Cancer immunotherapy adjuvants

Abstract

The present invention relates to a cancer immunotherapy adjuvant, wherein the adjuvant activates the function of immune factors without causing side effects in vivo when administered in combination with a cancer immunotherapy agent to exhibit an effect of enhancing the anticancer effect of the cancer immunotherapy agent, and can therefore be effectively used as a cancer immunotherapy adjuvant.

Description

Technical Field

[0001] The present invention relates to a cancer immunotherapy adjuvant. Background Art

[0002] Cancer is a disease characterized by abnormal, localized cell growth that has the potential to spread throughout the body. There are many types of cancer, including lung cancer, bladder cancer, prostate cancer, pancreatic cancer, cervical cancer, brain cancer, stomach cancer, colorectal cancer, and melanoma. In the past, the most common methods of treating oncological cancers (oncology) were surgery, radiation therapy, or chemotherapy. However, cancer immunotherapy has recently been shown to hold a lot of promise as an oncology treatment.

[0003] Cancer immunotherapy is a branch of oncology in which the immune system is used to treat cancer, rather than conventional treatments that directly remove or cure tumors. The therapy concept is based on the identification of a number of proteins on the surface of T cells that inhibit the immune function of these cells.

[0004] The most fundamental problem of tumor immunity is how to activate the immune system to recognize and eliminate antigens. In this regard, new methods of genetically engineering tumor cells to secrete specific cytokines have achieved significant progress in tumor immunity.

[0005] The theoretical background of transgenic tumor vaccines based on immunotherapy is that the host possesses antigens that can recognize tumors as external factors. Human T and B lymphocytes are able to distinguish almost infinite antigen differences in the form of antigen receptors during development. However, in order to be truly successful in tumor immunity, the following two criteria must be met. First, tumor cells must express new antigens (peptides) that are not expressed in normal cells. Second, immune cells must be properly activated to recognize these antigens.

[0006] Conventional immunotherapy using tumor cells into which cytokine genes have been introduced has shown its effectiveness in animal experiments using mice. Currently, studies are being conducted around the world to find that injecting tumor cells into mice into which specific cytokine genes have been introduced can eradicate new tumors and obtain tumor immunity in these mice.

[0007] Globally, more than 10 million people are diagnosed with cancer each year, a number that will increase to 15 million new cases per year by 2020. Cancer kills 6 million people each year, accounting for 12% of all deaths worldwide. There remains a need for methods that can treat cancer. These methods may provide a basis for pharmaceutical compositions for preventing or treating cancer in humans and other mammals.

[0008] In particular, as people realize the benefits of attacking cancer in a variety of ways, co-administration of cancer treatments has become increasingly common. Co-administration is useful even if resistance to anticancer drugs is shown. In addition, co-administration has the advantage of reducing the amount of anticancer agents administered by enhancing the efficacy of the anticancer agents. In this way, the anticancer efficacy can be improved while minimizing toxicity and side effects to various organs of the body. Although many effective combination therapies have been identified in the past few decades; given the continued high number of cancer deaths each year, there is a continuing need to identify effective therapies for anticancer treatment.

[0009] Patent reference 1 discloses a pharmaceutical composition for co-administration for preventing or treating cancer, which comprises a p53 activator and a c-Met inhibitor as active ingredients. Summary of the invention

[0010] Technical issues

[0011] The purpose of the present invention is to provide a cancer immunotherapy adjuvant.

[0012] Another object of the present invention is to provide a combination drug for cancer immunotherapy, which comprises a cancer immunotherapy agent; and a cancer immunotherapy adjuvant.

[0013] Another object of the present invention is to provide a pharmaceutical composition for enhancing the efficacy of cancer immunotherapeutics.

[0014] Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity.

[0015] Another object of the present invention is to provide a method for preventing or treating cancer.

[0016] Another object of the present invention is to provide a kit for anti-cancer treatment.

[0017] Means of solving the problem

[0018] To achieve the above objectives, in one aspect of the present invention, the present invention provides a cancer immunotherapy adjuvant, which comprises a compound represented by the following Formula 1, its isomer, its solvate, its hydrate, or its pharmaceutically acceptable salt as an active ingredient.

[0019] [Formula 1]

[0020]

[0021] (In Formula 1,

[0022] X is oxygen or sulfur;

[0023] represents a single bond or a double bond;

[0024] R1 is hydrogen, halogen, C 1-30 Alkyl, C 3-10 Cycloalkyl, C 2-30 Alkenyl, C 3-10 Cycloalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-15 Heterocycloalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-15 Heterocycloalkyl, C 2-30 Alkoxyalkyl, C 3-30 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkenyl, C 1-20 Alcohol, C 1-20 Enol, C 2-30 Acyl, C 1-10 Amide, C 1-10 Amine, C 2-15 Esters, sulfates, carboxyl groups, C 3-20 Carboxyalkyl, C 3-20 Carboxyalkenyl, C 3-20 Alkyl carboxyl, C 3-20 Alkenyl carboxyl, C 3-20 Alkylcarboxylalkyl, C 3-20 Alkyl carboxyl alkenyl, C 3-20 Alkenylcarboxyalkyl, C 4-20 Alkenyl carboxyl alkenyl, C 6-30 Aryl, C 6-30 Aralkyl, C 6-30 Alkyl, C 3-30 Heteroaryl or C containing nitrogen as a heteroatom 6-30 Arylcarbonyl;

[0025] R 21 C 2-30 Alkyl, C 3-10 Cycloalkyl, C 2-30 Alkenyl, C 3-10 Cycloalkenyl, C 2-30 Carboxyalkyl, C 2-30 Alkyl carboxyl, C 3-30 Carboxyalkenyl, C 3-30 Alkenyl carboxyl, C 3-30 Alkylcarboxylalkyl, C 3-30 Alkyl carboxyl alkenyl, C 3-30 Alkenylcarboxyalkyl, C 4-30 alkenylcarboxylalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-10 Heterocycloalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkyl, C 2-30 Alkoxyalkyl, C 3-30 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkenyl, C1-20 Alcohol, C 1-20 Enol, C 2-30 Acyl, C 1-10 Amide, C 1-10 Amine or C 2-15 ester;

[0026] R 22 is hydrogen, hydroxyl, halogen or C 1-10 alkyl;

[0027] R 23 is hydrogen, hydroxyl or C 1-10 alkyl;

[0028] R 21 Can be used with R 22 and R 23 The carbons bonded together form double bonds;

[0029] R 23 Can be used with R 21 and R 22 The carbons bonded together form double bonds;

[0030] When R 21 or R 23 When forming a double bond with the carbon, R 22 contains no atoms; and

[0031] R3 and R4 are independently hydrogen or C 1-10 alkyl).

[0032] In another aspect of the present invention, the present invention provides a combination drug for cancer immunotherapy, comprising a cancer immunotherapy agent; and a cancer immunotherapy adjuvant.

[0033] In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing the efficacy of cancer immunotherapeutics, comprising a compound represented by Formula I, its isomers, solvates, hydrates, or pharmaceutically acceptable salts thereof as an active ingredient.

[0034] In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing immunity, comprising a compound represented by Formula 1, an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0035] In another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the steps of administering a cancer immunotherapeutic agent; and a cancer immunotherapeutic adjuvant to a subject in need thereof.

[0036] In another aspect of the present invention, the present invention provides a kit for anti-cancer treatment, comprising a cancer immunotherapeutic agent; and a cancer immunotherapeutic adjuvant as active ingredients.

[0037] Beneficial effects: The cancer immunotherapy adjuvant according to the present invention, when administered in combination with a cancer immunotherapy agent, activates the function of immune factors without causing side effects in vivo to exhibit the effect of a kit for enhancing the anti-cancer effect of the cancer immunotherapy agent, and can therefore be effectively used as a cancer immunotherapy adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1a is a schematic diagram showing the process from the preparation of an experimental animal model to the killing of the animal model.

[0039] Figure 1b is a graph showing changes in tumor size in mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0040] Figure 1c is a graph showing the survival rate of mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0041] Figure 2a These are photos of mice before tumor and spleen removal after injection of MC38 colorectal cancer cell line and drug administration.

[0042] Figure 2b are photographs of spleens removed from mice injected with the MC38 colorectal cancer cell line and administered drugs.

[0043] Figure 2c are photographs of tumors removed from mice injected with the MC38 colorectal cancer cell line and administered drugs.

[0044] Figure 2d is a graph showing changes in body weight of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0045] Figure 2e is a graph showing tumor weights of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0046] Figure 2f is a graph showing changes in spleen weight of mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0047] Figures 3a to 3c The results of FACS of tumors of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0048] Figure 3d It is a graph showing the level of immune factors in each drug administration group by CD45.2+ marker.

[0049] Figure 3e The CD4 + Diagram of T cell levels.

[0050] Figure 3f The CD8 + Diagram of T cell levels.

[0051] Figure 3g is a graph showing the level of natural killer cells in each drug administration group.

[0052] Figure 3h is a graph showing the level of regulatory T cells in each drug administration group.

[0053] Figure 4a The CD4 + Graph of T cell proliferation capacity (% of total).

[0054] Figure 4b The CD8 + Graph of T cell proliferation capacity (% of total).

[0055] Figure 4c It is a graph showing the proliferation ability of natural killer cells in each drug administration group (in % of total number).

[0056] Figure 4d The CD4 + Graph showing T cell proliferation capacity (measured in MFI (mean fluorescence intensity)).

[0057] Figure 4e The CD8 + Graph showing T cell proliferation capacity (measured in MFI (mean fluorescence intensity)).

[0058] Figure 4f It is a graph showing the proliferation ability of natural killer cells in each drug administration group (measured in MFI (mean fluorescence intensity)).

[0059] Figure 5a The CD4 + CD107a levels in T cells are shown.

[0060] Figure 5b The CD8 + Graph of CD107a levels in T cells.

[0061] Figure 6a The CD4 + A graph of TNFα levels in T cells.

[0062] Figure 6b The CD8 + A graph of TNFα levels in T cells.

[0063] Figure 7a The CD4 + A graph of IFNγ levels in T cells.

[0064] Figure 7b The CD8 + A graph of IFNγ levels in T cells.

[0065] Figure 8a and 8b The results of FACS of tumors of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0066] Figure 8c The CD4 + Graph of CD107a levels on T cells.

[0067] Figure 8d The CD4 + Graph of TNFα levels in T cells.

[0068] Figure 8e The CD4 + Graph of IFNγ levels in T cells.

[0069] Figure 8f The CD8 + Graph of CD107a levels on T cells.

[0070] Figure 8g The CD8 + Graph of TNFα levels in T cells.

[0071] Figure 8h The CD8 + Plot of IFNγ levels in T cells.

[0072] Figure 9a The fluorescence photographs show the expression level of intratumoral adhesion junctions in each drug administration group.

[0073] Figure 9b This is a graph showing fluorescence density by quantifying the expression level of intratumoral adherens junctions in each drug administration group.

[0074] Fig.10 These are fluorescent photographs showing the expression levels of PDL1 and CD3 in each drug administration group.

[0075] Fig.11aare photographs showing the results of RT-PCR of the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in each drug administration group.

[0076] Fig.11b are photographs showing the results of RT-PCR of CXCL9, iNOS, and Gapdh expressions in each drug administration group.

[0077] Fig.11c are photographs showing graphically the mRNA expression levels of each group by RT-PCR.

[0078] Fig.12a is a schematic diagram showing the process of CD4 / 8+ T, NK depletion, MC38 colorectal cancer cell line injection and drug administration.

[0079] Figure 12b The diagram confirms the results of CD4 / 8+T, NK depletion, MC38 colorectal cancer cell line injection and drug administration by flow cytometry, and that the depletion of immune cells proceeded smoothly.

[0080] Fig.12c The graph shows the survival rate of mice depleted of CD4 / 8+T and NK cells after injection of MC38 colorectal cancer cell line and drug administration, and confirms that the survival rate of mice in the experimental group depleted of CD8+T cells is the lowest.

[0081] Fig.12d is shown with Fig.12c The same process, showing the growth rate of the tumor over time according to CD4 / 8+ T cell depletion after MC38 colorectal cancer cell line injection and drug administration, confirms that the tumor growth rate is highest in the experimental group mice depleted of CD8+ T cells.

[0082] Fig.12e It is a graph showing comparison of tumor size according to CD4 / 8+T and NK depletion in each experimental group after MC38 colorectal cancer cell line injection and drug administration, and confirming that the tumor size is largest in the CD8+T cell-depleted experimental group mice.

[0083] Fig.13a is a schematic diagram showing the process of injection and long-term drug administration of MC38 colorectal cancer cell line.

[0084] Fig.13b is a graph showing the survival rate of mice injected with MC38 colorectal cancer cell line and subjected to long-term drug administration.

[0085] Fig.13c is a graph showing tumor size in mice injected with MC38 colorectal cancer cell line and subjected to chronic drug administration. DETAILED DESCRIPTION

[0086] Hereinafter, the present invention is described in detail.

[0087] Embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. It will be appreciated by those skilled in the art that embodiments of the present invention are provided in order to more accurately explain the present invention. In addition, "comprising" an element throughout the specification does not exclude other elements, but may include other elements, unless otherwise specifically stated.

[0088] In one aspect of the present invention, the present invention provides a cancer immunotherapy adjuvant.

[0089] Particularly, the present invention provides a cancer immunotherapy adjuvant comprising a compound represented by the following Formula 1, an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0090] [Formula 1]

[0091]

[0092] In formula 1,

[0093] X is oxygen or sulfur;

[0094] represents a single bond or a double bond;

[0095] R1 is hydrogen, halogen, C 1-30 Alkyl, C 3-10 Cycloalkyl, C 2-30 Alkenyl, C 3-10 Cycloalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-15 Heterocycloalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-15 Heterocycloalkyl, C 2-30 Alkoxyalkyl, C 3-30 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkenyl, C 1-20 Alcohol, C 1-20 Enol, C 2-30 Acyl, C 1-10 Amide, C 1-10 Amine, C 2-15 Esters, sulfates, carboxyl groups, C 3-20 Carboxyalkyl, C 3-20 Carboxyalkenyl, C 3-20 Alkyl carboxyl, C 3-20 Alkenyl carboxyl, C 3-20 Alkylcarboxylalkyl, C 3-20 Alkyl carboxyl alkenyl, C 3-20 Alkenylcarboxyalkyl, C 4-20 Alkenyl carboxyl alkenyl, C6-30 Aryl, C 6-30 Aralkyl, C 6-30 Alkyl, C 3-30 Heteroaryl or C containing nitrogen as a heteroatom 6-30 Arylcarbonyl;

[0096] R 21 C 2-30 Alkyl, C 3-10 Cycloalkyl, C 2-30 Alkenyl, C 3-10 Cycloalkenyl, C 2-30 Carboxyalkyl, C 2-30 Alkyl carboxyl, C 3-30 Carboxyalkenyl, C 3-30 Alkenyl carboxyl, C 3-30 Alkylcarboxylalkyl, C 3-30 Alkyl carboxyl alkenyl, C 3-30 Alkenylcarboxyalkyl, C 4-30 alkenylcarboxylalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-10 Heterocycloalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkyl, C 2-30 Alkoxyalkyl, C 3-30 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkenyl, C 1-20 Alcohol, C 1-20 Enol, C 2-30 Acyl, C 1-10 Amide, C 1-10 Amine or C 2-15 ester;

[0097] R 22 is hydrogen, hydroxyl, halogen or C 1-10 alkyl;

[0098] R 23 is hydrogen, hydroxyl or C 1-10 alkyl;

[0099] R 21 Can be used with R 22 and R 23 The carbons bonded together form double bonds;

[0100] R 23 Can be used with R 21 and R 22 The carbons bonded together form double bonds;

[0101] When R 21 or R 23 When forming a double bond with the carbon, R 22 contains no atoms; and

[0102] R3 and R4 are independently hydrogen or C 1-10 alkyl.

[0103] X in Formula 1 may be oxygen.

[0104] R1 in Formula 1 can be hydrogen, halogen, C 1-10 Alkyl, C 3-8 Cycloalkyl, C 2-10 Alkenyl, C 3-8 Cycloalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-8 Heterocycloalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-10 Heterocycloalkyl, C 2-20 Alkoxyalkyl, C 3-20 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatom 3-8 Heterocycloalkenyl, C 1-10 Alcohol, C 1-10 Enol, C 2-20 Acyl, C 1-10 Amide, C 1-5 Amine, C 2-15 Esters, sulfates, carboxyl groups, C 3-20 Carboxyalkyl, C 3-20 Carboxyalkenyl, C 3-20 Alkyl carboxyl, C 3-20 Alkenyl carboxyl, C 3-20 Alkylcarboxylalkyl, C 3-20 Alkyl carboxyl alkenyl, C 3-20 Alkenylcarboxyalkyl, C 4-20 Alkenyl carboxyl alkenyl, C 6-20 Aryl, C 6-20 Aralkyl, C 6-20 Alkyl, C 3-20 Heteroaryl or C containing nitrogen as a heteroatom 6-20 Arylcarbonyl.

[0105] R1 in Formula 1 can be hydrogen, C 1-10 Alkyl, C 3-8 Cycloalkyl, C 2-10 Alkenyl, C 3-8 Cycloalkenyl, C containing oxygen as heteroatom 2-8 Heterocycloalkyl, C containing oxygen as heteroatom 3-10 Heterocycloalkyl, C 2-20 Alkoxyalkyl, C 3-10 Alkoxyalkyl, C 3-8 Heterocycloalkenyl, C 1-10 Alcohol, C 1-10 Enol, C 1-10 Amide, C 1-5 Amine, C 2-15Esters, sulfates, carboxyl groups, C 3-20 Carboxyalkyl, C 3-20 Carboxyalkenyl, C 3-20 Alkyl carboxyl, C 3-20 Alkenyl carboxyl, C 3-20 Alkylcarboxylalkyl, C 3-20 Alkyl carboxyl alkenyl, C 3-20 Alkenylcarboxyalkyl, C 4-20 Alkenyl carboxyl alkenyl, C 6-20 Aryl, C 6-20 Aralkyl, C 6-20 Alkyl, C 3-20 Heteroaryl or C containing nitrogen as a heteroatom 6-20 Arylcarbonyl.

[0106] In R1 of Formula 1, the cycloalkyl or heterocycloalkyl group may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, C 6-20 Aryl, C 7-20 Arylcarboxyl or its combination substitution; C 3-10 The cycloalkenyl or heterocycloalkenyl group may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 2-8 Alkyl carboxyl, C 3-8 Alkylcarboxylalkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, C 6-20 Aryl, C 7-20 Aryl carboxyl or a combination thereof; the aryl group may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, nitro, C 2-8 Alkylcarboxylamino or a combination thereof; Arylalkyl may be substituted with hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, nitro, C 2-8 Alkylcarboxylamino or a combination thereof; alkylaryl may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, nitro, C 2-8 Alkylcarboxylamino or a combination thereof; arylcarbonyl may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C1-5 Alkoxy, C 2-8 Alkoxyalkyl, nitro, C 2-8 Alkylcarboxylamino or a combination thereof; and the heteroaryl group may be substituted by hydroxyl, halogen, C 1-5 Alkyl, C 1-5 Alcohol, C 1-5 Alkoxy, C 2-8 Alkoxyalkyl, nitro, C 2-8 Alkylcarboxylamino or a combination thereof.

[0107] R in Formula 1 21 It can be a straight chain or branched C 2-15 Alkyl, C 3-10 Cycloalkyl, C 2-15 Alkenyl, C 3-10 Cycloalkenyl, C 2-15 Carboxyalkyl, C 2-15 Alkyl carboxyl, C 3-15 Carboxyalkenyl, C 2-15 Alkenyl carboxyl, C 3-15 Alkylcarboxyalkyl, C 3-15 Alkylcarboxyalkenyl, C 3-15 Alkenylcarboxyalkyl, C 2-30 Alkenylcarboxyalkenyl, C containing oxygen, sulfur or nitrogen as heteroatoms 2-10 Heterocycloalkyl, containing oxygen, sulfur or nitrogen as heteroatoms 3-10 Heterocycloalkyl, C 2-20 Alkoxyalkyl, C 3-30 Alkoxyalkyl, C containing oxygen, sulfur or nitrogen as heteroatoms 3-10 Heterocycloalkenyl, C 1-20 Alcohol, C 1-20 Enol, C 2-30 Acyl, C 1-10 Amide, C 1-10 Amine or C 2-15 ester.

[0108] R in Formula 1 23 C 1-5 Alkyl or can be with R 21 and R 22 The carbons bonded together form double bonds.

[0109] In formula 1 It can be a double bond.

[0110] Examples of the compound represented by Formula 1 according to the present invention include a compound represented by the following Formula 2:

[0111] [Formula 2]

[0112]

[0113] The compound represented by Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, wherein the salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt herein can be obtained from an inorganic acid, a non-toxic organic acid and an organic acid, the inorganic acid being for example hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid; the non-toxic organic acid being for example aliphatic monocarboxylates and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkandioates, aromatic acids and aliphatic and aromatic sulfonic acids; and the organic acid being for example trifluoroacetic acid, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid and fumaric acid, etc. Examples of pharmaceutically nontoxic salts are sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates , hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.

[0114] The acid addition salt according to the present invention can be prepared by conventional methods known to those skilled in the art. For example, the derivative represented by Formula 1 is dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane and acetonitrile, and an organic acid or an inorganic acid is added thereto to produce a precipitate. Then, the precipitate is filtered and dried to obtain a salt. Or the solvent and excess acid are distilled under reduced pressure, and dried to obtain a salt. Or the precipitate is crystallized in an organic solvent to obtain a salt.

[0115] Pharmaceutically acceptable metal salts can be prepared by using a base. Alkali metal or alkaline earth metal salts are obtained by the following process: dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution; filtering the insoluble compound salt; evaporating the remaining solution and drying it. In this case, the metal salt is preferably prepared in the form of a pharmaceutically suitable sodium salt, potassium salt or calcium salt. The corresponding silver salt is prepared by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (such as silver nitrate).

[0116] Furthermore, the present invention includes not only the compound represented by Formula 1, but also pharmaceutically acceptable salts thereof, and solvates, optical isomers, or hydrates that may be prepared therefrom.

[0117] The term "hydrate" refers to a compound of the present invention or a salt thereof comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate of the compound represented by Formula 1 of the present invention may include a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate may contain more than 1 equivalent of water, preferably 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by Formula 1 of the present invention, its isomer, or its pharmaceutically acceptable salt from water or an aqueous solvent.

[0118] The term "solvate" refers to a compound of the invention or a salt thereof that includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefore include those that are volatile, non-toxic and / or suitable for human administration.

[0119] The term "isomer" refers to a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but different structures or spaces. Such isomers include structural isomers such as tautomers, stereoisomers such as geometric isomers (trans, cis) and optical isomers (enantiomers). All of these isomers and mixtures thereof are also included within the scope of the present invention.

[0120] In the cancer immunotherapy adjuvant according to the present invention, the compound represented by Formula 1 or its pharmaceutically acceptable salt can be administered orally or parenterally in various dosage forms during clinical administration. More preferably, they can be parenteral preparations. The compound represented by Formula 1 or its pharmaceutically acceptable salt can be prepared for oral or parenteral administration by mixing with commonly used diluents or adjuvants (such as fillers, extenders, adhesives, wetting agents, disintegrants and surfactants). Oral solid preparations include tablets, pills, powders, granules and capsules. These solid preparations are prepared by mixing the compound represented by Formula 1 of the present invention or its pharmaceutically acceptable salt with one or more suitable adjuvants (such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.). In addition to simple adjuvants, lubricants such as magnesium stearate, talc, etc. can also be used. Liquid preparations for oral administration are suspensions, solutions, emulsions and syrups, and in addition to commonly used simple diluents (such as water and liquid paraffin), the above-mentioned preparations can also contain a variety of adjuvants, such as wetting agents, sweeteners, aromatics and preservatives. Preparations for parenteral administration are sterile aqueous solutions, water-insoluble excipients, suspensions and emulsions. In addition to the active compound, water-insoluble excipients and suspensions may also contain propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), injectable esters (eg ethyl oleate) and the like.

[0121] The cancer immunotherapy adjuvant including the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0122] In order to prepare a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as a preparation for parenteral administration, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or a buffer in water to produce a solution or a suspension, which is then formulated into an ampoule or a vial. The composition herein may be sterilized, and may additionally contain a preservative, a stabilizer, a wettable powder or an emulsifier, a salt for adjusting osmotic pressure and / or a buffer, and other therapeutically useful substances, and the composition may be formulated by conventional mixing, granulation or coating methods.

[0123] Orally administered preparations such as tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, lozenges, etc. In addition to active ingredients, these preparations may include diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (e.g., silicon dioxide, talc, stearate and its magnesium salt or calcium salt, and / or polyethylene glycol). Tablets may include binding agents such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone, and if necessary, disintegrants (e.g., starch, agarose, alginic acid or its sodium salt) or azeotropic mixtures and / or absorbents, coloring agents, flavoring agents and sweeteners may be additionally included therein.

[0124] Cancer immunotherapy adjuvants can enhance the efficacy of cancer immunotherapy agents, more specifically, they can enhance the efficacy of cancer immunotherapy agents by activating immune factors to assist the anti-cancer activity of cancer immunotherapy agents. The immune factors can be at least one selected from helper T cells, cytotoxic T cells, natural killer cells (NK cells) and cytokines.

[0125] The cancer immunotherapy adjuvant can be administered simultaneously or sequentially with the cancer immunotherapy agent. When administered sequentially, the cancer immunotherapy adjuvant can be administered after the cancer immunotherapy agent is administered, or the cancer immunotherapy adjuvant can be administered after the cancer immunotherapy agent is administered. However, this method of administration is only an example, and the method of administration can be changed to enhance the anti-cancer immune effect. In one embodiment of the present invention, the cancer immunotherapy adjuvant is administered by intravenous injection every day, and the cancer immunotherapy agent is administered 3 times per week by intraperitoneal injection, but is not always limited thereto.

[0126] Cancer immunotherapy adjuvants can activate one or more immune factors selected from helper T cells, cytotoxic T cells, natural killer cells (NK cells) and cytokines. Cancer immunotherapy adjuvants show the effect of enhancing the anti-cancer effect of cancer immunotherapy agents by activating immune factors.

[0127] At this time, the cancer immunotherapy adjuvant can be used to prevent or treat cancer by being administered in combination with a cancer immunotherapy agent.

[0128] The cancer may be at least one selected from the following: pseudomyxoma, intrahepatic bile duct cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Watt cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, nasal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, juvenile brain cancer, juvenile lymphoma, juvenile leukemia, small intestine cancer, meningioma, esophageal cancer, glioma , renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureter cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric cancer, gastrointestinal stromal cancer, Wilms cancer, breast cancer, triple-negative breast cancer, sarcoma, penile cancer, pharyngeal cancer, choriocarcinoma of pregnancy, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal cancer, vaginal cancer, spinal cord cancer, vestibular schwannoma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer and thymus cancer.

[0129] Cancer immunotherapy adjuvants can be co-administered with conventionally known and well-known cancer immunotherapeutics of those skilled in the art without limitation. For example, cancer immunotherapy adjuvants can be co-administered with one or more cancer immunotherapeutics, the cancer immunotherapeutics selected from: anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, anti-VISTA, anti-BTLA, anti-TIM3, anti-HVEM, anti-CD27, anti-CD137, anti-OX40, anti-CD28, anti-PDL2, anti-GITR, anti-ICOS, anti-SIRPα, anti-ILT2, anti-ILT3, anti-ILT4, anti-ILT5, anti-EGFR, anti-CD19 and anti-TIGIT, but not always limited thereto.

[0130] In another aspect of the present invention, the present invention provides a combination drug for cancer immunotherapy.

[0131] In particular, the present invention provides a combination drug for cancer immunotherapy, which comprises a cancer immunotherapy agent and a cancer immunotherapy adjuvant.

[0132] The specific descriptions of the cancer immunotherapy agent, cancer immunotherapy adjuvant and combination drug are the same as the specific description of the cancer immunotherapy adjuvant.

[0133] In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing the efficacy of cancer immunotherapeutics.

[0134] In particular, the present invention provides a pharmaceutical composition for enhancing the efficacy of a cancer immunotherapeutic agent, comprising a compound represented by Formula 1, an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0135] [Formula 1]

[0136]

[0137] In Formula 1, the specific descriptions of R1 to R4 and X are the same as those of Formula 1 in the cancer immunotherapy adjuvant.

[0138] In addition, the specific description of the pharmaceutical composition for enhancing the efficacy of cancer immunotherapy is the same as the specific description of the cancer immunotherapy adjuvant.

[0139] In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing immunity.

[0140] In particular, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises a compound represented by Formula 1, an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0141] [Formula 1]

[0142]

[0143] In Formula 1, the specific descriptions of R1 to R4 and X are the same as those of Formula 1 in the cancer immunotherapy adjuvant.

[0144] In addition, the specific description of the pharmaceutical composition for enhancing immunity is the same as the specific description of the cancer immunotherapy adjuvant.

[0145] In another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the step of administering a cancer immunotherapeutic agent and a cancer immunotherapeutic adjuvant to a subject in need thereof.

[0146] The cancer immunotherapy adjuvant and the cancer immunotherapy agent can be administered in combination or at different times.

[0147] In another aspect, the present invention provides uses of cancer immunotherapy adjuvants and immunotherapeutic agents in preventing or treating cancer.

[0148] In another aspect of the present invention, the present invention provides a combination therapy for treating cancer, comprising the step of administering a cancer immunotherapy adjuvant and a cancer immunotherapy agent to a subject in need thereof.

[0149] In another aspect of the present invention, the present invention provides a kit for preventing or treating cancer, comprising a cancer immunotherapeutic agent and a cancer immunotherapeutic adjuvant as active ingredients.

[0150] Hereinafter, the present invention will be described in detail through the following Examples and Experimental Examples.

[0151] However, the following embodiments and experimental examples are only used to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0152] Example 1: Preparation of cancer immunotherapy adjuvant (SAC-1004)

[0153] [Formula 2]

[0154]

[0155] Based on Korean Patent Publication No. 10-2011-0047170, the compound SAC-1004 represented by Formula 2, ie, the pharmaceutical composition for co-administration with a cancer immunotherapeutic agent of the present invention, was prepared according to the following Reaction Formula 1.

[0156] [Reaction 1]

[0157]

[0158] 13.4 mg of SAC-1003 was dissolved in 1 mL of tetrahydrofuran, and 26 mg of tri-O-acetyl-D-glucal (Aldrich) and 0.012 mL of boron trifluoride ether complex (Aldrich) were added thereto under an argon atmosphere, and then stirred at 0°C for 10 hours. After the reaction solution was warmed to room temperature, 5 mL of ether was added for dilution, washed with sodium bicarbonate aqueous solution, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography using a mixed eluent of ethyl acetate / hexane (1:10) to obtain the target compound SAC-1004 (11 mg, 56%): 1 H-NMR(300MHz, CDCl3)δ5.89-5.80(m,2H),5.37-5.27(m,2H),5.17-5.14(m,2H),4 .23-4.16(m,3H),3.66(s,3H),3.56(m,1H),2.38-2.28(m,4H),2.17-0.53(m,37H).

[0159] <Experimental methods>

[0160] 1. Preparation of experimental animal models

[0161] MC-38 colorectal cancer cells (5×10 5 MC-38 cells) were injected subcutaneously into 7-week-old C57BL / 6 male mice. Seven days after colorectal cancer cell injection, the average tumor volume of MC-38 subcutaneous tumors was about 40 mm 3 The above-mentioned injection method of mouse tumors was also applied to all four groups below. The experimental groups were then divided into a vehicle administration control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 single administration group, and an anti-PD1 single administration group, and the experiment was performed.

[0162] Figure 1a is a schematic diagram showing the process from the preparation of an experimental animal model to the killing of the animal model.

[0163] Specifically, the administration was performed as follows. SAC-1004 (vascular leakage inhibitor) was dissolved in DMSO in PBS, and 1 mg was administered per kg of mouse body weight per day by intravenous injection. Anti-PD1 (immune checkpoint inhibitor) and 200 ng rat IgG2a were administered 3 times per week by intraperitoneal injection at a concentration of 200 ng / mouse. As described above, each group was administered SAC-1004 and anti-PD1 for 7 days, and tumors and spleens were removed on the 8th day.

[0164] 2. RNA isolation, cDNA synthesis and PCR analysis

[0165] Total RNA was isolated from the extracted tumor tissue using Trizol reagent. The concentration of the isolated RNA was determined by measuring the absorbance at 260 / 280 nm using nano-drop (ND-1000, Thermo Fisher Scientific), and cDNA was synthesized by including 2 μg of RNA and making the total volume 20 μl.

[0166] 3. Fluorescence Activated Cell Sorting (FACS) of Mouse Tumors

[0167] After separating cells from tumor tissue using collagenase, culture medium was added to the cells, centrifuged, and the precipitated cells were collected and counted. Live cells were stained at 37°C for about 30 minutes. After staining only live cells, they were washed three times with PBS. Calibration was performed by live / dead cell staining. In addition, FACS was performed using Canto. In this way, the activity ratio of T cells can be compared.

[0168] T cells participate in adaptive immune responses and are divided into helper T cells (CD4 + T cells), cytotoxic T cells (CD8 + T cells), and CD4 + T cells but suppress regulatory T cells (Treg cells) of cytotoxic T cells. In addition, there are natural killer cells (NK cells) with cytotoxicity in the innate immune response, so the levels of these cells in each group were measured.

[0169] CD4 antibody was used to measure CD4 + CD8 antibody is used as a marker for T cells (which are helper T cells). + T cells (which are cytotoxic T cells). In addition, CD25 antibody and Foxp 3 The antibody was used to measure regulatory T cells, and the NK1.1 antibody was used to measure natural killer cells.

[0170] 4. IHC staining

[0171] Tumor tissues were immediately removed and then incubated in 4% PFA O / N (stored at 4°C). Tumor tissues were sequentially immersed in 15% to 30% sucrose until sinking, and then OCT samples were taken on dry ice. 20 μm sections were stained with primary and secondary antibodies respectively.

[0172] 5. T cell function analysis

[0173] T cell function is measured in two main ways. One method is the cytokine production capacity analysis, which measures the secretion of pro-inflammatory cytokines IFNγ or TNFα in T cells. Another method is the method of measuring cytotoxicity, in which perforin or granzyme B is inserted into tumor cells to directly connect to and kill tumor cells.

[0174] Experimental Example 1: Analysis of changes in tumor size in mice injected with MC38 colorectal cancer cell line and administered with drugs

[0175] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the changes in tumor size according to the above-mentioned drug administration for 7 days in the experimental animal model were analyzed.

[0176] Figure 1b is a graph showing changes in tumor size in mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0177] Figure 2a These are photos of mice before tumor and spleen removal after injection of MC38 colorectal cancer cell line and drug administration.

[0178] Figure 2c are photographs of tumors removed from mice injected with the MC38 colorectal cancer cell line and administered drugs.

[0179] In particular, the tumor size of the anti-PD1 single administration group and the SAC-1004 single administration group was reduced compared with the control group, and the tumor size of the SAC-1004 and anti-PD1 co-administration group was reduced compared with the single administration group. The results were statistically tested by two-way analysis of variance.

[0180] The above results indicate that the preventive or therapeutic effect on cancer is significantly increased when SAC-1004 and an immunotherapeutic agent are administered in combination rather than alone.

[0181] Experimental Example 2: Analysis of the survival rate of mice injected with MC38 colorectal cancer cell line and administered drugs

[0182] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the survival rate of mice according to the above drug administration for 7 days in the experimental animal model was analyzed.

[0183] Figure 1c is a graph showing the survival rate of mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0184] In particular, the survival rate of mice according to drug administration in the anti-PD1 single administration group was higher than that in the control group or the SAC-1004 single administration group, and the survival rate of mice in the SAC-1004 and anti-PD1 co-administration group was higher than that in the anti-PD1 single administration group. The results were statistically tested by two-way analysis of variance.

[0185] The above results indicate that the preventive or therapeutic effect on cancer is significantly increased when SAC-1004 and an immunotherapeutic agent are administered in combination rather than alone.

[0186] Experimental Example 3: Analysis of body weight changes in mice injected with MC38 colorectal cancer cell line and administered drugs

[0187] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, changes in body weight of mice administered the above drugs for 7 days in the experimental animal model were analyzed.

[0188] Figure 2d is a graph showing changes in body weight of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0189] In particular, body weights measured were similar in all groups, indicating that no abnormalities were observed in the mice even after long-term drug administration.

[0190] The above results indicate that the pharmaceutical composition for co-administration of the present invention does not produce side effects in vivo even when administered alone or in combination, and is a material with guaranteed safety.

[0191] Experimental Example 4: Comparison of spleen size and weight of mice injected with MC38 colorectal cancer cell line and administered drugs

[0192] The spleen sizes of mice treated with the above drugs for 7 days in the experimental animal model were analyzed in a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group.

[0193] Figure 2b are photographs of spleens removed from mice injected with the MC38 colorectal cancer cell line and administered drugs.

[0194] Figure 2f is a graph showing changes in spleen weight of mice injected with MC38 colorectal cancer cell line and administered with drugs.

[0195] In particular, the spleen weight of the mice in the SAC-1004 alone administration group was higher than that of the control group mice, the spleen weight of the mice in the anti-PD1 alone administration group was higher than that of the SAC-1004 alone administration group mice, and the spleen weight of the mice in the SAC-1004 and anti-PD1 co-administration group was higher than that of the anti-PD1 alone administration group mice. However, this was not statistically significant when verified by one-way ANOVA.

[0196] The above results indicate that the pharmaceutical composition for co-administration of the present invention does not produce side effects in vivo even when administered alone or in combination, and is a material with guaranteed safety.

[0197] Experimental Example 5: Comparison of tumor weights in mice injected with MC38 colorectal cancer cell line and administered drugs

[0198] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the tumor weights of mice treated with the above drugs for 7 days in the experimental animal model were analyzed.

[0199] Figure 2e is a graph showing tumor weights of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0200] In particular, the tumor weight of mice in the anti-PD1 single administration group was lower than that of mice in the control group, the tumor weight of mice in the SAC-1004 single administration group was lower than that of mice in the anti-PD1 single administration group, and the tumor weight of mice in the SAC-1004 and anti-PD1 co-administration group was lower than that of mice in the SAC-1004 single administration group. The results were statistically tested by one-way analysis of variance.

[0201] The above results indicate that the preventive or therapeutic effect on cancer is significantly increased when SAC-1004 and a cancer immunotherapeutic agent are administered in combination rather than alone.

[0202] Experimental Example 6: FACS analysis of mouse tumors

[0203] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the tumors of mice treated with the above-mentioned drugs for 7 days in the experimental animal model were subjected to FACS (fluorescence-activated cell sorting) analysis, and the activity ratio of T cells in each drug administration group was compared.

[0204] CD4 antibody is used to measure CD4 + T cells, the CD4 + T cells are helper T cells, and CD8 antibodies are used to measure CD8 + T cells, the CD8 + T cells are cytotoxic T cells. In addition, CD25 antibody and Foxp 3 The antibody was used to measure regulatory T cells, and the NK1.1 antibody was used to measure natural killer cells.

[0205] Figures 3a to 3c The results of FACS of tumors of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0206] Figure 3d It is a graph showing the level of immune factors in each drug administration group by CD45.2+ marker.

[0207] Figure 3e The CD4 + Diagram of T cell levels.

[0208] Figure 3f The CD8 + Diagram of T cell levels.

[0209] Figure 3g is a graph showing the level of natural killer cells in each drug administration group.

[0210] Figure 3h is a graph showing the level of regulatory T cells in each drug administration group.

[0211] In particular, in the SAC-1004 and anti-PD1 co-administration group, the levels of cytotoxic T cells and helper T cells were significantly high, the level of natural killer cells was also high, but the level of regulatory T cells that inhibit cytotoxic T cells was reduced. The results were statistically tested by one-way analysis of variance.

[0212] Therefore, it was confirmed that when SAC-1004 and a cancer immunotherapeutic agent were administered in combination, the effect of activating T cells was greater than that of either administration alone.

[0213] The above results show that when SAC-1004 and cancer immunotherapeutics are administered in combination, the effect of activating immune-related factor T cells is better than when administered alone. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutics by activating immune factors.

[0214] Experimental Example 7: Analysis of the proliferation capacity of immune factors in mouse tumors

[0215] In a total of 4 groups consisting of a control group, a group co-administered with SAC-1004 and anti-PD1, a group administered with SAC-1004 alone, and a group administered with anti-PD1 alone, the proliferation capacity of immune factors in mouse tumors administered with the above drugs for 7 days in an experimental animal model was analyzed. To measure the proliferation capacity, the proliferation marker Ki67 was used.

[0216] Figure 4a The CD4 + Graph of T cell proliferation capacity (% of total).

[0217] Figure 4b The CD8 +Graph of T cell proliferation capacity (% of total).

[0218] Figure 4c It is a graph showing the proliferation ability of natural killer cells in each drug administration group (in % of total number).

[0219] Figure 4d The CD4 + Graph showing T cell proliferation capacity (measured in MFI (mean fluorescence intensity)).

[0220] Figure 4e The CD8 + Graph showing T cell proliferation capacity (measured in MFI (mean fluorescence intensity)).

[0221] Figure 4f It is a graph showing the proliferation ability of natural killer cells in each drug administration group (measured in MFI (mean fluorescence intensity)).

[0222] In particular, the proliferation capacity of helper T cells, cytotoxic T cells, and natural killer cells in the SAC-1004 and anti-PD1 co-administration group was higher than that in the other groups.

[0223] The above results show that when SAC-1004 is administered in combination with a cancer immunotherapeutic agent, the effect of enhancing the proliferation ability of immune-related factors is better than when SAC-1004 and the cancer immunotherapeutic agent are administered alone. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutic agents by activating immune factors.

[0224] Experimental Example 8: Analysis of T cell cytotoxicity in mouse tumors

[0225] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the cytotoxicity of T cells in mouse tumors to which the above drugs were administered for 7 days in an experimental animal model was analyzed.

[0226] To measure cytotoxicity, NK cell degranulation and CD8 + CD107a, a marker for T cell activation, was used and treated with peptides and PMA / ionomycin to activate T cells. In the peptide-treated group, peptides expressed in tumors were added in vitro. In the PMA / ionomycin-treated group, all T cells were activated by stimulating T cell calcium signals.

[0227] Figure 5a The CD4 + CD107a levels in T cells are shown.

[0228] Figure 5b The CD8 + Graph of CD107a levels in T cells.

[0229] In particular, in the group not treated with peptide and PMA / ionomycin, the level of CD107a in T cells was low, and the level of CD107a in T cells in the PMA / ionomycin treated group was higher than that in the peptide treated group. In particular, when treated with PMA / ionomycin, the level of CD107a in CD8+T cells in the SAC-1004 and anti-PD1 co-administered group was high.

[0230] Although the activation of CD8+T cells increased relatively, the above results were not significant in all groups. When SAC-1004 and cancer immunotherapeutic agents were administered in combination, the cytotoxic effect of activated T cells to kill cancer cells was better than when administered alone. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect by co-administration with cancer immunotherapeutic agents.

[0231] Experimental Example 9: Analysis of cytokine production capacity of T cells in mouse tumors

[0232] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the cytokine production capacity of T cells in mouse tumors to which the above-mentioned drugs were administered for 7 days in the experimental animal model was analyzed.

[0233] (9-1)

[0234] To measure the cytokine production capacity, the levels of proinflammatory cytokines can be compared, and the proinflammatory cytokine marker TNFα can be used. As in Experimental Example 8, the level of TNFα was measured after treatment with peptides and PMA / ionomycin to activate T cells.

[0235] Figure 6a The CD4 + A graph of TNFα levels in T cells.

[0236] Figure 6b The CD8 + A graph of TNFα levels in T cells.

[0237] In particular, when the peptide and PMA / ionomycin were not treated, there was no significant difference in the TNFα level among the drug-administered groups, but when the peptide and PMA / ionomycin were treated, the TNFα level in the SAC-1004 and anti-PD1 co-administered group was significantly increased.

[0238] The above results show that when SAC-1004 and cancer immunotherapeutics are administered in combination rather than alone, the effect of activating immune-related factor T cells is superior. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutics by activating immune factors.

[0239] (9-2)

[0240] IFNγ is also a pro-inflammatory cytokine marker. Similarly, IFNγ levels were measured after treatment with peptides and PMA / ionomycin to activate T cells.

[0241] Figure 7a The CD4 + A graph of IFNγ levels in T cells.

[0242] Figure 7b The CD8 + A graph of IFNγ levels in T cells.

[0243] In particular, when the peptide and PMA / ionomycin were not treated, there was no significant difference in the IFNγ level of each drug administration group, but when the peptide and PMA / ionomycin were treated, the IFNγ level of the SAC-1004 and anti-PD1 co-administration group was significantly increased.

[0244] The above results show that when SAC-1004 and cancer immunotherapeutics are administered in combination rather than alone, the effect of activating immune-related factor T cells is superior. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutics by activating immune factors.

[0245] Experimental Example 10: Analysis of cytokine production capacity of T cells in mouse spleen

[0246] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 single administration group, and an anti-PD1 single administration group, FACS analysis, cytotoxicity analysis, and cytokine production capacity analysis were performed using CD107a, TNFα, and IFNγ markers to compare the function of T cells in the spleen of mice treated with the above drugs for 7 days in the experimental animal model. In addition, as in Experimental Examples 8 and 9, treatment with peptides and PMA / ionomycin was performed to activate T cells.

[0247] Figure 8a and 8b The results of FACS of tumors of mice injected with MC38 colorectal cancer cell line and administered drugs.

[0248] Figure 8cThe CD4 + Graph of CD107a levels on T cells.

[0249] Figure 8d The CD4 + Graph of TNFα levels in T cells.

[0250] Figure 8e The CD4 + Plot of IFNγ levels in T cells.

[0251] Figure 8f The CD8 + Graph of CD107a levels on T cells.

[0252] Figure 8g The CD8 + Graph of TNFα levels in T cells.

[0253] Figure 8h The CD8 + Plot of IFNγ levels in T cells.

[0254] In particular, when CD4 + T cells and CD8 + Upon T cell activation, the levels of CD107a, TNFα, and IFNγ increased in the SAC-1004 and anti-PD1 co-administration group.

[0255] The above results show that when SAC-1004 and cancer immunotherapeutic agents are administered in combination rather than alone, the cytotoxic effect of killing cancer cells is superior and the effect of activating immune-related factor T cells is also superior. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutic agents by activating immune factors.

[0256] Experimental Example 11: Analysis of adhesion junction expression in mouse tumors

[0257] The expression of adhesion junctions in the tumors of mice treated with the above drugs in the experimental animal model was analyzed in a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, except that the drugs were administered for 14 days, by daily administration of SAC-1004 for 7 days and by administration of anti-PD1 3 times a week.

[0258] Figure 9aThe fluorescence photographs show the expression level of intratumoral adhesion junctions in each drug administration group.

[0259] Figure 9b This is a graph showing fluorescence density by quantifying the expression level of intratumoral adherens junctions in each drug administration group.

[0260] In particular, Figure 9a The results of IHC staining are shown, DAPI is used for nucleus staining, CD31 is used for blood vessel staining, and VE-cadherin (adhesion protein) is used to observe adhesion junctions. At this time, if VE-cadherin is well expressed, drug delivery is good and anticancer effect is enhanced, and endothelial cell adhesion is good, blood vessels are stable, and T cells infiltrate the tumor well.

[0261] like Figure 9a and Figure 9b As shown, the proportion of intratumoral VE-cadherin in the SAC-1004 alone administration group was higher than that in the control group and the anti-PD1 alone administration group, and the expression of intratumoral VE-cadherin in the SAC-1004 and anti-PD1 co-administration group was higher than that in the SAC-1004 alone administration group.

[0262] The above results show that when SAC-1004 and a cancer immunotherapeutic agent are administered in combination rather than alone, the cancer immunotherapeutic agent is well delivered, T cells penetrate well into the tumor, and the anticancer effect is enhanced. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anticancer effect of a cancer immunotherapeutic agent.

[0263] Experimental Example 12: Analysis of PDL1 expression in mouse tumors

[0264] In a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group, the expression of PDL1 (programmed death ligand 1) in mouse tumors to which the above drugs were administered for 7 days in an experimental animal model was analyzed. PDL1 is a ligand expressed in tumors, and CD3 is used as a T cell marker.

[0265] Fig.10 These are fluorescent photographs showing the expression levels of PDL1 and CD3 in each drug administration group.

[0266] In particular, Fig.10 The results of IHC staining are shown, and the increase in PDL1 indicates that anti-PD1 is well delivered and immune activation occurs. The expression levels of PDL1 and CD3 in the SAC-1004 and anti-PD1 co-administration group were higher than those in the anti-PD1 single administration group.

[0267] The above results show that when SAC-1004 and a cancer immunotherapeutic agent are administered in combination rather than alone, the cancer immunotherapeutic agent is well delivered, immune activation occurs well, and the anti-cancer effect is enhanced. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of a cancer immunotherapeutic agent.

[0268] Experimental Example 13: Analysis of cytokine expression levels in mouse tumors

[0269] The expression levels of cytokines in the tumors of mice to which the above drugs were administered for 7 days in the experimental animal model were analyzed in a total of 4 groups consisting of a control group, a group co-administered with SAC-1004 and anti-PD1, a group administered with SAC-1004 alone, and a group administered with anti-PD1 alone. Pro-inflammatory cytokines and anti-inflammatory cytokines were used as cytokine markers, and RT-PCR was performed.

[0270] Fig.11a are photographs showing the results of RT-PCR of the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in each drug administration group.

[0271] Fig.11b are photographs showing the results of RT-PCR of CXCL9, iNOS, and Gapdh expressions in each drug administration group.

[0272] Fig.11c are photographs showing graphically the mRNA expression levels of each group by RT-PCR.

[0273] In particular, among the proinflammatory cytokines, IFNγ increased in the anti-PD1 administration group, and its increase in the SAC-1004 and anti-PD1 co-administration group was greater than that in the anti-PD1 administration group.

[0274] The above results show that when SAC-1004 and cancer immunotherapeutics are administered in combination rather than alone, the effect of activating immune-related factor T cells is superior. The above results confirm that the pharmaceutical composition for co-administration of the present invention can enhance the anti-cancer effect of cancer immunotherapeutics by activating immune factors.

[0275] Experimental Example 14: Growth of MC38 colorectal cancer cell line and CD4 / 8 expression during drug injection + Analysis of survival rate of mice after drug administration after T and NK cell depletion

[0276] In the four groups consisting of the control group, the group co-administered with SAC-1004 and anti-PD1, the group administered with SAC-1004 alone, and the group administered with anti-PD1 alone, the expression of CD4 / 8 + After T and NK cell depletion, the survival rate of mice treated with the above drugs for 7 days in the experimental animal model was analyzed.

[0277] Fig.12a is a schematic diagram showing the process of CD4 / 8+ T, NK depletion, MC38 colorectal cancer cell line injection and drug administration.

[0278] Figure 12b The diagram confirms the results of CD4 / 8+T, NK depletion, MC38 colorectal cancer cell line injection and drug administration by flow cytometry, and that the depletion of immune cells proceeded smoothly.

[0279] Fig.12c The graph shows the survival rate of mice depleted of CD4 / 8+T and NK cells after injection of MC38 colorectal cancer cell line and drug administration, and confirms that the survival rate of mice in the experimental group depleted of CD8+T cells is the lowest.

[0280] Fig.12d is shown with Fig.12c The same process, showing the growth rate of the tumor over time according to CD4 / 8+ T cell depletion after MC38 colorectal cancer cell line injection and drug administration, confirms that the tumor growth rate is highest in the experimental group mice depleted of CD8+ T cells.

[0281] Fig.12e It is a graph showing comparison of tumor size according to CD4 / 8+T and NK depletion in each experimental group after MC38 colorectal cancer cell line injection and drug administration, and confirming that the tumor size is largest in the CD8+T cell-depleted experimental group mice.

[0282] The above results indicate that the removal of CD8 + T cells showed the fastest tumor growth rate, and depletion of NK cells and CD4 + The experimental group with T cells showed the fastest tumor growth rate. This suggests that co-administration of SAC-1004 and anti-PD1 is a CD8 + T cell-dependent induction process.

[0283] Experimental Example 15: Analysis of results of long-term drug administration

[0284] The survival rate after long-term drug injection was analyzed in a total of 4 groups consisting of a control group, a SAC-1004 and anti-PD1 co-administration group, a SAC-1004 alone administration group, and an anti-PD1 alone administration group.

[0285] Fig.13a is a schematic diagram showing the process of injection and long-term drug administration of MC38 colorectal cancer cell line.

[0286] Fig.13b is a graph showing the survival rate of mice injected with MC38 colorectal cancer cell line and subjected to long-term drug administration.

[0287] Fig.13c is a graph showing tumor size in mice injected with MC38 colorectal cancer cell line and subjected to chronic drug administration.

[0288] Even after long-term drug treatment, the same results were consistently shown, thus confirming the reliability of the above experimental procedures and demonstrating the absence of drug toxicity despite long-term drug administration.

[0289] Preparation Example 1: Preparation of powder

[0290] Compound 2g represented by Formula 1

[0291] Lactose 1g

[0292] All the above ingredients are mixed into powder according to the conventional method for preparing powder, and the powder is packed into a sealed package.

[0293] Preparation Example 2: Preparation of tablets

[0294]

[0295] All the above combinations are mixed and prepared into tablets by a conventional method for preparing tablets.

[0296] Preparation Example 3: Preparation of capsules

[0297]

[0298] All the above ingredients are mixed to form capsules according to a conventional method for preparing capsules, and the capsules are filled into gelatin capsules.

[0299] Preparation Example 4: Preparation of injection

[0300]

[0301] All the above components are mixed to prepare an injection by a conventional method for preparing an injection.

[0302] Preparation Example 5: Preparation of Ointment

[0303]

[0304] All the above components are mixed to prepare an ointment by a conventional method for preparing an ointment.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for enhancing the efficacy of a cancer immunotherapy agent for colorectal cancer, the pharmaceutical composition comprising a compound represented by the following formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: [Formula 2] The cancer immunotherapeutic agent is anti-PD1.

2. Use of a compound represented by the following formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing immunity in colorectal cancer, wherein the compound is administered in combination with anti-PD1: [Formula 2]

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