Pharmaceutical composition for treating cancer comprising simultaneous inhibitor of histone deacetylase 6 and histone deacetylase 10

Abstract

The present invention relates to: a pharmaceutical composition for treating cancer, the pharmaceutical composition containing a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10); an anticancer adjuvant for cancer immunotherapy; a composition for increasing the expression of PD-L1 in cancer cells; methods for treating cancer and for increasing expression of PD-L1 in cancer cells by using the simultaneous inhibitor of HDAC6 and HDAC10; and use of the simultaneous inhibitor of HDAC6 and HDAC10 for cancer treatment, as an anticancer adjuvant for cancer immunotherapy, and for increasing expression of PD-L1 in cancer cells.

Description

Pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 and histone deacetylase 10

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0173635, filed with the Korean Intellectual Property Office on December 4, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention was made under the support of the Ministry of Health and Welfare under the task identification number 1465039353 and task number HR20C0025020023. The research management specialized organization of the task is the Korea Health Industry Development Institute, the research project name is "Research-oriented hospital promotion R&D", the research project name is "Development of immune cell therapy biomarkers and new targets based on ultra-precision multi-omics analysis system", the main organization is Samsung Seoul Hospital, and the research period is from January 1, 2023 to December 31, 2023.

[0003] In addition, the present invention was made under the support of the Ministry of Science and ICT under the task identification number 1711188753 and task number 2020R1A2C2006408, and the research management specialized institution of the said task is the National Research Foundation of Korea, the research project name is "Individual Basic Research", the research project name is "Development of immune microenvironment control technology through macrophage-mediated immune profiling of colorectal cancer patients", the main institution is Samsung Seoul Hospital, and the research period is from 2023.03.01 to 2024.02.29.

[0004] The present invention relates to a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (hereinafter, HDAC6) and histone deacetylase 10 (hereinafter, HDAC10), an anticancer adjuvant for cancer immunotherapy, a composition for increasing PD-L1 expression in cancer cells, a method for treating cancer using a simultaneous inhibitor of HDAC6 and HDAC10, a method for increasing PD-L1 expression in cancer cells, a use of a simultaneous inhibitor of HDAC6 and HDAC10 for treating cancer, an anticancer adjuvant for cancer immunotherapy, and a use for increasing PD-L1 expression in cancer cells.

[0005] Histone deacetylases (HDACs) are enzymes that regulate the balance of acetylation and deacetylation of histone and non-histone proteins by catalyzing the hydrolysis of ε-amide bonds of lysine residues, and play an important role in gene expression and differentiation and in maintaining cellular homeostasis.

[0006] Histone deacetylases can bind to gene promoters by means of corepressors or multi-protein transcriptional complexes, where they regulate transcription through chromatin modifications without directly binding to DNA.

[0007] These HDACs are involved in the regulation of numerous cellular processes; histone acetyltransferases (HATs) and HDACs affect transcriptional activity by acetylating or deacetylating lysine residues at the N-terminus of histone proteins, and are known to regulate the posttranscriptional acetylation of at least 50 non-histone proteins, such as α-tubulin, Hsp90, p53, c-Myc, NF-κB, and E2P.

[0008] There are 18 encoded human HDACs, which are classified into class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), class III (SIRT 1-7), and class IV (HDAC11).

[0009] Overexpression of HDACs in various cancer cells promotes cancer cell proliferation by suppressing key growth-inhibiting genes. Therefore, HDACs are important drug targets for anticancer drug development, and the development of inhibitors is actively underway.

[0010] HDAC inhibitors are generally classified into four types based on their chemical structures: hydroxamic acids, benzamides, cyclic peptides, and short-chain fatty acids. So far, the U.S. FDA has approved four HDAC inhibitors as anticancer agents: SAHA (vorinostat), FK-228 (romidepsin), PXD101 (belinostat), and LBH589 (panobinostat), and the China Food and Drug Administration has approved HBI-8000 (chidamide) for the treatment of T-cell lymphoma.

[0011] Additionally, about 20 promising HDAC inhibitors are in clinical or preclinical stages for various cancers, but most of them have been found to cause many side effects, including fatigue, nausea, vomiting, and cardiac toxicity.

[0012] Furthermore, many studies are developing new drugs that may be effective in immunotherapy for colorectal cancer (CRC). Unfortunately, most patients with CRC are of the microsatellite stable (MSS) type, which is different from the microsatellite instable (MSI) type, which has an unstable tumor microenvironment (TME), and therefore do not respond to immunotherapy.

[0013] Accordingly, the present inventors investigated whether specific HDAC isoforms can regulate immune-related genes in the colon cancer tumor microenvironment and whether selective HDAC inhibitors are effective in treating colon cancer.

[0014] As a result, we confirmed that HDAC6 and 10 are important epigenetic regulators of PD-L1 in colon cancer, and confirmed that PD-L1 expression was upregulated in colon cancer cell lines when treated with a simultaneous HDAC6 and 10 inhibitor, confirming that it is effective in immunotherapy for colon cancer patients.

[0015] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (hereinafter, HDAC6) and histone deacetylase 10 (hereinafter, HDAC10).

[0016] Another object of the present invention is to provide an anticancer adjuvant for cancer immunotherapy comprising a simultaneous inhibitor of HDAC6 and HDAC10.

[0017] Another object of the present invention is to provide a composition for increasing PD-L1 expression in cancer cells, comprising a simultaneous inhibitor of HDAC6 and HDAC10.

[0018] Another object of the present invention relates to a method for treating cancer using a simultaneous inhibitor of HDAC6 and HDAC10.

[0019] Another object of the present invention is to provide a method for increasing PD-L1 expression in cancer cells using a simultaneous inhibitor of HDAC6 and HDAC10.

[0020] Another object of the present invention relates to the use of a simultaneous inhibitor of HDAC6 and HDAC10 for the treatment of cancer.

[0021] Another object of the present invention relates to the use of a simultaneous HDAC6 and HDAC10 inhibitor as an adjuvant anticancer agent for cancer immunotherapy.

[0022] Another object of the present invention relates to the use of a simultaneous inhibitor of HDAC6 and HDAC10 for increasing PD-L1 expression in cancer cells.

[0023] The present invention relates to a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (hereinafter, HDAC6) and histone deacetylase 10 (hereinafter, HDAC10), an anticancer adjuvant for cancer immunotherapy, a composition for increasing PD-L1 expression in cancer cells, a method for treating cancer using a simultaneous inhibitor of HDAC6 and HDAC10, a method for increasing PD-L1 expression in cancer cells, a use of a simultaneous inhibitor of HDAC6 and HDAC10 for treating cancer, an anticancer adjuvant for cancer immunotherapy, and a use for increasing PD-L1 expression in cancer cells.

[0024] Hereinafter, the present invention will be described in more detail.

[0025] One aspect of the present invention relates to a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

[0026] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be, but is not limited to, Bufexamac.

[0027] In the present invention, the pharmaceutical composition for treating cancer may additionally include an anti-PD-L1 antibody.

[0028] The term "PD-L1" in this specification stands for Programmed Death-Ligand 1, also known as CD274 or B7-H1. PD-L1 is present on the surface of cells and plays a role in preventing T cells from attacking them. Some tumor cells have large amounts of PD-L1, which allows the tumor cells to evade the immune system, the body's natural defense system.

[0029] The term 'anti-PD-L1 antibody' in this specification means an antibody capable of specifically binding to PD-L1.

[0030] In the present invention, the anti-PD-L1 antibody may be at least one selected from the group consisting of durvalumab, atezolizumab, avelumab, or any fragment, derivative, conjugate, variant, radioisotope-labeled complex or biosimilar thereof, but is not limited thereto.

[0031] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0032] In the present invention, the pharmaceutical composition for treating cancer may have a target cell line that is an MSS cell line, for example, SW620, SW480, LS513, HT29, LS1034 or CT26.

[0033] In the present invention, the pharmaceutical composition for treating cancer may additionally include one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0034] In the present invention, the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier.

[0035] The term "pharmaceutically acceptable" as used herein means, as commonly used in the pharmaceutical field, that when administered, it does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.

[0036] In the present invention, any carrier commonly used in the art may be used as the carrier. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, or combinations thereof.

[0037] In the present invention, the pharmaceutical composition for treating cancer can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.

[0038] In the present invention, the dosage of the pharmaceutical composition for treating cancer may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art. For example, the daily dosage may be 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 0.1 to 100 mg / kg, or 0.1 to 50 mg / kg. Administration may be once a day or divided into several doses, and the scope of the present invention is not limited thereby.

[0039] In the present invention, the subject may be a mammal, for example, a human or a mouse, but is not limited thereto.

[0040] Another aspect of the present invention relates to an anticancer adjuvant for cancer immunotherapy comprising a simultaneous inhibitor of HDAC6 and HDAC10.

[0041] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be, but is not limited to, Bufexamac.

[0042] In the present invention, the anticancer adjuvant for cancer immunotherapy may additionally include an anti-PD-L1 antibody.

[0043] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0044] In the present invention, the target cell line of the anticancer adjuvant for cancer immunotherapy may be an MSS cell line, for example, SW620, SW480, LS513, HT29, LS1034 or CT26.

[0045] In the present invention, the anticancer adjuvant for cancer immunotherapy may additionally include one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0046] In the present invention, the anticancer adjuvant for immunotherapy can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes.

[0047] In the present invention, the dosage of the anticancer adjuvant for immunotherapy may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art. For example, the daily dosage may be 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 0.1 to 100 mg / kg, or 0.1 to 50 mg / kg. Administration may be once a day or divided into several doses, and the scope of the present invention is not limited thereby.

[0048] In the present invention, the subject may be a mammal, for example, a human or a mouse, but is not limited thereto.

[0049] Another aspect of the present invention relates to a composition for increasing PD-L1 expression in cancer cells, comprising a simultaneous inhibitor of HDAC6 and HDAC10.

[0050] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be, but is not limited to, Bufexamac.

[0051] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0052] In the present invention, the target cell line of the anticancer adjuvant for cancer immunotherapy may be an MSS cell line, for example, SW620, SW480, LS513, HT29, LS1034 or CT26.

[0053] Another aspect of the present invention relates to a method for treating cancer comprising administering to a subject a simultaneous inhibitor of HDAC6 and HDAC10.

[0054] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be, but is not limited to, Bufexamac.

[0055] In the present invention, the method for treating cancer may be to administer an anti-PD-L1 antibody to a subject simultaneously or sequentially with a simultaneous inhibitor of HDAC6 and HDAC10.

[0056] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0057] In the present invention, the cancer treatment method may be such that the target cell line is an MSS cell line, for example, SW620, SW480, LS513, HT29, LS1034 or CT26.

[0058] In the present invention, the subject may be a mammal, for example, a human or a mouse, but is not limited thereto.

[0059] In the present invention, administration may be via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.

[0060] In the present invention, the administration dosage may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the administration route and period, and may be appropriately selected by a person skilled in the art. For example, the daily dosage may be 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 0.1 to 100 mg / kg, or 0.1 to 50 mg / kg. Administration may be once a day or divided into several doses, and the scope of the present invention is not limited thereby.

[0061] In the present invention, the cancer treatment method may be, but is not limited to, administering to a subject a simultaneous HDAC6 and HDAC10 inhibitor at intervals of 1 to 3 days, or at intervals of 1 to 2 days, for example, at intervals of 1 day, and simultaneously administering an anti-PD-L1 antibody at intervals of 2 to 4 days, or at intervals of 2 to 3 days, for example, at intervals of 3 days.

[0062] In the present invention, the cancer treatment method may be, but is not limited to, administering to a subject a simultaneous HDAC6 and HDAC10 inhibitor at a dose of 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 1 to 200 mg / kg, 10 to 200 mg / kg, 50 to 200 mg / kg, 75 to 200 mg / kg, 100 to 200 mg / kg, 125 to 200 mg / kg, or 150 to 200 mg / kg, for example, 150 mg / kg per dose.

[0063] In the present invention, the cancer treatment method comprises administering to a subject a simultaneous HDAC6 and HDAC10 inhibitor at a dose of 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 1 to 150 mg / kg, 1 to 100 mg / kg, 1 to 75 mg / kg, 1 to 50 mg / kg, 10 to 150 mg / kg, 10 to 100 mg / kg, 10 to 75 mg / kg, 10 to 50 mg / kg, 25 to 150 mg / kg, 25 to 100 mg / kg, 25 to 75 mg / kg, or 25 to 50 mg / kg, for example, 50 mg / kg, and administering an anti-PD-L1 antibody at a dose of 0.01 to 200 mg / kg, 0.1 to 200 mg / kg, 0.1 to 50 mg / kg, or 10 to 150 mg / kg, 10 to 100 mg / kg, 10 to 75 mg / kg, 10 to 50 mg / kg, 25 to 150 mg / kg, 25 to 100 mg / kg, 25 to 75 mg / kg, or 25 to 50 mg / kg, for example, 50 mg / kg, per time. The dose may be administered at 100 mg / kg, 0.1 to 50 mg / kg, or 0.1 to 20 mg / kg, for example, 10 mg / kg, but is not limited thereto.

[0064] Another aspect of the present invention relates to a method for increasing PD-L1 expression in cancer cells using a simultaneous inhibitor of HDAC6 and HDAC10.

[0065] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be, but is not limited to, Bufexamac.

[0066] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0067] In the present invention, the target cell line of the anticancer adjuvant for cancer immunotherapy may be an MSS cell line, for example, SW620, SW480, LS513, HT29, LS1034 or CT26.

[0068] Another aspect of the present invention relates to the use of a simultaneous inhibitor of HDAC6 and HDAC10 for the treatment of cancer.

[0069] Another aspect of the present invention relates to the use of a simultaneous HDAC6 and HDAC10 inhibitor as an adjuvant anticancer agent for cancer immunotherapy.

[0070] Another aspect of the present invention relates to the use of a simultaneous inhibitor of HDAC6 and HDAC10 for increasing PD-L1 expression in cancer cells.

[0071] Another aspect of the present invention relates to a combination kit for preventing or treating cancer comprising (i) a simultaneous inhibitor of HDAC6 and HDAC10 and (ii) an anti-PD-L1 antibody.

[0072] In one embodiment of the present invention, the (i) simultaneous HDAC6 and HDAC10 inhibitor and (ii) anti-PD-L1 antibody may be contained in the same container or in different containers.

[0073] The above kit may additionally include not only the active ingredients (i) a simultaneous inhibitor of HDAC6 and HDAC10 and (ii) an anti-PD-L1 antibody, but also containers, materials, package inserts, etc. commonly used in the art suitable for use as a combination kit for the prevention or treatment of cancer.

[0074] The above containers may include, but are not necessarily limited to, vials, syringes, bottles, etc.

[0075] The above materials may include, but are not necessarily limited to, diluents such as saline solution, filters, IV bags and lines, needles and syringes, etc.

[0076] The package insert may be instructions for using (i) a concurrent HDAC6 and HDAC10 inhibitor and (ii) an anti-PD-L1 antibody in combination to treat or delay the progression of cancer, or instructions for treating a subject for cancer using (i) a concurrent HDAC6 and HDAC10 inhibitor and (ii) an anti-PD-L1 antibody.

[0077] In the present invention, the simultaneous HDAC6 and HDAC10 inhibitor may be Bufexamac, but is not necessarily limited thereto.

[0078] In the present invention, the anti-PD-L1 antibody may be at least one selected from the group consisting of durvalumab, atezolizumab, avelumab, or any fragment, derivative, conjugate, variant, radioisotope-labeled complex or biosimilar thereof, but is not limited thereto.

[0079] In the present invention, the (i) HDAC6 and HDAC10 simultaneous inhibitor and (ii) anti-PD-L1 antibody may be administered simultaneously or sequentially.

[0080] The term “simultaneously” as used herein means that two preparations or pharmaceutical compositions are administered at the same time.

[0081] The term "sequentially" as used herein means that the active ingredients or pharmaceutical compositions are administered one after the other, rather than simultaneously. For example, "sequentially" administration may mean that one agent / pharmaceutical composition is administered within 1 minute, 5 minutes, 10 minutes, 30 minutes, or several hours, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, after the administration of another agent / pharmaceutical composition, provided that the circulating half-life of the first administered agent is sufficiently long to allow both agents to be present simultaneously in therapeutically effective amounts, but is not necessarily limited thereto. The time delay in administration between components may vary depending on the exact nature, interactions, and respective half-lives of the components.

[0082] In one embodiment of the present invention, the (i) simultaneous HDAC6 and HDAC10 inhibitor and (ii) anti-PD-L1 antibody may be administered at the same time or at different time intervals.

[0083] In one embodiment of the present invention, the (i) simultaneous HDAC6 and HDAC10 inhibitor and (ii) anti-PD-L1 antibody may be administered via the same route of administration or different routes of administration.

[0084] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

[0085] The present invention relates to a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (hereinafter, HDAC6) and histone deacetylase 10 (hereinafter, HDAC10), an anticancer adjuvant for cancer immunotherapy, a composition for increasing PD-L1 expression in cancer cells, a method for treating cancer using a simultaneous inhibitor of HDAC6 and HDAC10, a method for increasing PD-L1 expression in cancer cells, a use of a simultaneous inhibitor of HDAC6 and HDAC10 for treating cancer, an anticancer adjuvant for cancer immunotherapy, and a use for increasing PD-L1 expression in cancer cells.

[0086] Figure 1 shows the IC of Bufexamac, a simultaneous HDAC6 and HDAC10 inhibitor according to one embodiment of the present invention. 50 This is the result of measuring the value.

[0087] Figure 2 shows the results of measuring the amounts of H3K9ac, H3, and β-actin after 48 hours of treatment with Bufexamac, a simultaneous HDAC6 and HDAC10 inhibitor according to one embodiment of the present invention, at different concentrations.

[0088] Figures 3a and 3b show the results of measuring the change in the shape of M2 macrophages and the change in the mRNA expression of CD206, an M2 marker, after 48 hours of treatment with Bufexamac, a simultaneous inhibitor of HDAC6 and HDAC10 according to one embodiment of the present invention, at different concentrations.

[0089] Figures 4a to 4c show the results of measuring the tumor volume and weight and the body weight of mice when Bufexamac, a simultaneous HDAC6 and HDAC10 inhibitor, and an anti-PD-L1 antibody were treated alone or in combination according to an embodiment of the present invention in an animal experiment.

[0090] FIG. 5a shows the results of measuring changes in CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells in lymph node cells when Bufexamac, a simultaneous HDAC6 and HDAC10 inhibitor, and an anti-PD-L1 antibody were treated alone or in combination according to an embodiment of the present invention.

[0091] FIG. 5b shows the results of measuring changes in monocytic myeloid-derived suppressor cells (M-MDSC), polymorphonuclear leukocyte myeloid-derived suppressor cells (PMN-MDSC), macrophages, M1 macrophages, and M2 macrophages in bone marrow cells when Bufexamac, a simultaneous inhibitor of HDAC6 and HDAC10, and an anti-PD-L1 antibody were treated alone or in combination according to an embodiment of the present invention.

[0092] FIG. 6 is a result confirming an increase in mRNA expression of PD-L1 by Bufexamac, a simultaneous inhibitor of HDAC6 and HDAC10, in the human MSS CRC cell line SW620 according to an embodiment of the present invention.

[0093] FIG. 7 is a result confirming an increase in mRNA expression of PD-L1 by Bufexamac, a simultaneous inhibitor of HDAC6 and HDAC10, in the mouse MSS CRC cell line CT26 according to an embodiment of the present invention.

[0094] The present invention relates to a pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

[0095] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0096]

[0097] Experimental Example 1. Measurement of IC50 concentration

[0098] IC 50 The concentration is the concentration at which half of the cells die, and the IC is calculated by performing the WST assay according to the conditions in Table 1 below. 50 The values ​​were measured. Specifically, the CT26 colon cancer cell line was treated with HDAC6 inhibitor (Nexturastat A) and HDAC6 and HDAC10 simultaneous inhibitor (Bufexamac) at various concentrations for 72 hours, and the concentration at which half of the cells were killed was determined. The results are shown in Fig. 1.

[0099] 1. IC50 (WST assay)Cell lineCT26Cell number1×10 4 HDAC6 inhibitor (Nexturastat A)30 μM ~ 3-fold dilutionHDAC class IIb inhibitor (Bufexamac)1 mM ~ 3-fold dilutionIncubation time72 hrs

[0100]

[0101] As shown in Figure 1, Bufexamac IC 50 Silver 107.4 μM, Nexturastat A IC 50 The IC of Bufexamac was derived as 12.8 μM. 50The value was confirmed to be approximately 7 times higher than that of Nexturastat A. This means that Bufexamac has lower pharmacological activity compared to Nexturastat A, which has superior selectivity, and that animal testing should be conducted at a higher concentration than Nexturastat A.

[0102]

[0103] Experimental Example 2. Confirmation of HDAC inhibition effect

[0104] The HDAC inhibitory effect was confirmed through Western blotting according to the conditions in Table 2 below. Specifically, after 48 hours of treatment with HDAC6 inhibitor (Nexturastat A) and HDAC6 and HDAC10 simultaneous inhibitor (Bufexamac) at different concentrations, the amounts of H3K9ac, H3, and β-actin were measured, and the results are shown in Figure 2.

[0105] 2. HDAC inhibition effect (Histone acetylation; western blot)Cell lineCT26Cell number2×10 5 HDAC6 inhibitor (Nexturastat A)7 μMHDAC class IIb inhibitor (Bufexamac)10 μM, 50 μMIncubation time48 hrs

[0106]

[0107] As shown in Fig. 2, it was confirmed that when HDAC function is lost due to an HDAC inhibitor, H3K9 acetylation increases. As confirmed in Fig. 1, in the case of Bufexamac, which has lower pharmacological activity than Nexturastat A, the degree of H3K9 acetylation was slightly increased compared to the control group, confirming that Bufexamac also inhibits intracellular HDAC.

[0108]

[0109] Experimental Example 3. Confirmation of M2 Macrophage Regulation

[0110] The M2 macrophage regulatory effect was confirmed through RT-PCR according to the conditions in Table 3 below. Specifically, human monocytic cells, THP-1, were differentiated into macrophages by stimulating with PMA and then differentiated into M2 macrophages by treating with IL-4 and IL-13. The differentiated M2 macrophages were treated with various concentrations of an HDAC6 inhibitor (Nexturastat A) and a simultaneous HDAC6 and HDAC10 inhibitor (Bufexamac) for 48 hours. The changes in M2 macrophages were measured, and the results are shown in Figures 3a and 3b.

[0111] 3. M2 macrophage modulation (RT-PCR; CD206)Cell lineTHP-1 (w / PMA)Cell number3×10 5 M2 differentiation using IL-4, IL-13HDAC6 inhibitor (Nexturastat A)4 μMHDAC class IIb inhibitor (Bufexamac)5, 10, 50 μMIncubation time48 hrs

[0112]

[0113] As shown in Fig. 3a, M2 macrophages have an elongated shape compared to M0 macrophages, but round-shaped cells similar to M0 macrophages were observed under conditions treated with 4 μM Nexturastat A and 50 μM Bufexamac. This means that M2 macrophages lost their M2 macrophage characteristics due to the HDAC inhibitors Nexturastat A and Bufexamac. In addition, as shown in Fig. 3b, when RNA was isolated under the same conditions and the change in RNA expression of CD206, an M2 macrophage marker, was confirmed, the expression of CD206 was decreased by the HDAC inhibitors Nexturastat A and Bufexamac. This means that the HDAC inhibitors Nexturastat A and Bufexamac reduce M2 macrophages.

[0114]

[0115] Experimental Example 4. Animal Experiment Using Simultaneous HDAC6 and HDAC10 Inhibitors

[0116] 1×10 mouse MSS colon cancer cell line CT26 6 Tumors were formed by subcutaneous injection of the dog into BALB / C mice.

[0117] Animal experiments using simultaneous HDAC6 and HDAC10 inhibitors were conducted in four groups: control group, anti-PD-L1 10 mg / kg group, Bufexamac 50 mg / kg group, and Bufexamac 50 mg / kg + anti-PD-L1 group.

[0118] Bufexamac (Selleckchem) was used as a simultaneous inhibitor of HDAC6 and HDAC10, and #BE0101 (Bioxcell) was used as an anti-PD-L1 (anti-PD-L1 antibody). The cell line was CT26 (1 × 10 6) were used, and BALB / C mice were used. The mice were divided into 5 control group, 5 anti-PD-L1 10 mg / kg group, 5 Bufexamac 50 mg / kg group, and 5 Bufexamac 50 mg / kg + anti-PD-L1 group, and the treatment conditions were as follows.

[0119]

[0120] [Processing Conditions]

[0121] - anti-PD-L1 10 mg / kg group: administered intraperitoneally once every three days

[0122] - Bufexamac 50 mg / kg group: administered intraperitoneally daily

[0123] - Bufexamac 50 mg / kg + anti-PD-L1 group 10 mg / kg group: anti-PD-L1 administered intraperitoneally once every three days, and Bufexamac administered intraperitoneally daily.

[0124]

[0125] The tumor volume and weight of each group were measured, and the results are shown in Figures 4a and 4b. As a result, as can be seen in Figures 4a and 4b, when the HDAC6 and HDAC10 simultaneous inhibitor (Bufexamac) was treated alone at 50 mg / kg, the change in tumor volume and weight was not significant compared to the control group, but when the HDAC6 and HDAC10 simultaneous inhibitor was treated in combination with an anti-PD-L1 antibody, a significantly superior tumor volume and weight reduction effect was observed.

[0126] In addition, when treated with a simultaneous HDAC6 and HDAC10 inhibitor in an animal experiment, the body weight of the mice was measured and shown in Fig. 4c, confirming that the weight of the animals was not reduced by the drug.

[0127]

[0128] Experimental Example 5. Lymph Node Cell Analysis

[0129] In addition, when HDAC6 and HDAC10 simultaneous inhibitors, anti-PD-L1 antibodies, and HDAC6 and HDAC10 simultaneous inhibitors and anti-PD-L1 antibodies were treated together in animal experiments, changes in CD3+ T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells in lymph node cells were measured using flow cytometry (FACS), and the results are shown in Fig. 5a.

[0130] As shown in Fig. 5a, CD8+ T cells were found to increase more significantly when treated with anti-PD-L1 antibodies and HDAC6 and HDAC10 simultaneous inhibitors together than when treated with anti-PD-L1 antibodies or HDAC6 and HDAC10 simultaneous inhibitors alone, compared to the other groups. This suggests that HDAC6 and HDAC10 simultaneous inhibitors can induce CD8+ T cell infiltration into tumors when used in combination with immunotherapy.

[0131] In addition, when HDAC6 and HDAC10 simultaneous inhibitors were treated with anti-PD-L1 antibodies, regulatory T cells (T reg ) was confirmed to be reduced.

[0132] Also, CD8 / T reg The ratio value was confirmed to increase when a simultaneous HDAC6 and HDAC10 inhibitor was treated together with an anti-PD-L1 antibody. This means that the combined treatment of a simultaneous HDAC6 and HDAC10 inhibitor and an anti-PD-L1 antibody induces changes in the intratumoral immune environment, with a decrease in regulatory T cells that play a role in suppressing T cells and an increase in CD8+ T cells that play a role in attacking tumors.

[0133]

[0134] Experimental Example 6. Bone Marrow Cell Analysis

[0135] In addition, when HDAC6 and HDAC10 simultaneous inhibitors, anti-PD-L1 antibodies, and HDAC6 and HDAC10 simultaneous inhibitors and anti-PD-L1 antibodies were treated together in animal experiments, changes in monocytic myeloid-derived suppressor cells (M-MDSC), polymorphonuclear leukocyte myeloid-derived suppressor cells (PMN-MDSC), macrophages, M1 macrophages, and M2 macrophages in bone marrow cells were measured using flow cytometry (FACS), and the results are shown in Fig. 5b.

[0136] As shown in Figure 5b, there was no change in myeloid-derived suppressor cells (MDSCs) and M2 macrophages due to the simultaneous HDAC6 and HDAC10 inhibitors, and the distribution of macrophages increased in all groups. M1 macrophages were found to increase compared to the other groups when treated with the simultaneous HDAC6 and HDAC10 inhibitors alone or together with anti-PD-L1 antibodies. This suggests that the increase in M1 macrophages, which play a pro-inflammatory role, similarly induces changes in the intratumoral immune environment.

[0137]

[0138] Experimental Example 7. Confirmation of increased PD-L1 expression by simultaneous HDAC6 and HDAC10 inhibitors in MSS colon cancer.

[0139] Human and mouse MSS CRC cell lines were treated with a simultaneous HDAC6 and HDAC10 inhibitor (Bufexamac), RNA was isolated, and increased expression of PD-L1 was confirmed by qRT-PCR. GAPDH was used as a control gene.

[0140] As can be seen in Figures 6 and 7, the mRNA expression of PD-L1 was statistically significantly increased in human MSS CRC cell line SW620 and mouse MSS CRC cell line CT26 when treated with 5 μM of Bufexamac, a simultaneous inhibitor of HDAC6 and HDAC10, compared to the control group.

[0141] From the above results, the inventors of the present invention predict that the synergistic effect on anticancer activity against MSS colon cancer when combined with a simultaneous HDAC6 and HDAC10 inhibitor and an anti-PD-L1 antibody compared to single treatment is due to the simultaneous HDAC6 and HDAC10 inhibitor inducing an increase in PD-L1 expression in MSS colon cancer and the anti-PD-L1 antibody reacting to the increased PD-L1 expression.

[0142]

[0143] Sintering

[0144] It was confirmed that the tumor growth inhibition effect was superior when treated with an anti-PD-L1 antibody compared to when treated alone with a simultaneous HDAC6 and HDAC10 inhibitor. Therefore, it is expected that the pharmaceutical composition comprising the simultaneous HDAC6 and HDAC10 inhibitor according to the present invention can be used as an effective cancer treatment composition.

[0145]

[0146] The present invention has confirmed that the tumor growth inhibition effect is significantly superior when combined with an anti-PD-L1 antibody than when combined with a simultaneous HDAC6 and HDAC10 inhibitor alone, and thus that there is a synergistic effect in combined treatment compared to single treatment. Therefore, it is expected that not only a pharmaceutical composition comprising a simultaneous HDAC6 and HDAC10 inhibitor according to the present invention, but also a pharmaceutical composition comprising a simultaneous HDAC6 and HDAC10 inhibitor and an anti-PD-L1 antibody can be effectively used for the prevention or treatment of cancer such as colon cancer.

Claims

1. A pharmaceutical composition for treating cancer comprising a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

2. A pharmaceutical composition for treating cancer, according to claim 1, which additionally contains an anti-PD-L1 antibody.

3. A pharmaceutical composition for treating cancer, wherein in paragraph 1, the cancer is at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

4. An anticancer adjuvant agent for cancer immunotherapy comprising a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

5. In the fourth paragraph, the anticancer adjuvant for cancer immunotherapy additionally contains an anti-PD-L1 antibody.

6. An anticancer adjuvant for cancer immunotherapy, wherein in paragraph 4, the cancer is at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

7. A composition for increasing PD-L1 expression in cancer cells, comprising a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

8. A composition for increasing PD-L1 expression in cancer cells, wherein the cancer in claim 7 is at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

9. A method for increasing PD-L1 expression in a cancer cell, comprising the step of contacting a subject with a simultaneous inhibitor of histone deacetylase 6 (HDAC6) and histone deacetylase 10 (HDAC10).

10. A method for increasing PD-L1 expression in cancer cells, wherein the cancer is at least one selected from the group consisting of colon cancer, pancreatic cancer, stomach cancer, and lung cancer.

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