate container, ovalbumin (OVA, low-endotoxin product 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) to a concentration of 5 mg / mL to obtain an OVA / PBS solution. After mixing water for injection, hyaluronic acid derivative aqueous solution, and OVA / PBS solution in that order, sucrose was added as a solid to make a 10% sucrose isotonic solution, and the mixture was thoroughly stirred at room temperature until a clear solution was obtained. The OVA concentration in the preparation was 0.15 mg / mL, and the hyaluronic acid derivative concentration was 3.0 mg / mL, and the 10% sucrose solution contained 10 mM PB (pH 7.4). The mixture was incubated at 75°C for 1 hour, and a composition containing an OVA-hyaluronic acid derivative complex was obtained.

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

[0329] [Antibody production test] The compositions containing the OVA-hyaluronic acid derivative complexes prepared in Example 6-1-1 and Comparative Example 6-1-1 were subcutaneously administered to BALB / c mice on days 0 and 7 so that the OVA was 20 μg, and serum was collected on day 10. At the time of administration of the OVA-hyaluronic acid derivative complexes, 50 μg of the adjuvant CpG1826 (manufacturer: INVIVOGEN, product code: tlrl-1826-1) was also subcutaneously administered. Antibody titers against OVA were measured using ELISA kits (Chondrex, Mouse Anti-OVA IgG Antibody Assay Kit, Mouse Anti-OVA IgG1 Antibody Assay Kit, Mouse Anti-OVA IgG2a Antibody Assay Kit) according to the attached protocol. The serum dilutions were 10, 50, 250, 1250, 6250, and 31250.

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

[0331] When serum dilution is 1250 times or more, there is no difference in antibody titer, but when the dilution rate is lower, it was confirmed that the composition containing the OVA-hyaluronic acid derivative complex of Example 6-1-1 can obtain a higher antibody titer than the composition containing the OVA-hyaluronic acid derivative complex of Comparative Example 6-1-1.This makes it clear that the hyaluronic acid derivative of Example 6-1 (A1 / A2=2.03) 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 internal production function of therapeutic antibodies is high.

[0332] It has been reported that antigen-loaded mature dendritic cells enhance antigen-specific IgG2a production through the induction of IFN-γ-producing type I helper T cells (Reference 5: Shigeharu Fujita et al., "Regulatory dendritic cells protect against allergic airway inflammation in a murine asthmatic model," J Allergy Clin Immunol 2008;121:95-104). Based on the report in Reference 5 and the results of Test Example 4, it is presumed that the pharmaceutical composition containing the OVA-hyaluronic acid derivative of Example 6-1-1 activated DC costimulatory molecules, resulting in strong expression of CD80 and CD86, which in turn enhanced the induction of antigen-specific T cells and IFN-γ production, thereby enhancing antigen-specific IgG2a production. Therefore, the pharmaceutical composition of Example 6-1-1 is expected to induce not only humoral immunity but also cellular immunity, and thus induce a stronger immune response as an infectious disease vaccine.

[0333] By using the pharmaceutical composition of this embodiment, it is possible to protect not only peptides but also medicinal ingredients of protein antigens from heating conditions, stably and efficiently deliver them to immune cells (particularly, cDC1s and macrophages) while maintaining an appropriate three-dimensional structure, and further improve, promote, or enhance the uptake of the medicinal ingredients into immune cells, and at the same time, it is presumed that the activity of immune cells (particularly, the expression of costimulatory molecules CD80 and CD86 in cDC1s) is improved, promoted, maintained, or enhanced. It is presumed that antigens against the epitope are produced by promoting the activation of helper T cells, which in turn promotes the activation and expansion of B cells.

[0334] It is believed that the use of the hyaluronic acid derivatives of the first to third embodiments can provide vaccines (mainly infectious disease vaccines) against foreign antigens that are superior to those using conventional hyaluronic acid derivatives, and at the same time, it is believed that pharmaceuticals containing superior therapeutic antibody-inducing antigens can be provided.

[0335] <Functional evaluation as a cancer vaccine (antitumor test of a cancer vaccine using an administration composition containing a hyaluronic acid derivative and an antigen)> [Test Example 5] To clarify the antitumor effect of cancer vaccines using hyaluronic acid derivatives with different area ratios A1 / A2, we used immune checkpoint inhibitor (ICI)-resistant fibrosarcoma cell line CMS5a tumor-bearing mice to create hyaluronic acid derivative cancer vaccines loaded with long-chain peptide antigens, which contain the CD8 epitope of the mutant ERK2 antigen (mERK2) expressed in CMS5a, conjugated with various hyaluronic acid derivatives, and examined their therapeutic effects.

[0336] The materials and methods are described below. (1) Cell RPMI1640 medium (supplemented with 2-mercaptoethanol) was purchased from Cell Science Institute. Fetal bovine serum (FBS) was purchased from Gibco. The mouse fibrosarcoma CMS5a cell line was provided by Memorial Sloan-Kettering Cancer Institute and passaged at Mie University. The mouse fibrosarcoma CMS5a cell line expresses a mutant ERK2 protein. A peptide containing the mutated portion of the mutant ERK2 protein (QYIHSANVL (SEQ ID NO: 4)) is recognized by CD8+ cytotoxic T cells in BALB / c mice. A T cell receptor (TCR) that recognizes this peptide has been isolated, and transgenic mice (DUC18 mice) have been generated by introducing the TCR gene.

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

[0338] (3) Long-chain peptide antigen The synthetic long-chain peptide antigen was purchased from Sigma Genosys and had the following sequence: NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1). The sequence comprises a 9-amino acid sequence (QYIHSANVL (SEQ ID NO: 4)) from the 16th Q to the 24th L as the CD8-positive cytotoxic T-cell-recognized epitope sequence of the mutant ERK2, and a 17-amino acid sequence (RGLQYIHSANVLHRDLK (SEQ ID NO: 5)) from the 13th R to the 29th K as the CD4-positive helper T-cell-recognized epitope sequence. The hyaluronic acid derivatives loaded with long-chain peptide antigens, which are complexed with the long-chain peptide antigens and various hyaluronic acid derivatives, are sometimes referred to as "long-chain peptide antigen-loaded HA nanogel cancer vaccines" or "HA nanogel cancer vaccines."

[0339] [Example 7-1] The hyaluronic acid derivative obtained in Comparative Example 6-1 was fractionated as follows. The lyophilized hyaluronic acid derivative (10k HA-C6-Chol-41.3%) was dissolved in water for injection at 5 mg / mL, transferred to a dialysis cassette (Floatalizer 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 dialyzate was measured by gel permeation chromatography. Figure 19 shows the resulting gel permeation chromatogram. The particle size distribution of the hyaluronic acid derivative was evaluated using the same method as in Example 1-1. The results are shown in Table 12.

[0340] [Table 12]

[0341] <Production of Pharmaceutical Composition> [Example 7-1-1] The concentration of the hyaluronic acid derivative in the aqueous solution of the hyaluronic acid derivative obtained in Example 7-1 was calculated to be 3.8 mg / mL from the area values ​​of the hyaluronic acid derivatives of known concentrations. Separately, in a separate container, the antigen peptide was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mg / mL to obtain a DMSO solution of the antigen peptide. The antigen peptide used was a peptide manufactured by Biologica having the following amino acid sequence:

[0342] NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)

[0343] At room temperature (about 25°C), the above hyaluronic acid derivative aqueous solution and the DMSO solution of the antigen peptide were mixed in a ratio of 100 parts by mass of hyaluronic acid derivative to 10 parts by mass of peptide, and the mixture was stirred at room temperature (about 25°C) for 2 hours to attempt the preparation of an antigen peptide-hyaluronic acid derivative complex. Visual inspection of the solution at this time revealed that a clear solution was obtained, as can be seen in the photograph labeled "Hyaluronic Acid Derivative" in Figure 11. This confirmed that the peptide had indeed been encapsulated.

[0344] Subsequently, solid purified sucrose (Fujifilm Wako Co., Ltd., for manufacturing purposes only, product number 198-18385) was added to the composition so that the sucrose concentration was 10% by mass relative to the total mass of the composition containing the peptide-hyaluronic acid derivative complex and sucrose, and the mixture was stirred at room temperature (approximately 25°C) for at least 1 hour. After confirming that the sucrose was fully dissolved, the mixture was diluted with 10% by mass sucrose-containing 10 mM phosphate buffer, pH 7.4, to a peptide concentration of approximately 0.30 mg / mL, and sterilized and filtered through 0.2 μm PES (polyethersulfone) (Pall Corporation, Acrodisc syringe filter, 25 mm diameter) to obtain a pharmaceutical composition containing a peptide-hyaluronic acid derivative complex. The peptide content in the pharmaceutical composition was quantified by high-performance liquid chromatography using the same method as in Example 2-1. As a result, the peptide content per 100 parts by mass of the pharmaceutical composition was 0.383 parts by mass. Furthermore, the pharmaceutical composition that contains the above-mentioned peptide-hyaluronic acid derivative complex is diluted with 10mM phosphate buffer solution containing 10% by mass of sucrose, pH 7.4, so that the peptide concentration is 250 μg / mL, to obtain the pharmaceutical composition of administration solution.The pharmaceutical composition that contains the above-mentioned peptide-hyaluronic acid derivative complex is measured by gel permeation chromatography according to the method described in Example 1-1, under the measurement conditions shown below.Figure 20 shows the obtained gel permeation chromatogram.

[0345] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 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) to a concentration of 50 mg / mL, and then diluted with 10% by mass sucrose-containing 10 mM phosphate buffer (pH 7.4) to a peptide concentration of 0.25 mg / mL.

[0347] [Test Example 6] Tumor growth test Mouse fibrosarcoma CMS5a cell line was cultured in RPMI1640 medium containing 10% FBS using a T75 culture flask (Corning). 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. The cells were then washed twice with RPMI1640 medium and resuspended at 1 × 10 6 The cells were suspended in RPMI1640 medium at a concentration of cells / 100 μL and subcutaneously implanted at a dose of 100 μL / cell into the right anterior dorsal region of BALB / c mice (5 mice per group).

[0348] The long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously to the right dorsal region of mice 7, 10, 13, and 16 days after tumor inoculation. 50 μg of the long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously along with 50 μg of CpG oligo DNA (Ajinomoto Bio-Pharma Services Gene Design) dissolved in PBS. The tumor area was then measured over time. When administering the long-chain peptide antigen of Comparative Example 7-1-1, 50 μg of the long-chain peptide was subcutaneously administered to the right dorsal region of the mice together with 50 μg of CpG oligo DNA (Ajinomoto Bio-Pharma Services Gene Design) dissolved in PBS 7, 10, 13, and 16 days after tumor inoculation. Thereafter, the tumor area was measured over time.

[0349] The tumor area values ​​(mean values) over time are shown in FIG. It was revealed that the pharmaceutical composition combining the long-chain peptide antigen-loaded HA nanogel cancer vaccine obtained by complexing the hyaluronic acid derivative (A1 / A2=6.62) of Example 7-1 with the long-chain peptide antigen and CpG oligoDNA suppresses the growth of CMS5a tumors more than the pharmaceutical composition combining the long-chain peptide antigen and CpG oligoDNA of Comparative Example 7-1.From this, it is believed that the use of a hyaluronic acid derivative with A1 / A2=6.62 can provide an excellent cancer vaccine.

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

[0351] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 1mL / min Injection volume: 50μL Detector: RI Temperature: 30℃

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

[0353]

Table 13

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

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

[0356] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 1mL / min Injection volume: 50μL Detector: RI Sample concentration: 1 mg / ml for the hyaluronic acid derivatives obtained in the synthesis examples, working examples, and comparative examples, and 2 mg / ml for the polyacrylic acid standard. Calibration curve conditions: The approximate equation used was third order: At3+Bt2+Ct+D.

[0357] [Table 14]

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

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

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

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

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

[0363] <Manufacturing conditions of a pharmaceutical composition that maintains an appropriate particle size distribution> [Examples 9-1 to 9-4, Comparative Examples 9-1 to 9-4] A 5 mg / mL aqueous solution of the hyaluronic acid derivative separated using the same method as in Example 1-1 was prepared, and the mixing ratio of the aqueous phase containing the hyaluronic acid derivative to the oil phase was examined in detail. A peptide drug, cyclosporine (Tokyo Chemical Industry Co., Ltd., product number: C2408), was weighed into a 6 mL clean vial and dissolved in DMSO (Fujifilm Wako Co., Ltd., product number: 045-24511) to a peptide concentration of 50 mg / mL. Next, 400 μL of 5 mg / mL hyaluronic acid derivative is added with 4 μL of 50 mg / mL cyclosporin, which is the oil phase containing the drug.Then, according to the following Table 15, the oil phase DMSO is mixed.After thorough mixing, according to the following Table 15, the water for injection is added so that the final hyaluronic acid derivative concentration is constant.Furthermore, after 24 hours of incubation at room temperature (25 ℃), measurement is carried out by gel permeation chromatography under the following conditions, and the area ratio A1 / A2 is calculated in the same manner as in Example 1-1 (Table 15).

[0364] (Measurement conditions) Apparatus: HLC8420-GPC (Tosoh Corporation) Column: G4000SWXL (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: 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, which has excellent medicinal properties, can be maintained by undergoing a mixing process 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. [Industrial Applicability]

[0367] According to the hyaluronic acid derivative of this embodiment, it is possible to provide a hyaluronic acid derivative that, when formulated with a medicinal ingredient, has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells.The pharmaceutical composition of this embodiment contains the hyaluronic acid derivative, and has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells.The manufacturing method of the pharmaceutical composition of this embodiment uses the hyaluronic acid derivative, and can obtain a pharmaceutical composition that has excellent delivery properties to immune cells in lymph nodes and the ability to activate said immune cells.

Claims

1. A hyaluronic acid derivative into which a steryl group has been introduced, The weight-average molecular weight of the hyaluronic acid derivative calculated from a chromatogram obtained by gel permeation chromatography measurement based on a calibration curve prepared using polyacrylic acids having molecular weights of 2 kDa, 4 kDa, 8 kDa, 18 kDa, 40 kDa, and 150 kDa as standard substances is 110,000 or more and less than 500,000; The hyaluronic acid derivative is a hyaluronic acid derivative in which multiple molecules are associated with each other.

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

3. The hyaluronic acid derivative according to claim 1, wherein the steryl group is a cholesteryl group.

4. The hyaluronic acid derivative according to claim 1, wherein the introduction rate of steryl groups relative to the repeating units of the disaccharide constituting the hyaluronic acid derivative is 30% or more and 60% or less.

5. The hyaluronic acid derivative according to claim 1, 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.

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

7. A pharmaceutical composition comprising the hyaluronic acid derivative according to claim 1 and a medicinal ingredient.

8. The pharmaceutical composition according to claim 7, which is for the prevention or treatment of one or more diseases selected from the group consisting of cancer, infectious diseases, and immune diseases.

9. The pharmaceutical composition according to claim 7, comprising, as the medicinal ingredient, at least one selected from the group consisting of a cancer antigen, an antigen derived from an infectious disease, and an autoantigen in an immune disease, and further comprising an adjuvant.

10. The pharmaceutical composition according to claim 9 , comprising a cancer antigen or an antigen derived from an infectious disease as the medicinal ingredient.

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

12. The method for producing a pharmaceutical composition according to claim 11, 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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