JP7576899B1 - Complex - Google Patents

Abstract

The objective of the present invention is to provide a technique for improving the poor solubility of the compound represented by general formula (1) in water and suppressing the excessive cytokine release and bone marrow toxicity of the compound represented by general formula (1). The objective of the present invention is to provide a complex containing a modified polysaccharide having a hydrophobic group and the compound represented by general formula (1).

Description

The present invention relates to a complex.

The importance of immune cells present in cancer tissue, particularly macrophages (tumor-associated macrophages, abbreviated TAM), has been pointed out as a factor that determines the malignancy of cancer. When TAM are M2-like dominant, they act immunosuppressively and create a microenvironment favorable for cancer growth. If this can be changed to M1-like dominant, it is expected that they will have an inhibitory effect on cancer growth.

It has been reported that Toll-like receptor 7 and Toll-like receptor 8 (TLR7 and TLR8) activators polarize macrophages from M2-like to M1-like. In addition, various compounds are known as TLR7 and TLR8 activators currently under development (Non-Patent Document 1).

Expert Opin Drug Discov. 2021 Aug;16(8):869-880.

General TLR7/8 agonists cannot be administered systemically due to the issues of excessive cytokine release and bone marrow toxicity that occur when administered systemically. In particular, compounds of general formula (1) described below, such as Telratolimod, have relatively long hydrocarbon chains and are poorly soluble in water. Furthermore, in the course of research, the inventors have newly discovered that Telratolimod has the effect of excessive cytokine release and bone marrow toxicity through formulation studies.

The objective of the present invention is to provide a technology that suppresses the excessive cytokine release and bone marrow toxicity of various TLR7/8 activators and enables systemic administration.

In view of the above problems, the present inventors have conducted extensive research into various TLR7/8 agonists and have found that the above problems can be solved by a complex containing a modified polysaccharide having a hydrophobic group and a compound represented by general formula (1). Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

Item 1. A modified polysaccharide containing a hydrophobic group and a polysaccharide represented by the general formula (1):

[In the formula, ^R1 represents a linear alkyl group having 12 to 25 carbon atoms, ^R2 represents a linear alkyl group having 2 to 8 carbon atoms, ^{and R3} represents a linear alkylene group having 2 to 8 carbon atoms.]
A complex containing a compound represented by the formula:

Item 2. The complex according to Item 1, wherein the constituent polysaccharide of the modified polysaccharide includes pullulan.

Item 3. The complex according to item 1 or 2, wherein the hydrophobic group includes a hydrophobic group having a sterol skeleton.

Item 4. The complex according to any one of items 1 to 3, wherein the weight-average molecular weight of the modified polysaccharide is 5,000 to 2,000,000.

Item 5. The complex according to any one of Items 1 to 4, wherein R ¹ is a linear alkyl group having 15 to 19 carbon atoms, R ² is a linear alkyl group having 3 to 5 carbon atoms, and R ³ is a linear alkylene group having 3 to 5 carbon atoms.

Item 6. The complex according to item 5, wherein the compound is telratolimod.

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

Item 8. The complex according to item 7, wherein the compound is contained inside the gel particle.

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

Item 10. The complex according to any one of items 1 to 9, having a scattering intensity average particle size of 10 to 200 nm.

Item 11. A medicine containing the complex according to any one of items 1 to 10.

Item 12. The pharmaceutical according to Item 11, which is a cancer therapeutic agent.

Item 12A. A method for treating cancer, comprising administering a complex according to any one of items 1 to 10 to a subject (e.g., a subject having cancer).

Item 12B. A conjugate according to any one of items 1 to 10 for use in the treatment of cancer.

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

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

Item 13. The pharmaceutical agent according to item 11 or 12, which is a macrophage polarization agent.

Item 13A. A method for polarizing macrophages, comprising administering the complex according to any one of items 1 to 10 to a subject (e.g., a subject having cancer).

Item 13B. A complex according to any one of items 1 to 10 for use in macrophage polarization.

Item 13C. Use of the complex according to any one of items 1 to 10 for the manufacture of a macrophage polarization agent.

Item 13D. Use of the complex according to any one of items 1 to 10 as a macrophage polarization agent.

Item 14. A conjugate according to any one of items 1 to 10, or a pharmaceutical agent according to any one of items 11 to 13, used in combination with an immune checkpoint inhibitor.

According to the present invention, it is possible to provide a pharmaceutical composition for an antitumor agent, etc., which suppresses the excessive cytokine release effect and bone marrow toxicity of TLR7/8 agonists and enables systemic administration.

The results of Test Example 4 are shown. The vertical axis indicates the serum Telratolimod concentration, and the horizontal axis indicates the time elapsed since administration of the test drug. The legend indicates the test drug, with Naked Telratolimod referring to the test drug not encapsulated in cholesterol-modified pullulan nanogel (hereafter referred to as CHP nanogel), and CHP:Telratolimod referring to the test drug coated with CHP nanogel. 1 shows the results of Test Example 5. The vertical axis indicates the Telratolimod concentration contained in the tumor, and the horizontal axis indicates the test drug added. 1 shows the results of Test Example 6. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days since the day cancer cells were intradermally transplanted, which is set as day 0. The legend indicates the test drug. The results of Test Example 7 are shown. The graph on the left shows the measurement results of M1-like macrophages (vertical axis), and the graph on the right shows the measurement results of M2-like macrophages (vertical axis). The horizontal axis shows the test drug added. "No treatment" shows the case where no drug was added. 1 shows the results of Test Example 8. The graph information shows the measured cytokines. The vertical axis shows the cytokine concentration in serum, and the horizontal axis shows the administered test drug. The results of Test Example 9 are shown. RET stands for reticulocytes. The graph on the left shows the measurement results of the ratio of reticulocyte count to total red blood cells (vertical axis: %), and the graph on the right shows the measurement results of the blood concentration of reticulocyte count (vertical axis: × ¹⁰⁴ /μL). 1 shows the results of Test Example 10. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days since the day cancer cells were intradermally transplanted, which is set as day 0. The legend indicates the test drug. 1 shows the results of Test Example 11. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days elapsed. 1 shows the results of Test Example 15. The vertical axis shows the tumor infiltration ratio of CD8-positive T cells and NK cells, and the horizontal axis shows the test drug added. The results of Test Example 16 are shown. 1 shows the results of Test Example 17. The vertical axis shows the tumor volume, and the horizontal axis shows the number of days since the day cancer cells were intradermally transplanted, which is set as day 0. The legend indicates the test drug.

In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

1. Conjugate In one aspect, the present invention relates to a modified polysaccharide containing a hydrophobic group and a conjugate of the general formula (1):

The present invention relates to a complex (sometimes referred to as the "complex of the present invention" in this specification) containing a compound represented by the formula:

The compounds represented by general formula (1) are as follows:

In general formula (1), ^R1 represents a linear alkyl group having 12 to 25 carbon atoms. The linear alkyl group preferably has 14 to 23 carbon atoms, more preferably 14 to 21 carbon atoms, even more preferably 15 to 19 carbon atoms, still more preferably 16 to 18 carbon atoms, and particularly preferably 17 carbon atoms.

In the general formula (1), ^R2 represents a linear alkyl group having 2 to 8 carbon atoms. The linear alkyl group preferably has 2 to 6 carbon atoms, more preferably 3 to 5 carbon atoms, and particularly preferably 4 carbon atoms.

In the general formula (1), ^R3 represents a linear alkylene group having 2 to 8 carbon atoms. The linear alkylene group preferably has 2 to 6 carbon atoms, more preferably 3 to 5 carbon atoms, and particularly preferably 4 carbon atoms.

The compound represented by general formula (1) is particularly preferably telratolimod (a compound in which ^R1 is a linear alkyl group having 17 carbon atoms, ^R2 is a linear alkyl group having 4 carbon atoms, and ^R3 is a linear alkylene group having 4 carbon atoms).

The compound represented by general formula (1) may be in the form of a salt. The salt is not particularly limited as long as it is a pharma- ceutically acceptable salt, and may be, for example, an acid addition salt. Specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; 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 acidic amino acids such as aspartic acid and glutamic acid.

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

As the compound represented by general formula (1), a commercially available product can be used, or a product obtained according to a known manufacturing method can be used.

The compound represented by general formula (1) may be a single type or a combination of two or more types.

The modified polysaccharide is not particularly limited as long as it is a compound obtained by modifying a polysaccharide and contains a hydrophobic group as a modifying group. In the present invention, it has been found that the poor solubility in water of the compound of general formula (1) can be improved by complexing with a modified polysaccharide, and furthermore, the excessive cytokine release action and bone marrow toxicity of the compound of general formula (1) can be suppressed.

The polysaccharides constituting the modified polysaccharides (i.e., polysaccharides before modification) are not particularly limited as long as they are polymers in which the sugar residues are glycosidicly bonded. The sugar residues constituting the polysaccharides can be, for example, monosaccharides such as glucose, mannose, galactose, and fucose, or residues derived from sugars such as disaccharides and oligosaccharides. The sugar residues may be 1,2-, 1,3-, 1,4-, or 1,6-glycosidic bonds, and the bonds may be either α- or β-type bonds. The polysaccharides may be either linear or branched. The sugar residue is preferably a glucose residue, and examples of the polysaccharides include natural or synthetic pullulan, mannan, dextran, amylose, amylopectin, and the like. Among these, from the viewpoints of the effect of improving poor solubility, the effect of suppressing excessive cytokine release and bone marrow toxicity, the anticancer effect, the macrophage polarization effect, etc., pullulan, mannan, dextran, etc. are preferably used, more preferably pullulan, mannan, etc., and particularly preferably pullulan, etc.

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

As polysaccharides, commercially available products can be used, or those obtained according to known manufacturing methods can be used.

The hydrophobic group is not particularly limited as long as it is a group having hydrophobicity. From the viewpoints of the poor solubility improving effect, the effect of suppressing excessive cytokine release and bone marrow toxicity, the anticancer effect, the macrophage polarization effect, etc., preferred examples of the hydrophobic group include hydrophobic groups having a sterol skeleton, hydrocarbon groups, etc., and particularly preferred examples include hydrophobic groups having a sterol skeleton.

The sterol skeleton is an alcohol with a hydroxy group bonded to the cyclopentahydrophenanthrene ring shown in formula (I). The symbols A to D in formula (I) represent each ring that makes up the cyclopentahydrophenanthrene ring.

In the sterol skeleton, the cyclopentahydrophenanthrene ring may have a double bond, and the position of the hydroxyl group is not particularly limited. Preferably, the sterol has a hydroxyl group bonded to the C-3 position and a double bond to the B ring, or the stanol has a hydroxyl group bonded to the C-3 position and is composed of a saturated ring. Examples of hydrophobic groups having a sterol skeleton include groups derived from compounds in which the sterol skeleton is modified, for example, by substituting a hydrocarbon group (e.g., a linear or branched alkyl group having 1 to 20 carbon atoms) at the ring carbon. Here, the "group derived from" refers to a group obtained by removing a functional group such as a hydrogen atom or a hydroxyl group from a certain compound.

Examples of hydrophobic groups having a sterol skeleton include cholesterol-derived groups, cholestanol-derived groups, lanosterol-derived groups, ergosterol-derived groups, β-sitosterol-derived groups, campesterol-derived groups, stigmasterol-derived groups, and brassicasterol-derived groups. Among these, preferred are sterol-derived groups such as cholesterol-derived groups, cholestanol-derived groups, lanosterol-derived groups, and ergosterol-derived groups, and more preferred are cholesterol-derived groups.

The hydrocarbon group as a hydrophobic group is not particularly limited, and examples thereof include chain (preferably linear) hydrocarbon groups (preferably alkyl groups) having 8 to 50 carbon atoms (preferably 10 to 30, more preferably 12 to 20).

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

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

The hydrophobic group can be linked to the polysaccharide directly or indirectly (e.g., via a linker).

A preferred modified polysaccharide is, for example, one in which the primary hydroxyl groups of, for example, 1 to 10 (preferably 1 to 5) saccharide units per 100 saccharide residues constituting the polysaccharide are represented by the formula (II): -O-( _CH2 ) _xCONH ( _CH2 ) _yNH -CO-O-R (II) (wherein R represents a hydrophobic group or a hydrocarbon group having a sterol skeleton; x represents 0 or 1; and y represents any positive integer), and y is preferably 1 to 8.

The modified polysaccharides can be synthesized according to or in accordance with known methods (e.g., International Publication No. WO00/12564). One example is the following method. First, a diisocyanate compound represented by OCN-R ^A NCO (wherein R ^A is a hydrocarbon group having 1 to 50 carbon atoms) is reacted with a hydroxyl-containing hydrocarbon or sterol having 12 to 50 carbon atoms to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl-containing hydrocarbon or sterol having 12 to 50 carbon atoms is reacted. Next, the resulting isocyanate group-containing hydrophobic compound is further reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide containing a hydrocarbon group or a steryl group having 12 to 50 carbon atoms as a hydrophobic group. The resulting reaction product is purified with a ketone solvent to produce a high-purity hydrophobic group-containing polysaccharide.

The modified polysaccharides may be a single type or a combination of two or more types.

The mass ratio of the modified polysaccharide to the compound of general formula (1) (mass of modified polysaccharide/mass of the compound of general formula (1)) is preferably 1 or more, more preferably 2 to 20, even more preferably 3 to 10, and even more preferably 3 to 8. By setting the mass ratio within the above range, it is possible to exert even greater anti-cancer effects and macrophage polarization effects.

The complex of the present invention can be a gel particle. "Gel particle" refers to a polymer gel particle having a hydrogel structure. A hydrogel is a three-dimensional network structure formed by crosslinking hydrophilic polymers, which swells with water. In the complex of the present invention, which is a gel particle, the modified polysaccharide self-organizes through physical crosslinks formed based on hydrophobic interactions caused by hydrophobic groups to form a three-dimensional network structure. When the complex of the present invention is a gel particle, it is preferable that the compound of general formula (1) is contained inside the gel particle.

The shape of the complex of the present invention is not particularly limited, but is typically spherical.

The complex of the present invention is preferably nano-sized (preferably a nanogel particle), and the scattering intensity average particle size is, for example, 200 nm or less, preferably 10 to 200 nm, more preferably 20 to 200 nm, even more preferably 60 to 190 nm, even more preferably 80 to 180 nm, and particularly preferably 90 to 160 nm. The particle size can be measured by dynamic light scattering.

The complex of the present invention may contain other substances in addition to the modified polysaccharide. Examples of the other substances include proteins, peptides, nucleic acids, sugars, low molecular weight compounds, high molecular weight compounds, inorganic substances, and complexes thereof. More specifically, examples of the other substances include drugs such as adjuvants, cancer antigens, anticancer drugs, and nucleic acid medicines.

Adjuvants can be selected from inactivated bacteria or bacterial extracts, nucleic acid lipopolysaccharides, lipopeptides, synthetic low molecular weight compounds, etc., and preferably imidazoquinolines (e.g., R848 and imiquimod), saponins (e.g., QuilA and QS21), STING agonists (e.g., cyclic di-GMP), monophosphoryl lipids, lipopeptides, etc. are used. In addition to the above, other adjuvants include, for example, taxane drugs, anthracycline drugs, JAK/STAT inhibitors, indole deoxygenase (IDO) inhibitors, and tryptophan deoxygenase (TDO) inhibitors. These inhibitors include compounds that have antagonistic effects against the factor, as well as neutralizing antibodies against the factor.

Cancer antigens include antigenic polypeptides, and are preferably antigenic polypeptides. Antigenic polypeptides are antigens or partial peptides thereof that are highly expressed in cancer cells, and in some cases, are expressed only by cancer cells. Antigenic polypeptides may be expressed within or on the surface of cancer cells.

Antigen polypeptides include, but are 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, C D20, 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), M AGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, GAGE-3, GA GE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC) , fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, and partial peptides thereof.

Examples of anticancer agents include alkylating agents, metabolic antagonists, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeted drugs, hormone agents, and biological agents.

Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosoureas, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.

Examples of metabolic antagonists include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, and mercaptopurine.

Examples of microtubule inhibitors include alkaloid anticancer drugs such as vincristine, and taxane anticancer drugs such as docetaxel and paclitaxel.

Examples of antibiotic anticancer drugs include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and zinostatin stimalamer.

Topoisomerase inhibitors include CPT-11, irinotecan, and nogitecan, which have topoisomerase I inhibitory effects, and etoposide and sobuzoxane, which have topoisomerase II inhibitory effects.

Examples of platinum agents include cisplatin, nedaplatin, oxaliplatin, and carboplatin.

Examples of hormonal agents include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane.

Examples of biological agents include interferon α, β and γ, interleukin 2, ubenimex, dried BCG, and exotoxins. A specific example of an exotoxin is Pseudomonas Exotoxin (PE), an exotoxin (protein) produced by bacteria. PE has an enzyme activity called NAD+-diphthamide-ADP-ribosyl transferase, which inhibits protein synthesis and exerts strong toxicity. To date, PE conjugated to an antibody called CD22 antibody has been approved as an anticancer drug.

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, and tretinoin. In addition to the molecular targeted drugs specified here, the present invention may also include immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, etc.), anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab, cemiplimab, etc.), anti-CTLA-4 antibodies (e.g., ipilimumab, tremelimumab, etc.), 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, vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibodies), and other angiogenesis-targeting inhibitors, various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, proteasome inhibitors, and antibody-anticancer drug combinations. These inhibitors also include antibodies.

A nucleic acid drug is an active pharmaceutical ingredient that is a nucleic acid, and is not particularly limited in that respect.

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 per 100 parts by mass of the compound of general formula (1) and the modified polysaccharide combined.

In a preferred embodiment, the complex of the present invention can be obtained in a state of being dissolved in water or a solvent containing a smaller amount of organic solvent (for example, a solvent containing 1% by mass or less, 0.5% by mass or less, 0.2% by mass or less, 0.1% by mass or less, 0.01% by mass or less, or 0.001% by mass or less of organic solvent relative to 100% by mass of solvent). The complex of the present invention can preferably be in a state of being dissolved in water (aqueous solution form).

The concentration of the compound of general formula (1) in the complex solution of the present invention (particularly, a solution using the above-mentioned solvent) 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 invention, a solution containing the compound of general formula (1), which is poorly soluble in water, at a relatively high concentration as described above can be obtained.

The concentration of the modified polysaccharide in the complex solution of the present invention (particularly a solution using the above-mentioned solvent) 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.

The complex of the present invention can be produced, for example, by stirring a solution (solution 1) containing the compound of general formula (1) and the modified polysaccharide at a relatively low temperature. This method is excellent in terms of increasing the concentration of the compound of general formula (1) in the complex of the present invention.

The solvent of solution 1 contains water. In addition, in order to dissolve the compound of general formula (1), the solvent of solution 1 preferably contains an organic solvent. The organic solvent is not particularly limited as long as it can dissolve the compound of general formula (1) and has compatibility with water, and preferably includes dimethyl sulfoxide or N,N-dimethylformamide. 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, and even more preferably 97.5 to 98.5 v/v% or more. The content of 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%, and even more preferably 1.5 to 2.5 v/v%.

The concentration of the compound of general 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 general formula (1) in solution 1 (modified polysaccharide mass/compound mass) is preferably 1 to 20, more preferably 2 to 10, and even more preferably 3 to 7.

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

The water used for solution 1 may contain a buffer such as a phosphate buffer.

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. By stirring under these conditions, a complex with a more uniform shape and particle size can be obtained.

After stirring, the mixture can be subjected to a purification process, if necessary. Examples of purification processes include filter filtration and ultrafiltration. Preferably, ultrafiltration is used to concentrate the mixture and remove the organic solvent. This process makes it possible to adjust the mass ratio of the modified polysaccharide to the compound of the general formula (1) described above.

2. Uses The complex of the present invention has an anticancer effect and a macrophage polarization effect (polarization effect from M2-like to M1-like). Therefore, the complex of the present invention can be used as an active ingredient of medicines, reagents, etc. (sometimes referred to as "drugs of the present invention" in this specification), specifically as an active ingredient of cancer therapeutic agents, macrophage (preferably TAM) polarization agents, etc.

The drug of the present invention is not particularly limited as long as it contains the complex of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharma- ceutically acceptable. The other components include components having pharmacological action as well as additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

The drug of the present invention may contain, in addition to the complex of the present invention, other substances that the complex of the present invention may contain (e.g., adjuvants, cancer antigens, anticancer drugs, drugs such as nucleic acid drugs, etc.). The drug of the present invention may also be used in combination with other substances that the complex of the present invention may contain (e.g., adjuvants, cancer antigens, anticancer drugs, drugs such as nucleic acid drugs, etc.). In particular, as shown in the below-described Example (Test Example 10) regarding the effect of combination with an anti-PD-1 antibody, combination with an immune checkpoint inhibitor is useful from the viewpoint of antitumor effect, and examples of such immune checkpoint inhibitors include anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, etc.), anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab, cemiplimab, etc.), and anti-CTLA-4 antibodies (e.g., ipilimumab, tremelimumab, etc.).

The mode of use of the drug of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of drug. Depending on the application, the drug of the present invention can be used, for example, in vitro (e.g., added to the medium of cultured cells) or in vivo (e.g., administered to animals).

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

When the drug of the present invention targets cancer, the target cancer is not particularly limited, and examples thereof 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, Burkitt's 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, and follicular lymphoma. Examples of cancers that may be present in the body include endometrial cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, malignant melanoma, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, thecoma, male germinoma, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, osteoblastoma, leiomyosarcoma, thyroid sarcoma, and Wilms' tumor.

The drugs of the present invention may be in any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; parenteral preparations such as injectable preparations (e.g., drip injections (e.g., intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections); topical preparations (e.g., ointments, poultices, and lotions); suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

The route of administration of the drug of the present invention is not particularly limited as long as the desired effect is obtained, and examples of such route include oral administration, enteral administration such as tube feeding and enema administration, and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

The content of the active ingredient (complex of the present invention) in the drug of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

When the drug of the present invention is administered to an animal, the dosage is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is usually, in terms of the weight of the active ingredient, generally 0.1 to 1000 mg/kg body weight per day in oral administration, preferably 0.5 to 500 mg/kg body weight per day, and 0.01 to 100 mg/kg body weight per day in parenteral administration, preferably 0.05 to 50 mg/kg body weight per day. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc.

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Preparation Example 1. Preparation of cholesterol-modified pullulan According to the previous report (Macromolecules 1993, 23, 3062-3068), cholesterol-modified pullulan (hereinafter referred to as CHP) was prepared by introducing 1.2 cholesterol molecules per 100 monosaccharides into pullulan having a weight-average molecular weight of 100,000.

Example 1. Examination of inclusion of various TLR7 and TLR8 agonists in CHP nanogel
We investigated whether nine TLR7 and TLR8 agonists (Telratolimod, Motolimod, Vesatolimod, Resiquimod, Imiquimod, CL075, CU-CPT17e, CU-CPT9a, PF-4878691) could be encapsulated in CHP nanogel. CHP was dissolved in 6M urea in PBS(-) and the compound solution was added. Then, dialysis was performed stepwise against PBS(-).

<Results>
Of the nine compounds, only one (Telratolimod) could be stably encapsulated and dissolved in CHP nanogel.

<Considerations>
The Telratolimod concentration in the nanogel preparation obtained in Example 1 was a low concentration of 0.112 mg/ml. Further improvement of the Telratolimod concentration was expected to show sufficient pharmacological efficacy.

Example 2. Preparation of Telratolimod-encapsulated CHP nanogel
Telratolimod-encapsulated CHP nanogel (hereafter, CHP:Telratolimod) was prepared as follows. 160 mg of CHP 80K (NOF) and 32 mg of Telratolimod (ACHEMBLOCK) were mixed and dissolved in 3.2 mL of N,N-dimethylformamide (hereafter, DMF). The above-mentioned DMF solution was added to 160 mL of PBS and stirred overnight at 4°C. The filtrate was passed through a 0.8 μm filter and concentrated and purified with TFF (Pall, MWCO; 30K) to remove DMF. After recovery, it was passed through a 0.45 μm filter to obtain a CHP:Telratolimod solution (scattering intensity average particle size: 149 nm, ELSZ-2000ZS, Otsuka Electronics Co., Ltd.). A calibration curve was prepared by HPLC (Column: YMC-Pack C4, 150X2.0 mmI.D. S-3 μm, 12 nm, flow rate 0.3 mL/min 60% B line, A line: 0.1% FA in H2O, B line: 0.1% FA in CH3CN) using a standard sample of Telratolimod. The Telratolimod concentration in the CHP:Telratolimod solution was calculated by decomposing CHP with an aqueous sodium periodate solution, and then quantifying the resulting aldehyde group with Schiff's reagent (CHP: 10.2 mg/mL, Telratolimod: 1.17 mg/mL).

<Considerations>
The Telratolimod concentration in the nanogel formulation obtained using the newly discovered method in Example 2 was 1.17 mg/ml, approximately 10 times higher than that achieved by previous methods. By using this method to create a nanogel formulation of a poorly soluble drug, we were successful in increasing the drug concentration. The formulation prepared in Example 2 was used in the following Test Examples 1-17.

Test Example 1. Preparation of TAM derived from human peripheral blood CD14 positive monocytes derived from human peripheral blood, which had been cryopreserved in advance, were put to sleep, seeded in 500μL of basal medium (RPMI, 10% fetal bovine serum, 50ng/mL M-CSF) at 2 x ¹⁰⁵ cells/well in a 24-well tissue culture plate, and cultured for three days in an incubator at 37℃ and 5% _CO2 , and then cultured for another three days with 500μL of basal medium added. After that, the culture supernatant of human cancer cells prepared separately was replaced with 500μL of TAM differentiation induction medium containing 10% fetal bovine serum, 50ng/mL IL-4, 50ng/mL IL-6, 50ng/mL IL-10, 50ng/mL IL-13, and 5% human AB type male donor defibrinized serum (Veritas), and cultured for two days.

Test Example 2. Polarization test of TAM derived from human peripheral blood
Telratolimod solution or CHP:Telratolimod solution adjusted to a final concentration of 30 μM in terms of Telratolimod was added to the TAM prepared in Test Example 1, incubated for two days, and TNF-α in the culture supernatant was measured by ELISA (BioLegend). The control was one to which only the medium was added.

<Results>
The results are shown in Table 1. Compared to telratolimod, CHP:telratolimod produced higher TNF-α. This suggests that encapsulation of telratolimod in CHP nanogels allows telratolimod to be more efficiently taken up by TAMs, causing polarization into M1-like macrophages, resulting in increased TNF-α production.

Test Example 3. Cancer cell phagocytosis test of TAM derived from human peripheral blood
HeLa cells were labeled with fluorescent label 1 (CellTracker Green CMTPX Dye, ThermoFisher) for 30 minutes, washed twice with PBS(-), and cultured overnight in medium (RPMI supplemented with 10% fetal bovine serum) containing 1.25 μM doxorubicin.

TAM was prepared in the same manner as in Test Example 1, and Telratolimod or CHP:Telratolimod adjusted to a final concentration of 10 μM in Telratolimod equivalent was added to the basal medium and incubated for two days. After labeling with fluorescent labeling agent 2 (CellTracker Red CMTPX Dye, ThermoFisher) for 30 minutes, the cells were washed twice with PBS(-). The above-mentioned doxorubicin-treated HeLa cells were added to the TAM at a half ratio and co-cultured overnight. The cells were then harvested and the number of cells co-positive for fluorescent labels 1 and 2 was measured by flow cytometry. As a control, only medium was added.

<Results>
The results are shown in Table 2. Compared to Telratolimod, CHP:Telratolimod had improved phagocytic activity against HeLa cancer cells. This indicates that encapsulation of Telratolimod in CHP nanogel enabled TAM to be more efficiently taken up and polarized into M1-like macrophages, resulting in enhanced phagocytic activity.

Test Example 4. Measurement of blood concentration transition in mouse homogeneous tumor transplantation model The mouse homogeneous tumor transplantation model was prepared by administering ¹⁰⁶ mouse colon cancer cell line MC38 into the back skin of a mouse (C57BL/6JJmsSlc, female). Mice were used 7 days after cell administration. CHP:Telratolimod prepared in Example 2 and Telratolimod dissolved in a solubilizing solvent (17.1% polyethylene glycol 300, 4.3% Tween 80, 8.6% polyoxyethylene hydrogenated castor oil) were injected into the tail vein at 84.5 μg, and the blood Telratolimod concentration transition was measured. Five mice were administered in each group and blood was collected. After administration of each test drug through the tail vein, 20 μL of serum collected at 1 minute, 30 minutes, 1 hour, 2 hours, and 6 hours was added with 80 μL of isopropyl alcohol and subjected to bead crushing with a stainless steel ball with a diameter of 5 mm for 1 minute. After adding 400μL of 80% isopropyl alcohol and 20% acetonitrile, the mixture was sonicated for 3 minutes and centrifuged at 12000g for 5 minutes to collect the supernatant. The collected supernatant was analyzed by LS-MS/MS to measure the Telratolimod concentration. For the calibration curve, a 1.17mg/mL Telratolimod standard solution was prepared in DMSO, and a dilution series of 10, 20, 100, 200, 1000, and 2000 times was prepared, followed by 10-fold dilution with fetal bovine serum. Cabozantinib (Selleck) was used as the internal standard for correction.

<Results>
The 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 confirmed to persist in the blood longer, with its half-life in the blood being extended by more than four times.

Test Example 5. Measurement of intratumoral Telratolimod concentration in a mouse homogeneous tumor transplant model Using a mouse homogeneous tumor transplant model prepared in the same manner as in Test Example 4, the CHP:Telratolimod solution containing 84.5 μg of Telratolimod prepared in Example 2 and the Telratolimod solution prepared in Test Example 4 were administered via the tail vein, and the tumor was excised one hour later to measure the Telratolimod concentration in the tumor. Five mice were used for each group. 80 μL of isopropyl alcohol was added to 20 μg of the excised tumor pieces, and beads were crushed with a stainless steel ball with a diameter of 5 mm for 1 minute, and the subsequent treatment was performed in the same manner as described in Test Example 4.

<Results>
The results are shown in Figure 2. When encapsulated in CHP nanogel, Telratolimod increased its accumulation in the tumor by approximately three-fold.

Test Example 6. Antitumor evaluation in mouse allogeneic tumor transplant model (1)
A mouse homogeneous tumor transplant model of MC38 was prepared in the same manner as in Test Example 4. On the 7th, 9th, 11th, 14th, 16th, and 18th days after cell transplantation, the CHP:Telratolimod solution prepared in Example 2 and the Telratolimod solution prepared in Test Example 4 were administered via tail vein injection to 5 mice per group (Telratolimod amount: 84.5 μg). The tumor diameter was measured on the 7th, 9th, 11th, 14th, 16th, 18th, and 19th days. The tumor volume was calculated by (major axis x minor axis x minor axis)/2. As a negative control, the same solution as the solvent for each drug was administered. Vehicle 1 refers to the solubilizing solvent in Test Example 4, and Vehicle 2 refers to PBS(-).

<Results>
The results are shown in Figure 3. In the CHP:Telratolimod group, tumor size remained almost unchanged from the start of drug administration, and by day 19, tumor growth was inhibited by 92% compared to the vehicle control. On the other hand, the Telratolimod group only exhibited a 49% tumor growth inhibition effect compared to the vehicle control. This shows that encapsulation with CHP nanogel dramatically enhanced the antitumor effect of Telratolimod.

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

<Results>
The results are shown in Figure 4. The expression of iNOS, an M1-like macrophage marker, was significantly increased in both the Telratolimod and CHP:Telratolimod groups, and the CHP:Telratolimod group increased iNOS more significantly than the Telratolimod group. In addition, the M2-like macrophage marker ARG1 showed a tendency to decrease in both the Telratolimod and CHP:Telratolimod groups, but it decreased significantly only in the CHP:Telratolimod group. These results indicate that the CHP:Telratolimod group polarized tumor macrophages from M2-like to M1-like more than the Telratolimod group.

Test Example 8. Evaluation of cytokine release in a mouse allogeneic tumor transplant model
Allograft model mice were used after 10 days from intradermal administration of ¹⁰⁶ mouse colon cancer cell lines MC38 to mice (C57BL/6JJmsSlc, female). Telratolimod solution prepared in Test Example 4 and CHP:Telratolimod solution prepared in Example 2 were administered at 84.5 μg of Telratolimod via tail vein injection to five mice per group, and blood was collected 2 hours later. Serum was obtained by centrifugation, and the changes in the amount of each cytokine were measured using LEGENDplex Mouse Inflammation Panel (BioLegend). Significance tests were performed using One-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

<Results>
The results are shown in Figure 5. The telratolimod solution group showed a rapid increase in IL-6, MCP-1, and IFN-β, suggesting the possibility of cytokine release syndrome. On the other hand, the increases in these levels were significantly reduced in the CHP:telratolimod group, indicating that cytokine release syndrome was suppressed.

Test Example 9. Blood test in a mouse allogeneic tumor transplant model
¹⁰⁶ mouse colon cancer cell line MC38 was administered intradermally to the back of mice (C57BL/6JJmsSlc, female), and 7, 9, and 11 days later, the Telratolimod solution prepared in Test Example 4 and the CHP:Telratolimod solution prepared in Example 2 were administered by tail vein injection to 5 mice per group at a dose of 84.5 μg of Telratolimod. One day after the final administration, blood was collected and changes in red blood cell and reticulocyte counts were measured. Significance tests were performed using one-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

<Results>
The results are shown in Figure 6. A significant decrease in reticulocytes was observed in the Telratolimod solution group, whereas no decrease was observed in the CHP:Telratolimod solution group. Reticulocytes are an indicator of red blood cell production in the bone marrow, and while the Telratolimod solution group suggested toxicity to the bone marrow, the CHP:Telratolimod solution group showed a significant decrease in reticulocytes, suggesting that encapsulation with CHP nanogel does not cause bone marrow suppression.

Test Example 10. Antitumor evaluation in mouse allogeneic tumor transplantation model (2)
The mouse allogeneic tumor transplantation model was prepared by injecting ¹⁰⁶ mouse colon cancer cell lines CT26 into the back of mice (BALB/cCrSlc, female). On the 7th, 9th, 11th, 14th, 16th, and 18th days after cell transplantation, the CHP:Telratolimod solution prepared in Example 2 and the Telratolimod solution prepared in Test Example 4 were injected by tail vein injection, and the anti-PD-1 antibody (RMP1-14, Bio X Cell) solution was intraperitoneally administered to 5 mice per group on the 7th, 11th, 14th, and 18th days after cell transplantation (Telratolimod amount 42 μg, anti-PD-1 antibody 100 μg). In addition, in Group 1, the CHP:Telratolimod solution and the anti-PD-1 antibody were used in combination, and the tumor diameter was measured on the 7th, 9th, 11th, 14th, 16th, 18th, and 21st days. The tumor volume was calculated by (longer diameter × shorter diameter × shorter diameter) / 2. The negative control was an untreated group.

<Results>
The results are shown in Figure 7. The CHP:Telratolimod group experienced tumor regression from the start of drug administration. On the other hand, the antitumor effect of the Telratolimod group was limited, indicating that the antitumor effect of Telratolimod was dramatically enhanced by encapsulation with CHP nanogel. In addition, the antitumor effect of the anti-PD-1 antibody was also limited, but the tumor completely disappeared when CHP:Telratolimod was used in combination with the anti-PD-1 antibody. This result indicates that a high antitumor effect can be expected by combining CHP:Telratolimod with cancers in which the antitumor effect of the anti-PD-1 antibody is limited (so-called cold tumors).

Test Example 11. Preparation of M2-like macrophages derived from human peripheral blood CD14-positive monocytes derived from human peripheral blood that had been cryopreserved in advance were awake, seeded in 24-well tissue culture plates at 2 x ¹⁰⁵ cells/well in 500μL of basal medium (RPMI, 10% fetal bovine serum, 50ng/mL M-CSF), and cultured for three days in an incubator at 37℃ and 5% _CO2 , and then cultured for another three days with 500μL of basal medium added. After that, the basal medium was replaced with 500μL of M2-like macrophage differentiation induction medium containing 50ng/mL IL-4, 50ng/mL IL-6, 50ng/mL IL-10, and 50ng/mL IL-13, and cultured for two days.

Test Example 12. Polarization test of M2-like macrophages derived from human peripheral blood
Telratolimod solution or CHP:Telratolimod solution adjusted to a final concentration of 3 μM in Telratolimod equivalent was added to the M2-like macrophages prepared in Test Example 11 and incubated for one day, and TNF-α, IL-1β, IL-6, IL-12p40 (p40 subunit of IL-12), IL-23, and CXCL10 in the culture supernatant were measured using a flow cytometer bead assay kit (LEGENDplex ^TM , BioLegend). Only medium was added as a control.

<Results>
The results are shown in Table 3. Compared to telratolimod, CHP:telratolimod produced higher levels of TNF-α, IL-1β, IL-6, IL-12p40, IL-23, and CXCL10. This suggests that encapsulation of telratolimod in CHP nanogels allows telratolimod to be more efficiently taken up by M2-like macrophages, causing their polarization into M1-like macrophages, resulting in increased production of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-12 (p40 subunit), IL-23, and CXCL10) produced by M1-like macrophages.

Test Example 13. Cancer cell phagocytosis test of human peripheral blood-derived M2-like macrophages
HeLa cells were labeled with fluorescent label 1 (CellTracker Green CMTPX Dye, ThermoFisher) for 30 minutes, washed twice with PBS(-), and cultured overnight in medium (RPMI supplemented with 10% fetal bovine serum) containing 2.5 μM doxorubicin.

Telratolimod or CHP:Telratolimod adjusted to a final concentration of 10 μM in Telratolimod equivalent in basal medium was added to the M2-like macrophages prepared in Test Example 11 and incubated for one day. After labeling with fluorescent labeling agent 2 (CellTracker Red CMTPX Dye, ThermoFisher) for 30 minutes, the cells were washed twice with PBS(-). The above-mentioned HeLa cells were added to the M2-like macrophages at a half ratio and co-cultured overnight. The cells were then collected and the number of cells co-positive for fluorescent labels 1 and 2 was measured by flow cytometry. Only medium was added as a control.

<Results>
The results are shown in Table 4. Compared to Telratolimod, CHP:Telratolimod had improved phagocytic activity against cancer cells, HeLa cells. This indicates that by being encapsulated in CHP nanogel, Telratolimod was more efficiently taken up by TAMs, causing polarization into M1-like macrophages, resulting in increased phagocytic activity.

Test Example 14. Antitumor evaluation in mouse allogeneic tumor transplantation model (3)
Among the CHP:Telratolimod administration group in Test Example 10, mice (n=4) in which the tumor had completely regressed on the 21st day were continued to be bred, and it was confirmed that the tumor did not recur in all cases until the 96th day. Thereafter, on the 97th day, 10 ⁶ mouse colon cancer cell line CT26 was transplanted into the dorsal skin on the opposite side to the side where the cancer cell line was transplanted in Test Example 10, and tumor growth was measured until the 120th day. As a control, five mice (BALB/cCrSlc, female) of the same age that had not previously been transplanted with the CT26 cancer cell line were prepared, and 10 ⁶ cancer cell line CT26 was transplanted into the dorsal skin to measure tumor growth.

<Results>
The results are shown in Figure 8. As a control, mice of the same age that had never been transplanted with the CT26 cancer cell line showed tumor growth in all cases (Figure 8, bottom). On the other hand, in mice that showed complete regression after the first cancer cell transplant, tumors did not take root in all cases, and the transplanted cancer cells were completely rejected (Figure 8, top). This result indicates that CHP:Telratolimod induces immune memory against cancer cells, suggesting that remission achieved with CHP:Telratolimod may not lead to relapse.

Test Example 15. Analysis of tumor-infiltrating CD8 positive T cells and natural killer cells (NK cells) in a mouse homogeneous tumor transplantation model A mouse homogeneous tumor transplantation model of MC38 was prepared in the same manner as in Test Example 6, and the CHP:Telratolimod solution prepared in Example 2 was administered to five mice via tail vein injection on the 7th and 9th days after cell transplantation (Telratolimod amount: 84.5 μg). As a control, five mice were prepared in a group not administered CHP:Telratolimod. The tumor was excised one day after the final administration, and CD8 positive T cells and NK cells infiltrating the tumor were detected by flow cytometry. A significant difference test was performed using One-way ANOVA.

<Results>
The results are shown in Figure 9. Administration of CHP:Telratolimod significantly increased the number of CD8 positive T cells and NK cells infiltrating into the tumor. This result indicates that CHP:Telratolimod has the effect of increasing the infiltration of CD8 positive T cells and NK cells, which are the main effector cells involved in anti-tumor activity, into the tumor, and suggests that this is one of the mechanisms of action of the high anti-tumor effect of CHP:Telratolimod in Test Example 6.

Test Example 16. Analysis of Cancer Antigen-Specific CD8-Positive T Cells in a Mouse Allogeneic Tumor Transplant Model A mouse allogeneic tumor transplant model of MC38 was prepared in the same manner as in Test Example 6, and the CHP:Telratolimod solution prepared in Example 2 was administered to five mice by tail vein injection on the 7th, 9th, and 12th days after cell transplantation (Telratolimod amount: 84.5 μg). As a control, five mice were prepared in a group not administered CHP:Telratolimod. One day after the final administration, the spleen was removed to prepare splenocytes. The cells were stimulated with a peptide of MuLV p15E, a cancer antigen expressed in MC38 cells, or a peptide of TRP2, a cancer antigen expressed in melanoma, as a negative control, and then permeabilized. CD8-positive T cells were analyzed by flow cytometry using an anti-IFN-γ antibody. Significance tests were performed using unpaired t-tests.

<Results>
The results are shown in Figure 10. In mice administered CHP:Telratolimod, the number of CD8 positive T cells that became IFN-γ positive upon stimulation with peptide from MuLV p15, a cancer antigen of MC38 cells, was significantly increased compared to the CHP:Telratolimod non-administration group. On the other hand, no change was observed in the number of CD8 positive T cells that became IFN-γ positive in the negative control TRP2 peptide stimulation compared to the non-administration group. This result indicates that CHP:Telratolimod has the effect of increasing CD8 positive T cells that specifically react with MC38 cancer cells, and suggests that this is one of the mechanisms of action of the high antitumor effect of CHP:Telratolimod in Test Example 6.

Test Example 17. Antitumor evaluation in mouse allogeneic tumor transplantation model (4)
The mouse allogeneic tumor transplantation model was prepared by injecting 5 x ¹⁰⁵ mouse melanoma cell line B16F10 into the dorsal skin of mice (C57BL/6NCrSlc, female). On the 7th, 9th, 11th, 14th, and 16th days after cell transplantation, the CHP:Telratolimod solution prepared in Example 2 was administered via tail vein injection, and the anti-PD-1 antibody (RMP1-14, Bio X Cell) solution and the anti-PD-L1 antibody (10F.9G2, Bio X Cell) solution were administered intraperitoneally, with 10 mice per group (Telratolimod amount 160 μg, anti-PD-1 antibody and anti-PD-L1 antibody 200 μg). In addition, the group using CHP:Telratolimod in combination with anti-PD-1 antibody and the group using CHP:Telratolimod in combination with anti-PD-L1 antibody were also evaluated. The tumor diameter was measured on days 7, 9, 11, 14, 16, and 18. The tumor volume was calculated by (long diameter x short diameter x short diameter)/2. The negative control was an untreated group.

<Results>
The results are shown in Figure 11. The CHP:Telratolimod group, anti-PD-1 antibody group, and anti-PD-L1 antibody group showed an anti-tumor effect of approximately 50%. On the other hand, the group administered CHP:Telratolimod in combination with anti-PD-1 antibody and anti-PD-L1 antibody showed an anti-tumor effect of approximately 75-80%. This result indicates that a high anti-tumor effect can be expected by combining CHP:Telratolimod with cancers in which the anti-tumor effect of anti-PD-1 antibody and anti-PD-L1 antibody is limited (so-called cold tumors).

Claims (12)

A modified polysaccharide containing a hydrophobic group, and a polysaccharide having the general formula (1):

[In the formula, ^R1 represents a linear alkyl group having 12 to 25 carbon atoms, ^R2 represents a linear alkyl group having 2 to 8 carbon atoms, ^{and R3} represents a linear alkylene group having 2 to 8 carbon atoms.]
The compound represented by the formula:
The constituent polysaccharides of the modified polysaccharide include pullulan; and
The hydrophobic group includes a hydrophobic group having a sterol skeleton .
Complex. The complex according to claim 1, wherein the weight average molecular weight of the modified polysaccharide is 5,000 to 2,000,000. 2. The complex according to claim 1, wherein R ¹ is a linear alkyl group having 15 to 19 carbon atoms, R ² is a linear alkyl group having 3 to 5 carbon atoms, and R ³ is a linear alkylene group having 3 to 5 carbon atoms. The conjugate of claim 3 , wherein the compound is Telratolimod. The composite of claim 1, which is a gel particle. The composite of claim 5 , wherein the compound is contained within the interior of the gel particle. The complex according to claim 1, wherein the mass ratio of the modified polysaccharide to the compound (modified polysaccharide mass/compound mass) is 2 to 20. The complex according to claim 1, having a scattering intensity average particle size of 10 to 200 nm. A medicine containing the complex according to any one of claims 1 to 8. The pharmaceutical composition according to claim 9 , which is a cancer therapeutic agent. The pharmaceutical according to claim 9 , which is a macrophage polarization agent. A conjugate according to any one of claims 1 to 8 , or a medicine containing said conjugate , which is used in combination with an immune checkpoint inhibitor.

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