Composite

Abstract

The purpose of the present invention is to provide a technique that improves the poor solubility of a compound of formula (1) in water and further inhibits the overexpression of cytokines and bone marrow toxicity of the compound of formula (1). This object is achieved by a complex comprising a modified polysaccharide containing a hydrophobic group and a compound represented by formula (1).

Description

TECHNICAL FIELD

[0001] The present application relates to a complex. BACKGROUND

[0002] The importance of immune cells, particularly macrophages (tumor-associated macrophages, abbreviated as "TAM"), in cancer tissues has been pointed out as a factor affecting the malignancy of cancer. When TAM is M2-dominant, they exert an immunosuppressive effect and create a microenvironment that is conducive to tumor growth. If the dominant type can be changed to M1-dominant, an effect of suppressing cancer growth can be expected.

[0003] It has been reported that an activator of Toll-like receptor 7 and Toll-like receptor 8 (abbreviated as TLR7 and TLR8, respectively) polarizes macrophages from M2-like to M1-like. In addition, it is currently known that various compounds are under development as TLR7 and TLR8 activators (NPL 1).

[0004] LIST OF REFERENCES NON-PATENT LITERATURE NPL 1: Expert Opin Drug Discov. 2021 Aug; 16 (8): 869-880. SUMMARY

[0005] PROBLEMS Since problems of cytokine over-release and myelotoxicity occur when administered systemically, general TLR7 / 8 agonists cannot be administered systemically. In particular, the compound of formula (1) described below, such as Telratolimod, has a relatively long hydrocarbon chain, and thus has poor water solubility. In the course of the study, the present inventors have also newly found, through formulation studies, that Telratolimod has a cytokine over-release effect and myelotoxicity.

[0006] An object of the present application is to provide a technology that suppresses the cytokine over-release effect and myelotoxicity of various TLR7 / 8 activators, enabling systemic administration.

[0007] SOLUTION TO THE PROBLEM To achieve the above object, the present inventors have intensively studied various TLR7 / 8 agonists, and as a result, have found that a complex including a modified polysaccharide containing a hydrophobic group and a compound represented by formula (1) can achieve the above object. As a result of further studies based on this finding, the present inventors have completed the present application. Specifically, the present application includes the following aspects.

[0008] 1. A complex including a modified polysaccharide containing a hydrophobic group and a compound represented by formula (1): wherein R 1 is a C 12-25 straight chain alkyl, R 2 is a C 2-8 straight chain alkyl, R 3 is a C 2-8 straight chain alkylene.

[0009] Item 2. The complex of item 1, wherein the polysaccharide forming the modified polysaccharide comprises a pullulan.

[0010] Item 3. The complex of item 1 or 2, wherein the hydrophobic group comprises a hydrophobic group having a sterol backbone.

[0011] Item 4. The complex of any one of items 1 to 3, wherein the modified polysaccharide has a weight average molecular weight of 5,000 to 2,000,000.

[0012] Item 5. The complex of any one of items 1 to 4, wherein R 1 is a C 15-19 straight chain alkyl, R 2 is a C 3-5 straight chain alkyl, R 3 is a C 3-5 straight chain alkylene.

[0013] Item 6. The complex of item 5, wherein the compound is Telratolimod.

[0014] Item 7. The complex of any one of items 1 to 6, which is a gel particle.

[0015] Item 8. The complex of item 7, wherein the compound is contained within the gel particle.

[0016] Item 9. The complex of any one of items 1 to 8, wherein the mass ratio of the modified polysaccharide to the compound (mass of the modified polysaccharide / mass of the compound) is 2 to 20.

[0017] Item 10. The complex of any one of items 1 to 9, which has an intensity average particle size of 10 to 200 nm.

[0018] Item 11. A medicament comprising the complex of any one of items 1 to 10.

[0019] Item 12. The medicament of item 11, which is a cancer therapeutic.

[0020] Item 12A. A cancer treatment method comprising administering to a subject (e.g., a subject with cancer) the complex according to any one of items 1 to 10.

[0021] Item 12B. Use of the complex according to any one of items 1 to 10 for cancer treatment.

[0022] Item 12C. Use of the complex according to any one of items 1 to 10 for the manufacture of a cancer therapeutic agent.

[0023] Item 12D. Use of the complex according to any one of items 1 to 10 as a cancer therapeutic agent.

[0024] Item 13. The drug described in Item 11 or 12 is a macrophage polarizing agent.

[0025] Item 13A. A macrophage polarization method comprising administering a complex according to any one of items 1 to 10 to a subject (e.g., a subject with cancer).

[0026] Item 13B. The complex according to any one of items 1 to 10, which is used for macrophage polarization.

[0027] Item 13C. Use of the complex according to any one of items 1 to 10 for the manufacture of a macrophage polarizer.

[0028] Item 13D. Use of the complex according to any one of items 1 to 10 as a macrophage polarizer.

[0029] Item 14. The complex according to any one of items 1 to 10, or the drug according to any one of items 11 to 13, used in combination with an immune checkpoint inhibitor.

[0030] Beneficial effects of the present invention The present invention can provide pharmaceutical compositions related to antitumor agents, which inhibit the excessive cytokine release and myelotoxicity of TLR7 / 8 agonists and enable them to be administered systemically. Attached Figure Description

[0031] Figure 1 The results of Experiment 4 are shown. The vertical axis indicates the concentration of Telratolimod in serum, and the horizontal axis indicates the time elapsed since the administration of the test drug. The legend indicates the test drug; "naked Telratolimod" refers to the test drug not encapsulated in cholesterol-modified pullulan nanogel (hereinafter referred to as "CHP nanogel"), and "CHP:Telratolimod" refers to the test drug encapsulated in CHP nanogel.

[0032] Figure 2 Results of Test Example 5 are shown. The vertical axis indicates the concentration of Telratolimod contained in the tumor, and the horizontal axis indicates the test drug added.

[0033] Figure 3 Results of Test Example 6 are shown. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days from the day of intradermal implantation of cancer cells (taken as day 0). The legend indicates the test drug.

[0034] Figure 4 Results of Test Example 7 are shown. The left graph shows the measurement results of Ml-like macrophages (vertical axis), and the right graph shows the measurement results of M2-like macrophages (vertical axis). The horizontal axis indicates the test drug added. "Untreated" indicates the case where no test drug was added.

[0035] Figure 5 Results of Test Example 8 are shown. The chart information shows the cytokines measured. The vertical axis indicates the cytokine concentration in the serum, and the horizontal axis indicates the test drug administered.

[0036] Figure 6 Results of Test Example 9 are shown. RET stands for reticulocyte. The left graph shows the measurement results of the proportion of reticulocyte count to total red blood cell count (vertical axis: %), and the right graph shows the measurement results of the blood concentration of reticulocytes (vertical axis: x 10 4 / μL).

[0037] Figure 7 Results of Test Example 10 are shown. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days from the day of intradermal implantation of cancer cells (taken as day 0). The legend indicates the test drug.

[0038] Figure 8 Results of Test Example 11 are shown. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days elapsed.

[0039] Figure 9 Results of Test Example 15 are shown. The vertical axis indicates the proportion of CD8-positive T cells and NK cells infiltrated into the tumor, and the horizontal axis indicates the test drug added.

[0040] Figure 10 Results of Test Example 16 are shown.

[0041] Figure 11 Results of Test Example 17 are shown. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days from the day of intradermal implantation of cancer cells (taken as day 0). The legend indicates the test drug. DETAILED DESCRIPTION

[0042] In the present specification, the terms "comprise" and "include" encompass the concepts of "comprising", "including", "consisting essentially of", and "consisting of".

[0043] 1. Complex In an embodiment, the present application relates to a complex comprising a modified polysaccharide containing a hydrophobic group and a compound represented by formula (1): (in the present specification, also referred to as "the complex of the present application"). It is described below.

[0044] The compound represented by formula (1) is described below.

[0045] In formula (1), R 1 is a C 12-25 linear alkyl group. The number of carbon atoms in the linear alkyl group is preferably from 14 to 23, more preferably from 14 to 21, even more preferably from 15 to 19, still even more preferably from 16 to 18, particularly preferably 17.

[0046] In formula (1), R 2 is a C 2-8 linear alkyl group. The number of carbon atoms in the linear alkyl group is preferably from 2 to 6, more preferably from 3 to 5, particularly preferably 4.

[0047] In formula (1), R 3 is a C 2-8 linear alkylene group. The number of carbon atoms in the linear alkylene group is preferably from 2 to 6, more preferably from 3 to 5, particularly preferably 4.

[0048] The compound represented by formula (1) is particularly preferably Telratolimod (in this compound, R 1 is a C 17 linear alkyl group, R 2 is a C4 linear alkyl group, and R 3 is a C4 linear alkylene group).

[0049] The compound represented by formula (1) can be in the form of a salt. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and for example, can be an acid addition salt. Specific examples of such a salt include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and with acidic amino acids such as aspartic acid and glutamic acid.

[0050] The compound represented by formula (1) can be in the form of a solvate. The solvate is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples include solvates with solvents such as water, ethanol, glycerol, and acetic acid.

[0051] The compound represented by formula (1) used can be a commercially available product, or a product obtained according to a known production method.

[0052] The compound represented by formula (1) can be used alone, or two or more in combination.

[0053] The modified polysaccharide refers to a compound obtained by modifying a polysaccharide, and is not particularly limited as long as it contains a hydrophobic group as a modifying group. In the present application, it has been found that the poor solubility in water of the compound of formula (1) can be improved by forming a complex with a modified polysaccharide, and the excessive cytokine release action and myelotoxicity of the compound of formula (1) can be further inhibited.

[0054] The polysaccharide forming the modified polysaccharide (i.e., the polysaccharide before modification) is not particularly limited as long as it is a polymer in which sugar residues are linked by glycosidic bonds. Usable examples of the sugar residue forming the polysaccharide include residues derived from sugars, such as monosaccharides (e.g., glucose, mannose, galactose, and fucose), disaccharides, or oligosaccharides. The sugar residues can be linked by 1,2-, 1,3-, 1,4-, or 1,6-glycosidic bonds, and the linkage thereof can be an α-linkage or a β-linkage. Furthermore, the polysaccharide can be linear or branched. A preferred sugar residue is a glucose residue. Preferred polysaccharides are, for example, naturally occurring or synthetic pullulan, mannan, dextran, amylose, amylopectin, and the like. Among these, from the effects of improving poor solubility, inhibiting excessive cytokine release action and myelotoxicity, anticancer effect, macrophage polarization effect, and the like, pullulan, mannan, dextran, and the like are preferred; more preferred are pullulan, mannan, and the like; particularly preferred are pullulan and the like.

[0055] The weight average molecular weight of the polysaccharide is not particularly limited as long as the modified polysaccharide can form a gel particle, but is, for example, 5,000 to 2,000,000. The weight average molecular weight of the polysaccharide is preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, even more preferably 40,000 to 250,000, and still even more preferably 80,000 to 125,000.

[0056] The polysaccharide used can be a commercially available product, or a product obtained according to a known production method.

[0057] The hydrophobic group is not particularly limited as long as it is a group having hydrophobicity. From the viewpoint of improving the effect of poor solubility, the effect of suppressing excessive release of cytokines, the effect of bone marrow toxicity, the anticancer effect, the macrophage polarization effect, and the like, the hydrophobic group is preferably a hydrophobic group having a sterol skeleton, a hydrocarbon group, or the like, and particularly preferably a hydrophobic group having a sterol skeleton.

[0058] The sterol skeleton is an alcohol having a hydroxyl group attached to a cyclopentahydrophenanthrene ring represented by formula (I). The symbols A to D in formula (I) each represent a respective ring forming the cyclopentahydrophenanthrene ring. In the sterol skeleton, the cyclopentahydrophenanthrene ring can have a double bond, and the bonding position of the hydroxyl group is not limited. A sterol having a hydroxyl group at the C-3 position and a double bond in the B ring, or a stanol having a hydroxyl group at the C-3 position and a saturated ring is preferable. Examples of the hydrophobic group having a sterol skeleton include a group derived from a compound having a modified sterol skeleton, for example, a ring-forming carbon is substituted with a hydrocarbon group (e.g., a C 1-20 The phrase "a group derived from" refers to a group of a compound from which a hydrogen atom or a functional group (e.g., a hydroxyl group) is removed.

[0059] Examples of the hydrophobic group having a sterol skeleton include a cholesterol-derived group, a cholestanol-derived group, a lanosterol-derived group, an ergosterol-derived group, a β-sitosterol-derived group, a campesterol-derived group, a stigmasterol-derived group, and a brassicasterol-derived group, and the like. Among these groups, a sterol-derived group, for example, a cholesterol-derived group, a cholestanol-derived group, a lanosterol-derived group, and an ergosterol-derived group is preferable; and a cholesterol-derived group is more preferable.

[0060] Examples of the hydrocarbon group as the hydrophobic group include, but are not particularly limited to, a C 8-50 (preferably a C 10-30 , more preferably a C 12-20 ) chain (preferably straight chain) hydrocarbon group (preferably an alkyl group).

[0061] The weight average molecular weight of the modified polysaccharide is not particularly limited as long as the modified polysaccharide can form a gel particle, but is, for example, 5,000 to 2,000,000. The weight average molecular weight of the modified polysaccharide is preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, even more preferably 40,000 to 250,000, and still even more preferably 80,000 to 125,000.

[0062] The number of hydrophobic groups contained in the modified polysaccharide is not particularly limited as long as the modified polysaccharide can form gel particles, for example, 1 to 10, preferably 1 to 5, per 100 saccharide residues forming the polysaccharide.

[0063] The hydrophobic group can be directly or indirectly (for example, through a linker) linked to the polysaccharide.

[0064] The modified polysaccharide is preferably, for example, a modified polysaccharide containing 1 to 10 (preferably 1 to 5) saccharide units per 100 saccharide residues forming the polysaccharide, whose primary hydroxyl group is represented by formula (II): -0-(CH2) x CONH(CH2) y NH-CO-O-R (II), wherein R is a hydrophobic group having a sterol skeleton or a hydrocarbon group, x is 0 or 1, and y is any positive integer. y is preferably 1 to 8.

[0065] The modified polysaccharide can be synthesized by or according to known methods (for example, WO 00 / 12564). For example, the following method can be used. First, a hydroxyl group-containing C 12-50 hydrocarbon or sterol is reacted with a diisocyanate compound represented by formula OCN-R A NCO (wherein R A is a C 1-50 hydrocarbon group) to produce a hydrophobic compound containing an isocyanato group, in which one molecule of the hydroxyl group-containing C 12-50 hydrocarbon or sterol is reacted. Then, the resulting hydrophobic compound containing an isocyanato group is further reacted with a polysaccharide to produce a polysaccharide containing a hydrophobic group, which contains a C 12-50 hydrocarbon group or a sterol group as the hydrophobic group. The resulting reaction product is purified with a ketone solvent, whereby a polysaccharide containing a hydrophobic group having a high purity can be produced.

[0066] The modified polysaccharide can be used alone or in combination of two or more.

[0067] The mass ratio of the modified polysaccharide to the compound of formula (1) (mass of the modified polysaccharide / mass of the compound of formula (1)) is preferably 1 or more, more preferably 2 to 20, even more preferably 3 to 10, and still even more preferably 3 to 8. The mass ratio in the above range makes it possible to exhibit even stronger anticancer effects and macrophage polarization effects.

[0068] The complex of the present application can be a gel particle. The term "gel particle" means a polymeric gel particle having a hydrogel structure. The term "hydrogel" means a three-dimensional network structure formed by cross-linking of a hydrophilic polymer and swelling with water. In the complex of the present application as a gel particle, the modified polysaccharide self-assembles by physical cross-linking formed by hydrophobic interaction based on a hydrophobic group, forming a three-dimensional network structure. When the complex of the present application is a gel particle, the compound of formula (1) is preferably contained within the gel particle.

[0069] The shape of the complex of the present application is not particularly limited, but is generally spherical.

[0070] The complex of the present application is preferably nanoscale (preferably a nanogel particle) having an average particle diameter of scattering intensity, for example, 200 nm or less, preferably 10 to 200 nm, more preferably 20 to 200 nm, even more preferably 60 to 190 nm, still even more preferably 80 to 180 nm, particularly preferably 90 to 160 nm. The particle diameter can be measured by a dynamic light scattering method.

[0071] The complex of the present application can contain a substance other than the modified polysaccharide. Examples of the other substance include proteins, peptides, nucleic acids, sugars, low molecular weight compounds, high molecular weight compounds, and minerals, and complexes thereof. More specifically, examples of the other substance include adjuvants, cancer antigens, anticancer agents, nucleic acid drugs, and other drugs.

[0072] The adjuvant can be selected from inactivated bacteria, bacterial extracts, nucleic acid lipopolysaccharides, lipopeptides, synthetic low molecular weight compounds, and the like. Preferably, imidazoquinolines (such as R848 and imiquimod), saponins (such as Quil A and QS21), STING agonists (such as cyclic di-GMP), monophosphoryl lipids, lipopeptides, and the like are used. In addition to the above, examples of the adjuvant also include taxane drugs, anthracycline drugs, JAK / STAT inhibitors, indoleamine deoxygenase (IDO) inhibitors, tryptophan deoxygenase (TDO) inhibitors, and the like. These inhibitors include compounds having antagonistic activity against these factors, as well as neutralizing antibodies against these factors.

[0073] The cancer antigen includes an antigenic polypeptide, and is preferably an antigenic polypeptide. The antigenic polypeptide is an antigen or a partial peptide thereof that is highly expressed in a cancer cell, and in some cases, it is expressed only by a cancer cell. The antigenic polypeptide can be expressed inside or on the surface of a cancer cell.

[0074] The antigenic polypeptide can be selected from, but not limited to, ERK1, ERK2, MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin B, colorectal associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad tumor antigen family, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, and partial peptides thereof.

[0075] Examples of the anticancer agent include an alkylating agent, a metabolic antagonist, a microtubule inhibitor, an antibiotic anticancer agent, a topoisomerase inhibitor, a platinum agent, a molecular targeted drug, a hormonal agent, a biological agent, and the like.

[0076] Examples of the alkylating agent include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, ramucirumab, and the like.

[0077] Examples of metabolic antagonists include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, catarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladrabine, doxifluridine, hydroxycarbamide, mercaptopurine, and the like.

[0078] Examples of microtubule inhibitors include alkaloid anticancer agents such as vincristine, and taxane anticancer agents such as docetaxel and paclitaxel.

[0079] Examples of antibiotic anticancer agents include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, dactinomycin, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, zinostatin stimalamer, and the like.

[0080] Examples of topoisomerase inhibitors include CPT-11, irinotecan, and nogitecan having topoisomerase I inhibitory activity; and etoposide and sobuzoxane having topoisomerase II inhibitory activity.

[0081] Examples of platinum agents include cisplatin, nedaplatin, oxaliplatin, carboplatin, and the like.

[0082] Examples of the hormone agents include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprolide, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, and the like.

[0083] Examples of the biological agents include interferon-α, -β, and -γ, interleukin 2, ubenimex, dry BCG, extracellular toxin, and the like. A specific example of the extracellular toxin includes exotoxin (protein) exotoxin (PE) produced by the bacterium Pseudomonas aeruginosa. PE has an enzyme activity of NAD+-diphthamide-ADP-ribosyltransferase, which inhibits protein synthesis and exhibits strong toxicity. At present, PE conjugated with an antibody named CD22 has been approved as an anticancer agent.

[0084] Examples of molecular targeted drugs include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tosituzumab, bortezomib, gemtuzumab ozogamicin, ibritumomab ozogamicin, ibritumomab tiuxetan, tamibarotene, tretinoin, and the like. In addition to the molecular targeted drugs specified herein, other examples include immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., nivolumab and pembrolizumab), anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab, and cemiplimab), and anti-CTLA-4 antibodies (e.g., ipilimumab and tremelimumab); inhibitors targeting angiogenesis such as human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, Bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, and vascular endothelial growth factor receptor 2 inhibitors (alpha-VEGFR-2 antibodies); various tyrosine kinase inhibitors such as MAP kinase inhibitors; inhibitors targeting cytokines, proteasome inhibitors, antibody-anticancer drug conjugates, and other molecular targeted drugs. These inhibitors also include antibodies.

[0085] The nucleic acid drug is an active pharmaceutical ingredient as a nucleic acid, which is not particularly limited in this regard.

[0086] The content of other substances is, for example, 0 to 10,000 parts by mass, 0 to 1,000 parts by mass, 0 to 500 parts by mass, 0 to 100 parts by mass, 0 to 50 parts by mass, or 0 to 10 parts by mass, based on 100 parts by mass in total of the compound of formula (1) and the modified polysaccharide.

[0087] In a preferred embodiment, the complex of the present application can be obtained in a state dissolved in water or a solvent having a small amount of an organic solvent (for example, the content of the organic solvent is 1 mass% or less, 0.5 mass% or less, 0.2 mass% or less, 0.1 mass% or less, 0.01 mass% or less, or 0.001 mass% or less, per 100 mass% of the solvent). The complex of the present application can preferably be in a state dissolved in water (in the form of an aqueous solution).

[0088] In the complex solution of the present application (particularly, a solution using the above solvent), the concentration of the compound of formula (1) is preferably 0.1 mg / mL or more, 0.2 mg / mL or more, 0.4 mg / mL or more, 0.6 mg / mL or more, 0.8 mg / mL or more, or 1.0 mg / mL or more. The upper limit of the concentration is not particularly limited, and is, for example, 10 mg / mL, 6 mg / mL, 4 mg / mL, or 2 mg / mL. According to the present application, a solution having a relatively high concentration of the compound of formula (1) that is poorly soluble in water can be obtained as described above.

[0089] In the complex solution of the present application (particularly, a solution using the above solvent), the concentration of the modified polysaccharide is preferably 1 to 30 mg / mL, 2 to 25 mg / mL, 4 to 20 mg / mL, 6 to 15 mg / mL, or 8 to 12 mg / mL.

[0090] The complex of the present application can be produced by, for example, stirring a solution (solution 1) containing the compound of formula (1) and the modified polysaccharide at a relatively low temperature. This method is excellent in improving the concentration of the compound of formula (1) in the complex of the present application.

[0091] The solvent of solution 1 contains water. In order to dissolve the compound of formula (1), the solvent of solution 1 preferably contains an organic solvent. The organic solvent is not particularly limited as long as it is a solvent capable of dissolving the compound of formula (1) and compatible with water. Preferably, dimethyl sulfoxide or N,N-dimethylformamide can be used. The content of water in the solvent of solution 1 is preferably 90.0 to 100 v / v%, more preferably 95.0 to 99.5 v / v%, even more preferably 97.0 to 99.0 v / v% or more, still even more preferably 97.5 to 98.5 v / v% or more. The content of the organic solvent in the solvent of solution 1 is preferably 0 to 10 v / v%, more preferably 0.5 to 5.0 v / v%, even more preferably 1.0 to 3.0 v / v%, still even more preferably 1.5 to 2.5 v / v%.

[0092] The concentration of the compound of formula (1) in Solution 1 is preferably 0.05 to 0.8 mg / mL, more preferably 0.1 to 0.4 mg / mL. The concentration of the modified polysaccharide in Solution 1 is preferably 0.2 to 4 mg / mL, more preferably 0.5 to 3 mg / mL. The mass ratio of the modified polysaccharide to the compound of formula (1) (mass of the modified polysaccharide / mass of the compound) in Solution 1 is preferably 1 to 20, more preferably 2 to 10, even more preferably 3 to 7.

[0093] Solution 1 can be preferably obtained by dissolving the compound of formula (1) and the modified polysaccharide in an organic solvent, and then adding water.

[0094] The water used for Solution 1 can contain a buffer such as a phosphate buffer.

[0095] The temperature of Solution 1 during stirring is preferably 1 to 10°C, more preferably 2 to 6°C. The stirring time of Solution 1 is preferably 4 to 24 hours, more preferably 6 to 16 hours. As a result of stirring under these conditions, a complex having more uniform shape and particle size can be obtained.

[0096] After stirring, if necessary, the resulting product can be subjected to purification treatment. Examples of the purification treatment include filter filtration, ultrafiltration, and the like. Preferably, ultrafiltration can be used for concentration and removal of the organic solvent. This process makes it possible to adjust the mass ratio of the modified polysaccharide to the compound of formula (1) described above.

[0097] 2. Use The complex of the present application has an anticancer effect and a macrophage polarization effect (polarization from M2-like to M1-like). Therefore, the complex of the present application can be used as an active ingredient of a drug, a reagent, or the like (also referred to as "a drug of the present application" in the present specification), and in particular, as an active ingredient of a cancer therapeutic agent, a macrophage (preferably TAM) polarizing agent, or the like.

[0098] The drug of the present application is not particularly limited as long as it includes the complex of the present application. If necessary, the drug of the present application can also contain other components. The other components are not particularly limited as long as they are pharmaceutically acceptable components. Examples of the other components include additives in addition to components having pharmacological effects. Examples of the additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickening agents, humectants, coloring agents, flavoring agents, chelating agents, and the like.

[0099] The pharmaceutical of the present application can include, in addition to the complex of the present application, other substances (e.g., drugs such as adjuvants, cancer antigens, anticancer agents, and nucleic acid drugs) that can be included in the complex of the present application described above. Furthermore, the pharmaceutical of the present application can be used in combination with other substances (e.g., drugs such as adjuvants, cancer antigens, anticancer agents, and nucleic acid drugs) that can be included in the complex of the present application described above. In particular, as shown in the following example (Test Example 10), which shows the effect of use in combination with an anti-PD-1 antibody, use in combination with an immune checkpoint inhibitor is useful in terms of antitumor effect. Examples include immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., nivolumab and pembrolizumab), anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab, and cemiplimab), and anti-CTLA-4 antibodies (e.g., ipilimumab and tremelimumab).

[0100] The mode of use of the pharmaceutical of the present application is not particularly limited, and an appropriate mode of use can be used depending on the type thereof. The pharmaceutical of the present application can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to an animal), depending on the use thereof.

[0101] The application target of the pharmaceutical of the present application is not particularly limited. Examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, deer, and the like. Furthermore, examples of cells include animal cells and the like. The type of cell is also not particularly limited, and examples include immune cells (macrophages are preferred), blood cells, hematopoietic stem / progenitor cells, gametes (sperm and eggs), fibroblasts, epithelial cells, vascular endothelial cells, neurons, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, and the like. Among these, macrophages are preferred, and TAMs are particularly preferred.

[0102] When the pharmaceutical of the present application targets cancer, the target cancer is not particularly limited. Examples include leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Berkit lymphoma, malignant lymphoma, diffuse lymphoma, and follicular lymphoma), myeloma (including multiple myeloma), breast cancer, colon cancer, kidney cancer, stomach cancer, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, urinary tract cancer, prostate cancer, villous cancer, pharyngeal cancer, laryngeal cancer, thecoma, male germ cell tumor, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, medulloblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, Wilms' tumor, and the like.

[0103] The pharmaceutical of the present application can have any dosage form, and examples include oral dosage forms such as tablets (including oral disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, and the like), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrup, liquids (including drinkable preparations, suspensions, and syrups), and jellies; and parenteral dosage forms such as injectable preparations (e.g., drip (intravenous drip, etc.), intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection), external preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, eye drops, eye ointments, nose drops, ear drops, and liposome preparations.

[0104] The route of administration of the pharmaceutical of the present application is not particularly limited, as long as the desired effect is obtained. Examples include enteral administration such as oral administration, tube feeding, and enema administration; parenteral administration such as intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intradermal, and intraperitoneal administration; and the like.

[0105] The content of the active ingredient (the complex of the present application) in the pharmaceutical of the present application varies depending on the mode of use, the application target, the state of the application target, and the like, and is not limited. For example, the content of the active ingredient is 0.0001 to 100 wt.%, preferably 0.001 to 50 wt.%.

[0106] The dose of the pharmaceutical of the present application when administered to an animal is not particularly limited as long as it is an effective amount to express a pharmaceutical effect. In the case of oral administration, the weight of the active ingredient is usually 0.1 to 1000 mg / kg body weight per day, preferably 0.5 to 500 mg / kg body weight per day. In the case of parenteral administration, the weight of the active ingredient is 0.01 to 100 mg / kg body weight per day, preferably 0.05 to 50 mg / kg body weight per day. The above-mentioned dose can be appropriately increased or decreased depending on the age, pathological condition, symptoms, and the like.

[0107] Examples The present application is described in detail below based on examples; however, the present application is not limited to these examples.

[0108] Preparation Example 1. Preparation of cholesterol-modified pullulan Cholesterol-modified pullulan (hereinafter referred to as CHP) was prepared by introducing 1.2 cholesterols per 100 monosaccharides in pullulan having a weight average molecular weight of 100,000 according to the previously reported literature (Macromolecules 1993, 23, 3062-3068).

[0109] Example 1. Investigation of encapsulation of various TLR7 and TLR8 agonists in CHP nanogel Nine TLR7 and TLR8 agonists (Telratolimod, Motolimod, Vesatolimod, Resiquimod, Imiquimod, CL075, CU-CPT17e, CU-CPT9a, and PF-4878691) were investigated whether they can be encapsulated in CHP nanogel. CHP was dissolved in a solution of 6 M urea in PBS (-), and a compound solution was added thereto. Then, stepwise dialysis against PBS (-) was performed.

[0110] Results Among the nine compounds, only one (Telratolimod) can be stably encapsulated in CHP nanogel and form a solution.

[0111] Consideration The concentration of Telratolimod in the nanogel prepared in Example 1 was as low as 0.112 mg / ml. Further increasing the concentration of Telratolimod is expected to exhibit sufficient therapeutic efficacy.

[0112] Example 2. Preparation of CHP nanogels encapsulating Telratolimod CHP nanogel encapsulating Telratolimod (hereinafter CHP:Telratolimod) was prepared as follows. 160 mg of CHP 80K (NOF Corporation) and 32 mg of Telratolimod (ACHEMBLOCK) were mixed and dissolved in 3.2 mL of N,N-dimethylformamide (DMF). The DMF solution was added to 160 mL of PBS and stirred at 4°C overnight. The filtrate was passed through a 0.8 μm filter and concentrated and purified using TFF (Pall, MWCO; 30K) to remove DMF. After collection, the resulting product was passed through a 0.45 μm filter to obtain a CHP:Telratolimod solution (average particle size of scattering intensity: 149 nm, ELSZ-2000ZS, Otsuka Electronics Co., Ltd.). Using a Telratolimod standard sample, a calibration curve was prepared by HPLC (column: YMC-Pack C4, 150 x 2.0 mm I.D. S-3 μm, 12 nm, flow rate: 0.3 mL / min, 60% B phase, A phase: 0.1% FA in H2O, B phase: 0.1% FA in CH3CN), and the concentration of Telratolimod in the CHP:Telratolimod solution was calculated by decomposing CHP in an aqueous sodium periodate solution and then quantifying the resulting aldehyde group with Schiff reagent (CHP: 10.2 mg / mL, Telratolimod: 1.17 mg / mL).

[0113] It is considered The concentration of Telratolimod in the nanogel obtained by the newly discovered method in Example 2 was 1.17 mg / ml, which was about 10 times higher than the concentration obtained by the previous method. The drug concentration was successfully improved by forming a poorly soluble drug into a nanogel by this preparation method. In Test Examples 1 to 17 below, the preparation prepared in Example 2 was used.

[0114] Test Example 1. Preparation of human peripheral blood-derived TAMs After thawing and recovering the previously cryopreserved human peripheral blood-derived CD14-positive mononuclear cells, 2 x 10 5Cells were seeded at 1 cell / well in 24-well tissue culture plates in 500 μL base media (RPMI, 10% fetal bovine serum, 50 ng / mL M-CSF) and incubated for 3 days at 37°C in a 5% CO2 incubator. After this time, 500 μL base media was added and the cells were incubated for a further 3 days. The media was then replaced with 500 μL TAM differentiation inducing media, which was prepared by adding 10% fetal bovine serum, 50 ng / mL IL-4, 50 ng / mL IL-6, 50 ng / mL IL-10, 50 ng / mL IL-13 and 5% human AB male donor defibrinated serum (Veritas) to a separate preparation of human cancer cell culture supernatant, and the cells were incubated for 2 days.

[0115] Example 2. Polarisation assay of human peripheral blood derived TAMs Telratolimod solution or CHP:Telratolimod solution (final concentration of Telratolimod adjusted to 30 μM) was added to the TAMs prepared in Example 1 and incubated for a further 2 days, after which time the culture supernatant was measured for TNF-a by ELISA (BioLegend). Controls were supplemented with media only.

[0116] Results The results are shown in Table 1. CHP:Telratolimod showed higher production of TNF-a compared to Telratolimod. This indicates that encapsulation of Telratolimod in CHP nanogels allows Telratolimod to be more effectively taken up by TAMs, leading to their polarisation to Ml -like macrophages, resulting in higher production of TNF-a.

[0117] Table 1 Example 3. Cancer cell phagocytosis assay of human peripheral blood derived TAMs HeLa cells were labelled with fluorescent marker 1 (CellTracker Green CMTPX Dye, ThermoFisher) for 30 minutes, then washed twice with PBS (-) and incubated overnight in media (RPMI supplemented with 10% fetal bovine serum) containing 1.25 μΜ doxorubicin.

[0118] TAMs were prepared in the same way as in Experiment 1, with the final concentration of Telratolimod adjusted to 10 mM Telratolimod or CHP:Telratolimod added to the base medium, followed by incubation for 2 days. After labelling with fluorescent marker 2 (CellTracker Red CMTPX Dye, ThermoFisher) for 30 minutes, cells were washed twice with PBS (-). Adriamycin-treated HeLa cells were added to the TAMs at half the proportion, and co-cultured overnight. Cells were then harvested, and the number of cells that were co-positive for fluorescent markers 1 and 2 was determined by flow cytometry. Controls were supplemented with medium only.

[0119] Results Results are shown in Table 2. CHP:Telratolimod enhanced phagocytic activity against HeLa cancer cells compared to Telratolimod. This suggests that encapsulating Telratolimod in CHP nanogels allowed Telratolimod to be more efficiently taken up by TAMs, leading to its polarisation into M1-like macrophages, resulting in enhanced phagocytic activity.

[0120] Table 2 Experiment 4. Measurement of blood concentration changes in a mouse allogeneic tumour xenograft model By adding 10 6A mouse allogeneic tumor transplantation model was prepared by intradermally administering mouse colon cancer cell line MC38 cells to the back skin of mice (C57BL / 6JJmsSlc, female). Mice at 7 days after cell administration were used. CHP:Telratolimod prepared in Example 2 or Telratolimod dissolved in a solubilizing solvent (17.1% polyethylene glycol 300, 4.3% Tween 80, and 8.6% polyoxyethylene hydrogenated castor oil) at 84.5 μg were administered by tail vein injection, and the change in the concentration of Telratolimod in the blood was measured. Blood was collected from 5 mice per group. After administration of each test drug by tail vein injection, 80 μL of isopropyl alcohol was added to 20 μL of serum collected at 1 minute, 30 minutes, 1 hour, 2 hours, and 6 hours after administration, and bead beating was performed for 1 minute using a 5-mm-diameter stainless steel ball. Subsequently, 400 μL of an 80% isopropyl alcohol and 20% acetonitrile solution was added and mixed, followed by ultrasonic treatment for 3 minutes, and then centrifugation at 12000 g for 5 minutes, and the supernatant was collected. The collected supernatant was analyzed by LS-MS / MS to measure the concentration of Telratolimod. To create a calibration curve, a Telratolimod standard solution was prepared as a 1.17 mg / mL DMSO solution, and serial dilutions of 10, 20, 100, 200, 1000, and 2000 times were prepared, and then diluted 10 times with fetal bovine serum, and cabozantinib (Selleck) was used as an internal standard for correction.

[0121] Results Results are shown in Figure 1 . The blood concentration of Telratolimod rapidly decreased within 30 minutes after administration, whereas Telratolimod encapsulated in CHP nanogel was more persistent in the blood, confirming a 4-fold or more extension of the blood half-life.

[0122] Test Example 5. Measurement of intratumoral Telratolimod concentration in a mouse allogeneic tumor transplantation model A mouse allogeneic transplantation model prepared in the same manner as in Test Example 4 was administered with CHP:Telratolimod solution containing 84.5 μg of Telratolimod prepared in Example 2 and Telratolimod solution prepared in Test Example 4 by tail vein injection, respectively. The tumor was excised 1 hour later, and the concentration of Telratolimod in the tumor was measured. Five mice were used per group. 80 μL of isopropyl alcohol was added to 20 μg of excised tumor tissue pieces, and bead beating was performed for 1 minute using a 5-mm-diameter stainless steel ball. Subsequent processing was performed in the same manner as in Test Example 4.

[0123] Results Results are shown in Figure 2 Intratumoral accumulation of Telratolimod was increased by about 3-fold by encapsulation in CHP nanogel.

[0124] Test Example 6. Anti-tumor evaluation in mouse allograft tumor transplantation model (1) The MC38 mouse allograft tumor transplantation model was prepared in the same manner as in Test Example 4. CHP:Telratolimod solution prepared in Example 2 and Telratolimod solution prepared in Test Example 4 were respectively administered to 5 mice in each group (amount of Telratolimod: 84.5 μg) by tail vein injection on days 7, 9, 11, 14, 16, and 18 after cell transplantation. Tumor size was measured on days 7, 9, 11, 14, 16, 18, and 19. Tumor volume was calculated by (long axis x short axis x short axis) / 2. As a negative control, the same solution as each drug solvent was administered. “Vehicle (1)” refers to the solubilizing solvent of Test Example 4, and “Vehicle (2)” refers to PBS (-).

[0125] Results Results are shown in Figure 3 In the CHP:Telratolimod-treated group, the tumor volume remained almost unchanged from the start of drug administration, and the tumor inhibition rate was 92% compared to the solvent control on day 19. On the other hand, in the Telratolimod-treated group, the tumor inhibition rate was 49% compared to the solvent control. These results indicate that encapsulation of Telratolimod in CHP nanogel significantly enhances the anti-tumor effect of Telratolimod.

[0126] Test Example 7. Evaluation of TAM polarization in mouse allograft tumor transplantation model The MC38 mouse allograft tumor transplantation model was prepared in the same manner as in Test Example 6, and CHP:Telratolimod solution prepared in Example 2 and Telratolimod solution prepared in Test Example 4 were respectively administered to 5 mice in each group (amount of Telratolimod: 84.5 μg) by tail vein injection on days 7, 9, and 11 after cell transplantation. One day after the final administration, the tumor was excised, and macrophages infiltrated into the tumor were measured by flow cytometry. Significance test was performed using one-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0127] Results Results are shown in Figure 4In both the Telratolimod and CHP:Telratolimod treatment groups, the expression of the M1-like macrophage marker iNOS was significantly increased. The iNOS level was significantly higher in the CHP:Telratolimod treatment group than in the Telratolimod treatment group. Furthermore, the expression of the M2-like macrophage marker ARG1 tended to decrease in both the Telratolimod and CHP:Telratolimod treatment groups, but only significantly decreased in the CHP:Telratolimod treatment group. These results indicate that, compared to the Telratolimod treatment group, more M2-like macrophages in the tumor polarized to M1-like macrophages in the CHP:Telratolimod treatment group.

[0128] Experimental Example 8. Evaluation of Cytokine Release in a Mouse Allogeneic Tumor Transplantation Model The allogeneic transplantation model mice used were 10 6 Ten days after intradermal administration of the MC38 mouse colorectal cancer cell line into the dorsal skin of female mice (C57BL / 6JJmsSlc), Telratolimod solution prepared in Example 4 and CHP:Telratolimod solution prepared in Example 2 (Telratolimod amount: 84.5 μg) were administered to five mice in each group via tail vein injection. Blood was collected two hours later. Serum was obtained by centrifugation, and changes in cytokine levels were measured using a LEGENDplex Mouse Inflammation Panel (BioLegend). Significance was tested using one-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0129] result The results are shown in Figure 5. In the Telratolimod solution-treated group, a rapid increase in IL-6, MCP-1, and IFN-β was observed, suggesting the possibility of cytokine release syndrome. On the other hand, in the CHP:Telratolimod-treated group, these increases were significantly reduced, indicating that cytokine release syndrome was suppressed.

[0130] Experimental Example 9. Blood Tests in a Mouse Allogeneic Tumor Transplantation Model 10 6MC38 mouse colorectal cancer cell line was administered intradermally to the dorsal skin of female mice (C57BL / 6JJmsSlc). At 7, 9, and 11 days, five mice in each group were administered Telratolimod solution prepared in Example 4 and CHP:Telratolimod solution prepared in Example 2 (Telratolimod amount: 84.5 μg) via tail vein injection. One day after the final administration, blood was collected, and changes in erythrocyte and reticulocyte counts were measured. Significance was assessed using one-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0131] result The results are shown in Figure 6 A significant decrease in reticulocytes was observed in the Telratolimod solution-treated group. However, this was not observed in the CHP:Telratolimod solution-treated group. Reticulocytes are an indicator of erythropoiesis in the bone marrow, and their presence in the Telratolimod solution-treated group suggested bone marrow toxicity. The significant decrease in reticulocytes in the CHP:Telratolimod solution-treated group, on the other hand, indicates that encapsulation in CHP nanogels does not induce bone marrow suppression.

[0132] Experimental Example 10. Antitumor Evaluation in a Mouse Allogeneic Tumor Transplantation Model (2) By 10 6 A mouse allogeneic tumor transplantation model was established by intradermal administration of CT26 colorectal cancer cell line to the dorsal skin of female BALB / cCrSlc mice. On days 7, 9, 11, 14, 16, and 18 post-transplantation, CHP:Telratolimod solution prepared in Example 2 and Telratolimod solution prepared in Example 4 were administered via tail vein injection, respectively. On days 7, 11, 14, and 18 post-transplantation, anti-PD-1 antibody (RMP1-14, Bio X Cell) solution was administered intraperitoneally to 5 mice in each group (Telratolimod: 42 μg, anti-PD-1 antibody: 100 μg). One group received a combination of CHP:Telratolimod solution and anti-PD-1 antibody. Tumor size was measured on days 7, 9, 11, 14, 16, 18, and 21. Tumor volume was calculated as (major axis × minor axis × minor axis) / 2. The negative control group was the untreated group.

[0133] result The results are shown in Figure 7In the CHP:Telratolimod treatment group, tumor regression was observed from the start of drug administration. On the other hand, the anti-tumor effect of the Telratolimod treatment group was limited, indicating that encapsulation in CHP nanogel significantly enhanced the anti-tumor effect of Telratolimod. The anti-tumor effect of anti-PD-1 antibody was also limited; however, when anti-PD-1 antibody was combined with CHP:Telratolimod, the tumor completely disappeared. These results suggest that combination with CHP:Telratolimod can be expected to have a strong anti-tumor effect in cancers in which the anti-tumor effect of anti-PD-1 antibody is limited (so-called cold tumors).

[0134] Test Example 11. Preparation of human peripheral blood-derived M2-like macrophages After recovering the previously cryopreserved human peripheral blood-derived CD14-positive monocytes, 2 x 10 5 cells / well were seeded in 500 μL of basal medium (RPMI, 10% fetal bovine serum, 50 ng / mL M-CSF) in a 24-well tissue culture plate and cultured for 3 days in a 37°C, 5% CO2incubator. After that, 500 μL of basal medium was further added and the cells were cultured for 3 days. Then, the medium was replaced with 500 μL of M2-like macrophage differentiation induction medium prepared by adding 50 ng / mL IL-4, 50 ng / mL IL-6, 50 ng / mL IL-10, and 50 ng / mL IL-13 to the basal medium, and the cells were cultured for 2 days.

[0135] Test Example 12. Polarization test of human peripheral blood-derived M2-like macrophages Telratolimod solution or CHP:Telratolimod solution (the final concentration of Telratolimod was adjusted to 3 μM) was added to the M2-like macrophages prepared in Test Example 11, followed by incubation for 1 day, and the culture supernatant was measured for TNF-a, IL-1β, IL-6, IL-12p40 (p40 subunit of IL-12), IL-23, and CXCL10 using a flow cytometry microsphere assay kit (LEGENDplex TM , BioLegend). The control was supplemented with only the medium.

[0136] Results Results are shown in Table 3. CHP:Telratolimod showed higher production of TNF-a, IL-1b, IL-6, IL-12p40, IL-23 and CXCL10 compared to Telratolimod. This indicates that encapsulating Telratolimod in CHP nanogels allows Telratolimod to be more efficiently taken up by M2-like macrophages, leading to their polarization to M1-like macrophages, resulting in higher production of proinflammatory cytokines (TNF-a, IL-1b, IL-6, IL-12 (p40 subunit), IL-23 and CXCL10) produced by M1-like macrophages.

[0137] Table 3 Example 13. Cancer cell phagocytosis assay of human peripheral blood-derived M2-like macrophages HeLa cells were labeled with fluorescent marker 1 (CellTracker Green CMTPX Dye, ThermoFisher) for 30 minutes, then washed twice with PBS (-) and cultured overnight in medium (RPMI supplemented with 10% fetal bovine serum) containing 2.5 mM doxorubicin.

[0138] Telratolimod or CHP:Telratolimod (final concentration of Telratolimod adjusted to 10 mM) was added to the base medium and M2-like macrophages prepared in Example 11 were incubated for 1 day. After labeling with fluorescent marker 2 (CellTracker Red CMTPX Dye, ThermoFisher) for 30 minutes, the cells were washed twice with PBS (-). HeLa cells were added to the M2-like macrophages at half the proportion and co-cultured overnight. Cells were then harvested and the number of fluorescent marker 1 and 2 co-positive cells was determined by flow cytometry. Control was supplemented with medium only.

[0139] Results Results are shown in Table 4. CHP:Telratolimod enhanced phagocytic activity against HeLa cancer cells compared to Telratolimod. This indicates that encapsulating Telratolimod in CHP nanogels allows Telratolimod to be more efficiently taken up by TAMs, leading to their polarization to M1-like macrophages, resulting in enhanced phagocytic activity.

[0140] Table 4 Example 14. Anti-tumor evaluation in mouse allogeneic tumor transplantation model (3) In the CHP:Telratolimod treatment group of Experiment 10, mice (n=4) whose tumors had completely regressed by day 21 were continued to be fed, and it was confirmed that no tumor recurrence occurred in any of the individuals until day 96. Subsequently, on day 97, 10 6 CT26 mouse colorectal cancer cell line was intradermally implanted into the skin on the side opposite to the back of the cancer cell line implanted in Example 10, and tumor growth was measured until day 120. As a control, five age-matched mice (BALB / cCrSlc, female) that had not previously received CT26 cancer cell line implantation were used. 10 6 CT26 cancer cell line cells were intradermally implanted into the back skin of mice, and tumor growth was measured.

[0141] result The results are shown in Figure 8 As a control, tumor growth was observed in all age-matched mice that had not previously been implanted with the CT26 cancer cell line. Figure 8 (Bottom). On the other hand, in mice that showed complete regression after the first transplantation of cancer cells, the tumor did not develop in any of the individuals, and the transplanted cancer cells were completely rejected. Figure 8 (Top). These results indicate that CHP:Telratolimod induces immune memory against cancer cells, suggesting that remission achieved using CHP:Telratolimod can prevent relapse.

[0142] Example 15. Analysis of tumor-infiltrating CD8-positive T cells and natural killer (NK) cells in a mouse allogeneic tumor transplantation model. The MC38 mouse allogeneic tumor transplantation model was established in the same manner as in Example 6. On days 7 and 9 post-transplantation, five mice were administered the CHP:Telratolimod solution prepared in Example 2 via tail vein injection (Telratolimod dosage: 84.5 μg). As a control, five mice were prepared in an untreated CHP:Telratolimod group. One day after the last administration, the tumor was resected, and the number of tumor-infiltrating CD8-positive T cells and NK cells was detected by flow cytometry. Significance was assessed using one-way ANOVA.

[0143] result The results are shown in Figure 9CHP:Telratolimod significantly increased the number of CD8-positive T cells and NK cells infiltrated into the tumor. These results indicate that CHP:Telratolimod has an effect of increasing CD8-positive T cell and NK cell infiltration, which are major effector cells involved in anti-tumor responses, suggesting that this is one of the mechanisms of the high anti-tumor effect of CHP:Telratolimod in Test Example 6.

[0144] Test Example 16. Analysis of cancer antigen-specific CD8-positive T cells in a mouse allogeneic tumor transplantation model A MC38 mouse allogeneic tumor transplantation model was prepared in the same manner as in Test Example 6. On days 7, 9, and 12 after cell transplantation, CHP:Telratolimod solution prepared in Example 2 was administered to 5 mice (amount of Telratolimod: 84.5 μg) by tail vein injection. As a control, 5 mice of a CHP:Telratolimod non-treated group were prepared. One day after the last administration, the spleen was excised, and splenocytes were prepared. The cells were stimulated with a peptide from MuLV p15E, which is a cancer antigen expressed in MC38 cells, or as a control, with a peptide from TRP2, which is a cancer antigen expressed in melanoma cells, and the cells were permeabilized. Then, CD8-positive T cells were analyzed by flow cytometry using an anti-IFN-γ antibody. Significance test was performed using an unpaired t-test.

[0145] Results Results are shown in Figure 10 Compared to the CHP:Telratolimod non-treated group, the number of CD8-positive T cells that turned IFN-γ positive after stimulation with a peptide from MuLV p15, which is a cancer antigen of MC38 cells, was significantly increased in mice administered with CHP:Telratolimod. On the other hand, as a negative control, stimulation with a TRP2 peptide did not show a change in the number of CD8-positive T cells that turned IFN-γ positive, compared to the non-treated group. These results indicate that CHP:Telratolimod has an effect of increasing CD8-positive T cells specific to the MC38 cancer cell response, suggesting that this is one of the mechanisms of the high anti-tumor effect of CHP:Telratolimod in Test Example 6.

[0146] Test Example 17. Anti-tumor evaluation in a mouse allogeneic tumor transplantation model (4) CHP:Telratolimod was administered to 5 mice (amount of Telratolimod: 84.5 μg) by tail vein injection at 7, 9, and 12 days after cell transplantation. As a control, 5 mice of a CHP:Telratolimod non-treated group were prepared. One day after the last administration, the tumor was excised, and the number of viable cells was counted. Significance test was performed using an unpaired t-test. 5A mouse melanoma cell line B16F10 cells were intradermally administered to the back skin of mice (C57BL / 6NCrSlc, female) to prepare a mouse allogeneic tumor transplantation model. On days 7, 9, 11, 14, and 16 after cell transplantation, the CHP:Telratolimod solution prepared in Example 2 was administered by tail vein injection, and an anti-PD-1 antibody (RMP1-14, Bio X Cell) solution and an anti-PD-L1 antibody (10F.9G2, Bio X Cell) solution were intraperitoneally administered to 10 mice in each group (amount of Telratolimod: 160 μg, amount of anti-PD-1 antibody and anti-PD-L1 antibody: 200 μg). In addition, a CHP:Telratolimod and anti-PD-1 antibody combination group and a CHP:Telratolimod and anti-PD-L1 antibody combination group were also evaluated. Tumor size was measured on days 7, 9, 11, 14, 16, and 18. Tumor volume was calculated by (long axis x short axis x short axis) / 2. The negative control was an untreated group.

[0147] Results Results are shown in Figure 11 The CHP:Telratolimod treatment group, anti-PD-1 antibody treatment group, and anti-PD-L1 antibody treatment group showed an anti-tumor effect of about 50%. On the other hand, the groups in which anti-PD-1 antibody or anti-PD-L1 antibody was administered in combination with CHP:Telratolimod showed an anti-tumor effect of about 75% to 80%. These results indicate that combination with CHP:Telratolimod can be expected to have a strong anti-tumor effect in cancers in which the anti-tumor effect of anti-PD-1 antibody and anti-PD-L1 antibody is limited (so-called cold tumors).

Claims

1. A complex comprising a modified polysaccharide containing a hydrophobic group and a compound of formula (1): in, R 1 C 12-25 Straight-chain alkyl, R 2 C 2-8 Straight-chain alkyl, R 3 C 2-8 Straight-chain alkylene groups.

2. The complex according to claim 1, wherein the polysaccharide forming the modified polysaccharide comprises pullulan.

3. The complex according to claim 1, wherein the hydrophobic group comprises a hydrophobic group having a sterol backbone.

4. The complex according to claim 1, wherein the weight-average molecular weight of the modified polysaccharide is from 5,000 to 2,000,000.

5. The complex according to claim 1, wherein R 1 C 15-19 Straight-chain alkyl, R 2 C 3-5 Straight-chain alkyl, R 3 C 3-5 Straight-chain alkylene groups.

6. The complex according to claim 5, wherein the compound is Telratolimod.

7. The complex according to claim 1, wherein it is a gel particle.

8. The complex according to claim 7, wherein the compound is contained within the gel particles.

9. The complex according to claim 1, wherein the mass ratio of the modified polysaccharide to the compound (mass of the modified polysaccharide / mass of the compound) is 2 to 20.

10. The composite according to claim 1, wherein the average particle size of its scattering intensity is 10 to 200 nm.

11. A medicament comprising the complex according to any one of claims 1 to 10.

12. The medicament according to claim 11, wherein it is a cancer therapeutic agent.

13. The drug according to claim 11, wherein it is a macrophage polarizing agent.

14. The complex according to any one of claims 1 to 10, or the medicament according to claim 11, used in combination with an immune checkpoint inhibitor.

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