Novel combination therapy for blood cancer

Abstract

The present invention provides a new means for treating blood cancer by 1) a two-drug combination of a DNA methyl transferase inhibitor and a pharmaceutical composition containing as an active ingredient compound (I), or a pharmaceutically acceptable salt or hydrate thereof, and 2) a three-drug combination of a DNA methyl transferase inhibitor, a BCL-2 inhibitor, and a pharmaceutical composition containing as an active ingredient compound (I), or a pharmaceutically acceptable salt or hydrate thereof.

Description

Novel combination therapy for blood cancer

[0001] The present invention relates to a novel therapeutic method for blood cancers. More specifically, the present invention relates to a novel therapeutic method that utilizes a combination of compound (I), a pharmaceutically acceptable salt or hydrate thereof, and a DNA methyltransferase inhibitor, or a combination of compound (I), a pharmaceutically acceptable salt or hydrate thereof, and a DNA methyltransferase inhibitor, further combined with a BCL-2 inhibitor.

[0002] Blood cancer is a general term for diseases in which blood cells become cancerous, broadly classified as leukemia, malignant lymphoma, and multiple myeloma. In recent years, various therapies and anticancer drugs have been developed and are used to treat these blood cancers. Myelodysplastic syndrome (MDS) is a disease in which abnormalities occur in hematopoietic stem cells in the bone marrow, preventing normal blood production. Approximately 30% of MDS patients are thought to progress to acute myeloid leukemia (AML). In recent years, abnormal DNA methylation, which induces the inactivation of tumor suppressor genes, has been considered one of the causes of cancer development and leukemogenesis. DNA methyltransferase inhibitors have been developed to suppress this abnormal DNA methylation. DNA methyltransferase inhibitors, such as azacitidine and decitabine, have a chemical structure similar to cytidine, and are incorporated into DNA in vivo. They inhibit DNA methyltransferase and suppress DNA methylation, thereby inducing DNA damage and apoptosis and exerting antitumor effects. DNA methyltransferase inhibitors such as azacitidine and decitabine are already being used as therapeutic agents for MDS and AML, but clinical problems include ineffectiveness in some patients and drug resistance resulting from loss of efficacy during treatment (Non-Patent Document 1). BCL-2 (B-cell lymphoma 2) is a member of the BCL-2 family that controls cell death and is known to inhibit apoptosis in some hematologic cancers. Several BCL-2 inhibitors, including venetoclax, have been reported to date. These BCL-2 inhibitors selectively bind to BCL-2, inhibiting its action, and are known to potently induce apoptosis and exhibit antitumor effects when used alone or in combination with other therapeutic agents (Non-Patent Document 2). Venetoclax has been approved as a treatment for AML patients in combination with DNA methyltransferase inhibitors such as azacitidine and decitabine. However, even with this combination therapy, serious side effects that make it difficult to continue treatment and insufficient therapeutic effects have become a problem, and the development of new treatment methods is desired (Non-Patent Document 3).CDC7 (cell division cycle 7) is a serine-threonine kinase and is an enzyme essential for initiating DNA replication during the cell cycle. CDC7 forms a complex with cofactors such as Dbf4 (ASK), which activate its phosphorylation activity, and phosphorylates its substrate, the MCM (minichromosome maintenance) protein. This phosphorylation is thought to cause Cdc45 and DNA polymerase to assemble on DNA, forming an MCM complex and initiating DNA replication (Non-Patent Document 4). In recent years, CDC7 has attracted attention as a target for anticancer drugs, and AS-0141, which selectively and potently inhibits CDC7, has been shown to have antitumor activity against various cancer cell lines (Non-Patent Document 5).

[0003] AS-0141 is a compound represented by the formula: ethyl 5-[(1H-pyrrolo[2,3-b]pyridin-3-yl)methylene]-4-oxo-2-{[4-(2,2,2-trifluoroethyl)piperazinyl]amino}-4,5-dihydrofuran-3-carboxylate [Compound (I)]. Compound (I) is disclosed in International Publication No. 2012 / 133802 (Patent Document 1) and Non-Patent Document 5. It has also been disclosed that the combination of compound (I) with an M-phase promoter such as a Wee1 inhibitor enhances anticancer activity (Patent Document 2). International Publication No. 2018 / 84266 discloses a method for treating cancer using compound (I) (Patent Document 3). International Publication No. 2020 / 68347 discloses a treatment method in which SRA141 (AS-0141) is administered in combination therapy (Patent Document 4).

[0004] International Publication No. WO 2012 / 133802 International Publication No. WO 2015 / 115355 International Publication No. WO 2018 / 84266 International Publication No. WO 2020 / 68347

[0005] Zhao G, et al. , Front Oncol. 2021 Sep 28;11:706030.Ashkenazi, A. , et al. , Nature Rev. Drug Discov. , 2017, 16, 273-284. Estey EH. , Am J Hematol. 2020 Nov;95(11):1368-1398. Sawa M, Masai H. , Drug Des Devel Ther. , 2008 Feb 6;2:255-264. Irie T, et al. , J Med Chem. 2021 Oct 14;64(19):14153-14164.

[0006] An object of the present invention is to provide a new therapeutic method for treating blood cancer patients that has a higher therapeutic effect and fewer side effects.

[0007] There have been no reports on the combination of compound (I) or a pharmaceutically acceptable salt or hydrate thereof with a DNA methyltransferase inhibitor, nor has there been any report that the combined use of compound (I) or a pharmaceutically acceptable salt or hydrate thereof with a DNA methyltransferase inhibitor enhances the anti-cancer effect against blood cancers. Furthermore, there have been no reports on the triple combination of compound (I) or a pharmaceutically acceptable salt or hydrate thereof with a DNA methyltransferase inhibitor and a BCL-2 inhibitor, nor has it been known that such triple combination enhances the anti-cancer effect against blood cancers. The present inventors have conducted extensive research to solve the above-mentioned problems, and as a result have found that the problems of the present invention can be solved by combining 1) compound (I) or a pharmaceutically acceptable salt or hydrate thereof with 2) a DNA methyltransferase inhibitor, and further by administering the combination of 1) and 2) in combination with 3) a BCL-2 inhibitor, due to the synergistic effect thereof, and have thus completed the present invention. More specifically, the present invention relates to the following: In this specification, unless otherwise specified, Compound (I) also includes its pharmaceutically acceptable salts or hydrates. The same applies to azacitidine and decitabine.

[0008] (1) A pharmaceutical composition comprising compound (I) as an active ingredient, which is administered to a patient in combination with a DNA methyltransferase inhibitor for use in treating blood cancer. (2) The pharmaceutical composition of (1), which is administered to a patient in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor for use in treating a blood cancer. (3) The pharmaceutical composition of (1) or (2), wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML). (4) The pharmaceutical composition of (1) or (2), wherein the blood cancer is myelodysplastic syndrome (MDS). (5) The pharmaceutical composition of (1) or (2), wherein the blood cancer is acute myeloid leukemia (AML).

[0009] (6) The pharmaceutical composition of (1) or (2), wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising azacitidine or decitabine as an active ingredient. (7) The pharmaceutical composition of (6), wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising azacitidine as an active ingredient. (8) The pharmaceutical composition of (6), wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising decitabine as an active ingredient. (9) The pharmaceutical composition of (7), wherein the pharmaceutical composition comprising azacitidine as an active ingredient and the pharmaceutical composition comprising compound (I) as an active ingredient are administered simultaneously or separately and sequentially. (10) The pharmaceutical composition of (8), wherein the pharmaceutical composition comprising decitabine as an active ingredient and the pharmaceutical composition comprising compound (I) as an active ingredient are administered simultaneously or separately and sequentially. (11) The pharmaceutical composition of (2), wherein the BCL-2 inhibitor is a pharmaceutical composition comprising venetoclax as an active ingredient. (12) The pharmaceutical composition according to any one of (9) to (11), wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

[0010] (13) The pharmaceutical composition according to (12), wherein the blood cancer is myelodysplastic syndrome (MDS). (14) The pharmaceutical composition according to (12), wherein the blood cancer is acute myeloid leukemia (AML). (15) A kit comprising a pharmaceutical composition containing a DNA methyltransferase inhibitor and compound (I) as active ingredients, respectively, for use in the combined administration of (1). (16) A kit comprising a pharmaceutical composition containing a DNA methyltransferase inhibitor, a BCL-2 inhibitor, and compound (I) as active ingredients, respectively, for use in the combined administration of (2). (17) A method for treating blood cancer, comprising administering to a patient a pharmaceutical composition containing compound (I) as an active ingredient in combination with a DNA methyltransferase inhibitor. (18) A method for treating blood cancer, comprising administering to a patient a pharmaceutical composition containing compound (I) as an active ingredient in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor. (19) The method for treating (17) or (18), wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML). (20) The method for treating (17) or (18), wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising azacitidine or decitabine as an active ingredient. (21) The method for treating (18), wherein the BCL-2 inhibitor is a pharmaceutical composition comprising venetoclax as an active ingredient.

[0011] The combination of compound (I), its pharmaceutically acceptable salt, or hydrate with a DNA methyltransferase inhibitor can induce cell death more efficiently than the use of compound (I) or the DNA methyltransferase inhibitor alone. CDC7 inhibitors cause genome instability in cancer cells, inducing DNA damage and apoptosis. Meanwhile, DNA methyltransferase inhibitors suppress abnormal DNA methylation in cancer cells, activating tumor suppressor genes and inducing DNA damage and apoptosis. Therefore, the combination of compound (I) with a DNA methyltransferase inhibitor is expected to enhance anticancer activity and provide a high therapeutic effect against various blood cancers. Furthermore, a triple combination of compound (I), its pharmaceutically acceptable salt, or hydrate with a DNA methyltransferase inhibitor, and a BCL-2 inhibitor is expected to provide a synergistic effect and provide a high therapeutic effect against various blood cancers.

[0012] Figure 1 shows the cell growth inhibition rate of a DNA methyltransferase inhibitor (azacytidine) in combination with Compound (I) on a human acute monocytic leukemia cell line (THP-1), demonstrating the interaction between the two compounds. Figure 2A shows the effect of the combination of a DNA methyltransferase inhibitor (azacytidine) and Compound (I) on THP-1 cells, as measured by changes in the expression levels of γH2AX, an indicator of DNA damage, and cleaved caspase-3, an indicator of apoptosis. Figure 2B shows the effect of the combination of a DNA methyltransferase inhibitor (decitabine) and Compound (I) on MV-4-11 cells, as measured by changes in the expression levels of γH2AX, an indicator of DNA damage, and cleaved caspase-3, an indicator of apoptosis. Figure 3 shows the induction of apoptosis in THP-1 cells by the combination of a DNA methyltransferase inhibitor (azacytidine) and compound (I). Figure 4A shows an isobologram (50% inhibition) illustrating the interaction of the DNA methyltransferase inhibitor (azacytidine) and compound (I) in THP-1 cells. Figure 4B shows an isobologram (50% inhibition) illustrating the interaction of the DNA methyltransferase inhibitor (azacytidine) and compound (I) in a human acute myeloid leukemia cell line (MV-4-11). Figure 4C shows an isobologram (50% inhibition) illustrating the interaction of the DNA methyltransferase inhibitor (azacytidine) and compound (I) in a human acute monocytic leukemia cell line (MOLM-14). Figure 4D is an isobologram (50% inhibition) showing the interaction of the DNA methyltransferase inhibitor (azacytidine) and compound (I) in combination on a human erythroleukemia cell line (TF-1). Figure 4E is an isobologram (50% inhibition) showing the interaction of the DNA methyltransferase inhibitor (decitabine) and compound (I) in combination on a human acute myeloid leukemia cell line (MV-4-11).Figure 4F is an isobologram (50% inhibition) showing the interaction of the DNA methyltransferase inhibitor (decitabine) and compound (I) in combination on a human acute monocytic leukemia cell line (MOLM-14). Figure 4G is an isobologram (50% inhibition) showing the interaction of the DNA methyltransferase inhibitor (decitabine) and compound (I) in combination on a human erythroleukemia cell line (TF-1). Figure 5 is an Fa-CI plot showing the interaction of the DNA methyltransferase inhibitor (azacitidine), a BCL-2 inhibitor (venetoclax), and compound (I) in combination on a human acute myeloid leukemia cell line (MV-4-11). Figure 6A shows that the combination of compound (I) and the DNA methyltransferase inhibitor (azacitidine) suppresses tumor growth in a mouse subcutaneous xenograft model of the human acute myeloid leukemia cell line (MV-4-11). Figure 6B shows that tumor growth is suppressed by the combination of compound (I) and a DNA methyltransferase inhibitor (decitabine) in a mouse subcutaneous xenograft model of human acute myeloid leukemia cell line (MV-4-11). Figure 7A shows that tumor growth is suppressed by the combination of compound (I) with a DNA methyltransferase inhibitor (azacitidine) and a BCL-2 inhibitor (venetoclax) in a mouse subcutaneous xenograft model of human acute myeloid leukemia cell line (MV-4-11). Figure 7B shows that tumor growth is suppressed by the combination of compound (I) with a DNA methyltransferase inhibitor (decitabine) and a BCL-2 inhibitor (venetoclax) in a mouse subcutaneous xenograft model of human acute myeloid leukemia cell line (MV-4-11).

[0013] (1) DNA Methyltransferase Inhibitors In the present invention, a DNA methyltransferase inhibitor refers to a pharmaceutical composition containing as an active ingredient a drug that inhibits DNA methyltransferase activity, and includes pharmaceutical compositions containing drugs that inhibit the physiological function of DNA methyltransferase, such as low-molecular-weight compounds, polypeptides, proteins, nucleic acids (e.g., siRNA, miRNA, aptamers), and other polymeric compounds. Also included are drugs that are activated in vivo to inhibit the physiological function of DNA methyltransferase, i.e., prodrugs of DNA methyltransferase inhibitors. Examples of drugs that inhibit DNA methyltransferase activity include azacitidine and decitabine, which have the following structures, and pharmaceutically acceptable salts, hydrates, and prodrugs thereof. (2) BCL-2 Inhibitors The BCL-2 inhibitor used in the present invention refers to a drug that inhibits the physiological function of BCL-2 in cells, and includes drugs that inhibit the physiological function of BCL-2, such as low molecular weight compounds, polypeptides, proteins, nucleic acids (e.g., siRNA, miRNA, aptamers), and other polymeric compounds. BCL-2 is a member of the BCL-2 family that controls cell death, and negatively regulates apoptosis. BCL-2 is activated in lymphatic B-cell tumors such as follicular lymphoma (DLBCL) and CLL, as well as in multiple myeloma and T-cell tumors. BCL-2 inhibitors induce apoptosis in these cancer cells, thereby exhibiting an antitumor effect. Therefore, even greater antitumor effects can be expected when used alone or in combination with other drugs. Examples of BCL-2 inhibitors include venetoclax, navitoclax, obatoclax, obatoclax mesylate, sabutoclax, APG-1252, AZD-0466, APG-2575, ABBV-167, S-65487, S-55746, and the like.

[0014] (3) Pharmaceutical composition containing compound (I) In the present invention, compound (I) is a furanone derivative (I) represented by ethyl 5-[(1H-pyrrolo[2,3-b]pyridin-3-yl)methylene]-4-oxo-2-{[4-(2,2,2-trifluoroethyl)piperazinyl]amino}-4,5-dihydrofuran-3-carboxylate, Compound (I) also encompasses its pharmaceutically acceptable salts, hydrates and prodrugs.

[0015] Compound (I) is disclosed in International Publication No. 2012 / 133802 (Patent Document 1) and Non-Patent Document 5, and is known as a compound that selectively and potently inhibits CDC7. Pharmaceutically acceptable salts of compound (I) used in the present invention include inorganic acid salts with hydrochloric acid, sulfuric acid, carbonic acid, phosphoric acid, etc., and organic acid salts with fumaric acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. Also included in the present invention are alkali metal salts with sodium, potassium, etc., alkaline earth metal salts with magnesium, calcium, etc., organic amine salts with triethylamine, ethanolamine, etc., basic amino acid salts with lysine, arginine, ornithine, etc., as well as ammonium salts.

[0016] Compound (I) or its pharmaceutically acceptable salt or hydrate used in the present invention can be prepared in the form of a conventional pharmaceutical preparation (pharmaceutical composition) suitable for oral, parenteral, or topical administration. Preparations for oral administration include solid preparations such as tablets, granules, powders, and capsules, as well as liquid preparations such as syrups. These preparations can be prepared by conventional methods. Solid preparations can be prepared using conventional pharmaceutical carriers such as lactose, starch such as cornstarch, crystalline cellulose such as microcrystalline cellulose, hydroxypropyl cellulose, calcium carboxymethylcellulose, talc, magnesium stearate, and the like. Capsules can be prepared by encapsulating the granules or powder thus prepared. Syrups can be prepared by dissolving or suspending the compound of the present invention or its pharmaceutically acceptable salt or hydrate in an aqueous solution containing sucrose, carboxymethylcellulose, or the like. Preparations for parenteral administration include injections such as intravenous drip infusions. Injection preparations can also be prepared by conventional methods and can be appropriately incorporated into isotonic agents (e.g., mannitol, sodium chloride, glucose, sorbitol, glycerol, xylitol, fructose, maltose, mannose), stabilizers (e.g., sodium sulfite, albumin), and preservatives (e.g., benzyl alcohol, methyl p-hydroxybenzoate).

[0017] (4) Combination Therapy In the combination therapy of the present invention, a pharmaceutical composition containing compound (I) as an active ingredient is administered to a patient with hematological cancer in combination with a DNA methyltransferase inhibitor as a two-drug combination, or in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor as a triple-drug combination. One aspect of the present invention is a pharmaceutical composition containing compound (I) as an active ingredient to be administered in combination with a DNA methyltransferase inhibitor, and also encompasses a kit containing pharmaceutical compositions containing a DNA methyltransferase inhibitor and compound (I) as active ingredients, respectively. The kit can also include instructions for use of both drugs.

[0018] One aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered simultaneously with a DNA methyltransferase inhibitor. Another aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered after administration of a DNA methyltransferase inhibitor. Another aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered before administration of a DNA methyltransferase inhibitor. One aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor. The pharmaceutical composition also includes a kit comprising pharmaceutical compositions comprising a DNA methyltransferase inhibitor, a BCL-2 inhibitor, and compound (I) as active ingredients, respectively. The kit may include instructions for use of both drugs. One aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered simultaneously with a DNA methyltransferase inhibitor and a BCL-2 inhibitor. Another aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered after administration of a DNA methyltransferase inhibitor and a BCL-2 inhibitor. Another aspect of the present invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered prior to administration of a DNA methyltransferase inhibitor and a BCL-2 inhibitor.

[0019] Azacitidine, decitabine, and venetoclax are already on the market and are used to treat blood cancers. Those skilled in the art can appropriately determine the dosage of azacitidine or decitabine in the combination therapy of the present invention based on the case where these are administered as single agents, taking into account the type of blood cancer, the patient's weight, age, and severity of the disease, etc. Furthermore, those skilled in the art can appropriately determine the dosage of a DNA methyltransferase inhibitor such as azacitidine and a BCL-2 inhibitor such as venetoclax in the triple combination therapy of the present invention based on the case where these are administered as combination therapy, taking into account the type of blood cancer, the patient's weight, age, and severity of the disease, etc. The dosage of Compound (I) used in the present invention can vary depending on the severity of the disease, the patient's age and weight, dosage form, etc., but is typically in the range of 10 mg to 400 mg per day for adults, and can be administered once, or in two or three divided doses, via oral or parenteral routes.

[0020] Test Example 1: Cell proliferation inhibition test of single agents (cell culture) Human acute monocytic leukemia cell line (THP-1), human acute myeloid leukemia cell line (MV-4-11), human acute monocytic leukemia cell line (MOLM-14), and human erythroleukemia cell line (TF-1) were cultured in RPMI 1640 medium (Roswell Park Memorial Institute medium, Nakarai) containing 10% fetal bovine serum (Thermo Scientific, SIGMA-ALDRICH, or Cytiva) and 1% penicillin-streptomycin (Nacalai) at 5% CO ２ The cells were cultured in an incubator. (Cytostatic test) THP-1 cells and MV-4-11 cells were cultured at 2 × 10 ４ MOLM-14 cells and TF-1 cells were cultured at 5 × 10 cells / well. ３Cells were seeded in a 96-well plate (cell plate), and test compounds diluted in medium were added to a final concentration of 3 nM to 30 μM (final DMSO concentration 0.3%). After 72 hours of incubation, Resazurin reagent (Nacalai) was added. After 3 hours, fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The IC50 inhibitory activity was calculated by setting the percentage of wells without compound and cells to 100% and wells without compound and cells to 0%. ５０ The results are shown in Table 1.

[0021] Example 1 Dose-response test of DNA methyltransferase inhibitor in the presence of compound (I) IC of DNA methyltransferase inhibitor (azacytidine) in the presence of compound (I) ５０ From the results of compound (I) alone, one dose of compound (I) was selected, and the DMSO-added group was used as a control. The DNA methyltransferase inhibitor was added to the cell plate at a concentration of 3 nM to 30 μM (final DMSO concentration, 0.4%), and the IC value in the presence of compound (I) was examined in the same manner as in Test Example 1. ５０ The IC value of azacitidine in the presence of compound (I) when THP-1 cells were used was calculated. ５０ The changes in the values ​​are shown in Figure 1 and Table 2. In this test, as shown in Figure 1 and Table 2, in THP-1 cells, a human acute monocytic leukemia cell line, in the presence of compound (I), the IC ５０ The value decreased. The results of Example 1 show that the combined use of Compound (I) of the present invention and a DNA methyltransferase inhibitor has a combined effect on blood cancer cells.

[0022] Example 2 Evaluation of DNA Damage and Apoptosis Induction The effect of the combination of a DNA methyltransferase inhibitor and Compound (I) was examined as changes in the expression levels of γH2AX, an indicator of DNA damage, and cleaved caspase-3, an indicator of apoptosis. THP-1 cells or MV-4-11 cells were cultured at 2 × 10 ５The cells were seeded at 1000 cells / well in a 12-well plate and incubated with CO ２ The cells were cultured overnight in an incubator. The day after seeding, compound (I) and azacytidine or decitabine were added to the cells, respectively, and incubated for an additional 24 hours. 24 hours after drug addition, the cells were lysed using RIPA buffer, and proteins were collected. Expression of each protein was examined by Western blotting using anti-γH2AX antibody and anti-cleaved caspase 3 antibody (Cell Signaling Technologies). As shown in Figures 2A and 2B, the presence of compound (I) promoted the induction of γH2AX and the cleavage of caspase 3 by a DNA methyltransferase inhibitor. This indicates that the combined use of compound (I) and a DNA methyltransferase inhibitor promotes DNA damage and cell death in hematological cancers.

[0023] Example 3 Evaluation of Apoptosis and Cell Death Induction The induction of apoptosis by the combination of a DNA methyltransferase inhibitor and Compound (I) was investigated. THP-1 cells were cultured at 1 × 10 ５ The cells were seeded in a 24-well plate at 1000 cells / well, and Compound (I) and azacytidine were added to the cells. ２ The cells were cultured in an incubator for 48 hours and then harvested. The cell suspension was mixed with an equal volume of Guava Nexin Reagent (Millipore) and analyzed using an SA3800 flow cytometer (Sony). As shown in Figure 3, the percentage of Annexin V-positive, 7-AAD-positive apoptotic dead cells was significantly increased in the group treated with Compound (I) compared to the group treated with azacitidine alone, demonstrating a strong combined effect between the two drugs.

[0024] Example 4 Evaluation of the combined drug effect of a DNA methyltransferase inhibitor and compound (I) by isobologram method The isobologram method is a method for evaluating whether the combined effect of two drugs used in combination is additive, synergistic, or antagonistic (Chou Cancer Res. 70(2):440-6(2010)). Using this isobologram method, the combined effect of a DNA methyltransferase inhibitor and compound (I) on the growth inhibition of blood cancer cells was analyzed. Based on the results of cell growth inhibition tests using single agents for each cell line, the IC ５０ The IC value is calculated based on the concentration corresponding to the ５０ The two drugs were mixed at six ratios: 1:0, 5:1, 3:1, 1:1, 1:5, and 0:1, and the final concentration of the mixture was adjusted to IC ５０ The resazurin reagent was added to the cell plates of each cell line at concentrations between 0.003 and 30 times the original concentration (final DMSO concentration: 0.3%). After 72 hours of culture, the resazurin reagent was added, and after 3 hours, the fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The IC value of each mixture ratio was calculated. ５０ The IC value of the single drug was calculated. ５０ From the values ​​and the mixing ratio of the two drugs, the concentrations of compound (I) and the DNA methyltransferase inhibitor required to exhibit 50% growth inhibitory activity were calculated, and the results are shown in Figure 4, where the vertical axis represents the concentration of the DNA methyltransferase inhibitor and the horizontal axis represents the concentration of compound (I).

[0025] (Evaluation by isobologram) The dose at which drug A and drug B show a certain effect alone is D Ａ , D Ｂ When the dose-response curves of both drugs are parallel, the dose at which the combined use of both drugs produces the same effect as when they are administered alone is represented by line D. Ａ D Ｂ If it is above, it is additive, line D Ａ D Ｂ If it is at the bottom left of Ａ D ＢIn this test, as shown in Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E, Figure 4F, and Figure 4G, the concentration of each mixture ratio required to show 50% growth inhibition was determined to be equal to the IC50 of the single agent. ５０ The results of Example 4 indicate that the combined use of Compound (I) of the present invention and a DNA methyltransferase inhibitor has a strong synergistic effect, as the results are located to the lower left of the straight line connecting the values.

[0026] Example 5 Evaluation of the combined drug effect of a DNA methyltransferase inhibitor and Compound (I) by median-effect analysis Median-effect analysis is a theory proposed by Chou and Talalay, and is a method for evaluating whether the combined effect of two drugs used in combination is additive, synergistic, or antagonistic (Chou Cancer Res., 70(2):440-6(2010)). When the drug concentration is D, the 50% inhibitory concentration (median-effect dose) is Dm, the fraction of suppressed cells is Fa (fraction affected), the fraction of unsuppressed cells is Fu (fraction unaffected), and m is a coefficient, D = Dm (Fa / Fu). １／ｍ The combination index (CI), which is a quantitative indicator of the combined effect, is calculated by the following equation: Ａ＋Ｂ ) Ａ , x% inhibitory concentration of A alone is (Dx) Ａ , the concentration of drug B when used in combination (D Ａ＋Ｂ ) Ｂ , x% inhibitory concentration of B alone is (Dx) Ｂ Then, CI = (D Ａ＋Ｂ ) Ａ / (Dx) Ａ + (D Ａ＋Ｂ ) Ｂ / (Dx) Ｂ is given by

[0027] As described above, CI is expressed as a function of Fa, and a correlation diagram called an Fa-CI plot is obtained by plotting Fa on the horizontal axis and CI on the vertical axis. A CI of less than 1 indicates synergism, 1 indicates additive action, and a CI of greater than 1 indicates antagonism. Using the experimental data of the isobologram in Example 4, the combined use coefficient CI of a DNA methyltransferase inhibitor and compound (I) in blood cancer cells when Fa = 0.5 was determined. As shown in Tables 3 and 4, the CI was less than 1, indicating that the combined use of a DNA methyltransferase inhibitor and compound (I) had a strong synergistic effect. Example 6 Evaluation of the effect of triple-drug combinations in which compound (I) was combined with a DNA methyltransferase inhibitor and a BCL-2 inhibitor The CI for triple-drug combinations was determined using a method similar to the Chou-Talalay median-effect analysis in Example 5. The concentration of drug A in the combined use was (D Ａ＋Ｂ＋Ｃ ) Ａ , x% inhibitory concentration of A alone is (Dx) Ａ , the concentration of drug B when used in combination (D Ａ＋Ｂ＋Ｃ ) Ｂ , x% inhibitory concentration of B alone is (Dx) Ｂ , the concentration of drug C when used in combination (D Ａ＋Ｂ＋Ｃ ) Ｃ , x% inhibitory concentration of C alone is (Dx) Ｃ Then, CI = (D Ａ＋Ｂ＋Ｃ ) Ａ / (Dx) Ａ + (D Ａ＋Ｂ＋Ｃ ) Ｂ / (Dx) Ｂ + (D Ａ＋Ｂ＋Ｃ ) Ｃ / (Dx) Ｃ The combined use index CI of a DNA methyltransferase inhibitor, a BCL-2 inhibitor and Compound (I) in blood cancer cells was determined when Fa=0.5. As shown in Figure 5 and Table 5, the CI was smaller when compound (I) was used in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor than when compound (I) was used in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor, indicating that the triple combination of compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor had a stronger synergistic effect. Example 7 Antitumor Effect of Combination Use of Compound (I) with a DNA methyltransferase inhibitor The drug combination effect of compound (I) with DNA methyltransferase inhibitors (azacytidine, decitabine) was examined using a nude mouse subcutaneous transplant model of the MV-4-11 cell line. (Preparation of Tumor-Bearing Model) MV-4-11 cells cultured in the same manner as in Test Example 1 were cultured at a cell density of 2.5 x 10 ７ The cells were adjusted to a concentration of 1000 cells / mL with HBSS (Nacalai Tesque) containing Matrigel (50%, BD) to prepare a cell preparation solution for transplantation. 0.1 mL of this cell preparation solution for transplantation was injected subcutaneously into the dorsal skin of BALB / c Slc-nu / nu mice (female, 5 weeks old, Japan SLC). 20 days after cancer cell transplantation, the tumor-bearing mice were divided into groups (n=8) so that their mean tumor volumes (see the calculation formula below) were similar. (Preparation of a sample solution for administration of the test substance) The dihydrochloride of compound (I) was dissolved in 0.1 M HCl / 0.5% methylcellulose solution (solvent A) to a concentration of 6 mg / mL in terms of the free form to prepare a sample solution of compound (I). Azacitidine was dissolved in a 5% (v / v) DMSO / 30% (w / v) PEG300 aqueous solution (solvent B) to a concentration of 0.5 mg / mL to prepare a sample solution for administration of azacitidine. Decitabine was dissolved in distilled water (solvent C) to a concentration of 0.025 mg / mL to prepare a sample solution for administration of decitabine. (Antitumor Effect Test) Each mouse (n=7-8) implanted with cancer cells was administered azacitidine (Table 6) or decitabine (Table 7) in an amount determined from the body weight on each day according to the administration schedule of each test substance, twice daily (BID, at least 5 hours apart) or once daily (QD), orally (PO) or intraperitoneally (IP). The tumor volume of each mouse was calculated using the following formula to evaluate the antitumor effect: Tumor volume = major axis × minor axis × minor axis × 0.5 Off indicates a day off medication. "Off" indicates a drug holiday. In this study, as shown in Figures 6A and 6B, tumor growth was most suppressed in the group treated with the combination of compound (I) and a DNA methyltransferase inhibitor (azacytidine, decitabine). The results of Example 7 indicate that the combination of compound (I) of the present invention and a DNA methyltransferase inhibitor also has a strong synergistic effect in vivo. Example 8 Antitumor Effect of Triple-Drug Combination of Compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor The effect of the triple-drug combination was examined using a nude mouse subcutaneously transplanted MV-4-11 cell line model in the same manner as in Example 7. (Preparation of Sample Solution for Administration of Test Substance) The dihydrochloride of compound (I) was dissolved in 0.1 M HCl / 0.5% methylcellulose solution (solvent A) to a concentration of 3 mg / mL in terms of the free form to prepare a sample solution of compound (I). Azacitidine was dissolved in a 5% (v / v) DMSO / 30% (w / v) PEG300 aqueous solution (solvent B) at 0.25 mg / mL and 0.125 mg / mL to prepare azacitidine administration sample solutions. Decitabine was dissolved in distilled water (solvent C) at 0.025 mg / mL to prepare a decitabine administration sample solution. Venetoclax was dissolved in a 5% (v / v) DMSO / 50% (w / v) PEG300 / 5% (w / v) Tween 80 aqueous solution (solvent D) at 0.5 mg / mL and 0.25 mg / mL to prepare a venetoclax administration sample solution. (Antitumor Effect Test) On the 19th day after the transplantation of cancer cells, the mice were divided into groups, and each of the cancer cell-transplanted mice (n=6-8) was orally (PO) or intraperitoneally (IP) administered with the test substance at a dose calculated from the body weight on each day according to the administration schedule of each test substance in Table 8 or Table 9, twice a day (BID, at least 5 hours apart) or once a day (QD), and the antitumor effect was evaluated. Off indicates a day off medication. "Off" indicates a drug holiday. In this test, as shown in Figures 7A and 7B, tumor growth was most suppressed in the triple-drug combination group of compound (I), a DNA methyltransferase inhibitor, and a BCL-2 inhibitor. The results of Example 8 demonstrate that the triple-drug combination of compound (I), a DNA methyltransferase inhibitor, and a BCL-2 inhibitor of the present invention also has a strong synergistic effect in vivo.

[0028] The present invention provides a more effective therapeutic method for blood cancer by combining two drugs, compound (I) and a DNA methyltransferase inhibitor, and by combining three drugs, compound (I), a DNA methyltransferase inhibitor, and a BCL-2 inhibitor, as well as a combination of pharmaceutical compositions or a kit for carrying out the therapeutic method.

Claims

1. Compound (I), administered to a patient in combination with a DNA methyltransferase inhibitor for use in treating hematological cancers: A pharmaceutical composition comprising a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient.

2. The pharmaceutical composition of claim 1, which is administered to a patient in combination with a BCL-2 inhibitor in addition to a DNA methyltransferase inhibitor for use in treating hematological cancers.

3. The pharmaceutical composition of claim 1 or 2, wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

4. The pharmaceutical composition of claim 1 or 2, wherein the blood cancer is myelodysplastic syndrome (MDS).

5. The pharmaceutical composition of claim 1 or 2, wherein the blood cancer is acute myeloid leukemia (AML).

6. The pharmaceutical composition according to claim 1 or 2, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising, as an active ingredient, azacitidine, a pharma- ceutically acceptable salt or hydrate thereof, or decitabine, a pharma- ceutically acceptable salt or hydrate thereof.

7. The pharmaceutical composition according to claim 6, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition containing azacitidine, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient.

8. The pharmaceutical composition according to claim 6, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising decitabine, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient.

9. The pharmaceutical composition according to claim 7, wherein a pharmaceutical composition comprising azacitidine, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient and a pharmaceutical composition comprising compound (I), a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient are both administered simultaneously or separately, sequentially.

10. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition comprising decitabine, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient and the pharmaceutical composition comprising compound (I), a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient are both administered simultaneously or separately, sequentially.

11. The pharmaceutical composition of claim 2, wherein the BCL-2 inhibitor is a pharmaceutical composition comprising venetoclax, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

13. The pharmaceutical composition according to claim 12, wherein the blood cancer is myelodysplastic syndrome (MDS).

14. The pharmaceutical composition according to claim 12, wherein the blood cancer is acute myeloid leukemia (AML).

15. A kit comprising a pharmaceutical composition containing, as active ingredients, a DNA methyltransferase inhibitor and compound (I) or a pharma- ceutically acceptable salt or hydrate thereof, for use in the combined administration of claim 1.

16. A kit comprising a pharmaceutical composition containing, as active ingredients, a DNA methyltransferase inhibitor, a BCL-2 inhibitor and compound (I) or a pharma- ceutical acceptable salt or hydrate thereof, for use in the combined administration of claim 2.

17. A method for treating blood cancer, comprising administering to a patient a pharmaceutical composition containing, as an active ingredient, compound (I) or a pharma- ceutically acceptable salt or hydrate thereof in combination with a DNA methyltransferase inhibitor.

18. A method for treating blood cancer, comprising administering to a patient a pharmaceutical composition containing compound (I) or a pharma- ceutical acceptable salt or hydrate thereof as an active ingredient, in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor.

19. The method of claim 17 or 18, wherein the blood cancer is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

20. The method of claim 17 or 18, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition comprising, as an active ingredient, azacitidine, a pharma- ceutically acceptable salt or hydrate thereof; or decitabine, a pharma- ceutically acceptable salt or hydrate thereof.

21. The method of treatment according to claim 18, wherein the BCL-2 inhibitor is a pharmaceutical composition comprising venetoclax, a pharma- ceutically acceptable salt or hydrate thereof as an active ingredient.

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