Novel combination therapy for hematologic cancer

Abstract

The present invention provides a novel therapeutic means for hematologic cancer based on: 1) a two-agent combination of a DNA methyltransferase inhibitor and a pharmaceutical composition comprising compound (I), a pharmaceutically acceptable salt or hydrate thereof as an active ingredient; and 2) a three-agent combination of a DNA methyltransferase inhibitor, a BCL-2 inhibitor, and a pharmaceutical composition comprising compound (I), a pharmaceutically acceptable salt thereof, or a hydrate thereof as an active ingredient.

Description

Technical Field

[0001] This invention relates to novel treatment methods for hematologic malignancies. More specifically, it relates to providing novel treatment methods based on the combination of: compound (I), a pharmaceutically acceptable salt or hydrate thereof, and a DNA methyltransferase inhibitor; or further combining a BCL-2 inhibitor into said combination of compound (I), a pharmaceutically acceptable salt or hydrate thereof, and a DNA methyltransferase inhibitor. Background Technology

[0002] Blood cancers are a general term for diseases in which blood cells become cancerous, broadly classified into leukemia or malignant lymphoma, multiple myeloma, etc. In recent years, various treatment methods and anticancer agents have been developed for the treatment of these blood cancers.

[0003] Myelodysplastic syndrome (MDS) is a disease in which hematopoietic stem cells in the bone marrow undergo abnormalities, resulting in an inability to produce normal blood. Approximately 30% of MDS patients will progress to acute myeloid leukemia (AML). In recent years, abnormal DNA methylation, such as inducing the inactivation of tumor suppressor genes, has been considered as one of the causes of cancer development or leukemia.

[0004] To inhibit this abnormal DNA methylation, DNA methyltransferase inhibitors have been developed. DNA methyltransferase inhibitors, such as azacitidine and decitabine, have chemical structures similar to cytidine, allowing them to integrate into DNA in vivo. They inhibit DNA methyltransferases, thereby suppressing DNA methylation, inducing DNA damage and apoptosis, and exerting anti-tumor effects.

[0005] DNA methyltransferase inhibitors such as azacitidine and decitabine have been used as treatments for MDS and AML, but the lack of efficacy or drug resistance during treatment has become a clinical challenge (Non-Patent Literature 1).

[0006] BCL-2 (B-cell lymphoma 2) is a member of the BCL-2 family that regulates cell death and is known to inhibit apoptosis in some hematologic malignancies. To date, several BCL-2 inhibitors, represented by venetoclax, have been reported. These inhibitors selectively bind to BCL-2 to inhibit its activity and are known to strongly induce apoptosis, demonstrating antitumor effects, whether as a single agent or in combination with other therapeutic agents (Non-Patent Literature 2). Venetoclax has been approved as a treatment for AML patients in combination with DNA methyltransferase inhibitors such as azacitidine or decitabine. However, this combination therapy still suffers from serious side effects that make continued treatment difficult or insufficient efficacy, thus prompting a desire to develop newer treatment methods (Non-Patent Literature 3).

[0007] CDC7 (cell division cycle 7) is a serine / threonine kinase essential for initiating DNA replication during the cell cycle. CDC7 forms a complex with cofactors such as Dbf4 (ASK) that activate its phosphorylation, and phosphorylates the MCM (minichromosome maintenance) protein, which serves as a substrate. It is believed that through this phosphorylation, Cdc45 and DNA polymerase aggregate on DNA, forming the MCM complex, thereby initiating DNA replication (Non-Patent Literature 4). In recent years, CDC7 has attracted much attention as a target for anticancer agents, and AS-0141, which selectively and potently inhibits CDC7, has shown antitumor activity against various cancer cell lines (Non-Patent Literature 5).

[0008] AS-0141 is a compound 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)].

[0009] Compound (I) is disclosed in International Publication No. 2012 / 133802 (Patent Document 1) and Non-Patent Document 5. Furthermore, the enhanced anticancer effect of compound (I) in combination with M-phase promoters such as Wee1 inhibitors is disclosed (Patent Document 2). A cancer treatment method comprising compound (I) is disclosed in International Publication No. 2018 / 84266 (Patent Document 3). A treatment method in which SRA141 (AS-0141) is administered in combination therapy is disclosed in International Publication No. 2020 / 68347 (Patent Document 4).

[0010] Existing technical documents Patent documents Patent Document 1: International Publication No. 2012 / 133802 Patent Document 2: International Publication No. 2015 / 115355 Patent Document 3: International Publication No. 2018 / 84266 Patent Document 4: International Publication No. 2020 / 68347 Non-patent literature Non-patent literature 1: Zhao G. et al., Front Oncol. 2021 Sep 28; 11: 706030 Non-patent literature 2: Ashkenazi A. et al., Nature Rev. Drug Discov., 2017, 16, 273-284 Non-patent literature 3: Estey EH., Am J Hematol. 2020 Nov; 95 (11): 1368-1398 Non-patent literature 4: Sawa M, Masai H., Drug Des Devel Ther., 2008 Feb 6; 2: 255-264 Non-patent literature 5: Irie T. et al., J Med Chem. 2021 Oct 14; 64 (19): 14153-14164. Summary of the Invention

[0011] The problem that the invention aims to solve The purpose of this invention is to provide a new treatment method for treating patients with blood cancer that is more effective and has fewer side effects.

[0012] Methods for solving problems There are no reports on the combination of compound (I), its pharmaceutically acceptable salt or hydrate, and a DNA methyltransferase inhibitor. There are also no reports on the enhanced anticancer effect against hematologic malignancies achieved by combining compound (I), its pharmaceutically acceptable salt or hydrate, and a DNA methyltransferase inhibitor. Furthermore, there are no reports on a three-dose combination of compound (I), its pharmaceutically acceptable salt or hydrate, a DNA methyltransferase inhibitor, and a BCL-2 inhibitor; the enhancement of the anticancer effect against hematologic malignancies by this three-dose combination is completely unknown.

[0013] In order to solve the aforementioned problem, the inventors conducted in-depth research and found that by combining 1) compound (I), its pharmaceutically acceptable salt or hydrate, with 2) a DNA methyltransferase inhibitor, and further combining 3) a BCL-2 inhibitor in addition to the above-mentioned combination of 1) and 2), the problem of the present invention was solved due to their synergistic effect, thus completing the present invention.

[0014] More specifically, this invention relates to the following. It should be noted that, unless otherwise specified, in this application specification, compound (I) also includes its pharmaceutically acceptable salts or hydrates. The same applies to azacitidine and decitabine.

[0015] (1) A pharmaceutical composition comprising compound (I) as an active ingredient, administered to a patient in combination with a DNA methyltransferase inhibitor for the treatment of hematologic malignancies. .

[0016] (2) (1) The pharmaceutical composition, which is given to patients in combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor, is used to treat blood cancers.

[0017] (3) The pharmaceutical composition of (1) or (2), wherein the hematologic malignancy is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

[0018] (4) The pharmaceutical composition of (1) or (2), wherein the hematologic cancer is myelodysplastic syndrome (MDS).

[0019] (5) The pharmaceutical composition of (1) or (2), wherein the hematologic cancer is acute myeloid leukemia (AML).

[0020] (6) A pharmaceutical composition of (1) or (2), wherein the DNA methyltransferase inhibitor is a pharmaceutical composition containing azacitidine or decitabine as an active ingredient.

[0021] (7) (6) pharmaceutical compositions, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition containing azacitidine as an active ingredient.

[0022] (8) (6) pharmaceutical compositions, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition containing decitabine as an active ingredient.

[0023] (9) (7) pharmaceutical compositions, wherein the pharmaceutical composition containing azacitidine as an active ingredient and the pharmaceutical composition containing compound (I) as an active ingredient are administered simultaneously or sequentially.

[0024] (10) (8) pharmaceutical compositions, wherein the pharmaceutical composition containing decitabine as an active ingredient and the pharmaceutical composition containing compound (I) as an active ingredient are administered simultaneously or sequentially.

[0025] (11) (2) pharmaceutical compositions, wherein the BCL-2 inhibitor is a pharmaceutical composition containing Veneclare as an active ingredient.

[0026] (12) The pharmaceutical composition of any one of (9) to (11), wherein the hematologic malignancy is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

[0027] (13) (12) The pharmaceutical composition wherein the hematologic cancer is myelodysplastic syndrome (MDS).

[0028] (14) (12) The pharmaceutical composition wherein the hematologic cancer is acute myeloid leukemia (AML).

[0029] (15) A kit comprising: a pharmaceutical composition comprising a DNA methyltransferase inhibitor and compound (I) as active ingredients, for combined administration of (1).

[0030] (16) A kit comprising: a pharmaceutical composition comprising a DNA methyltransferase inhibitor, a BCL-2 toxicant and compound (I) as active ingredients, for combined administration of (2).

[0031] (17) A treatment for blood cancer, wherein a pharmaceutical composition comprising compound (I) as an active ingredient is combined with a DNA methyltransferase inhibitor and administered to a patient.

[0032] (18) A treatment for blood cancer, wherein a pharmaceutical composition comprising compound (I) as an active ingredient is combined with a DNA methyltransferase inhibitor and a BCL-2 inhibitor and administered to a patient.

[0033] Treatment methods of (19) (17) or (18), wherein the hematologic malignancy is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).

[0034] (20) (17) or (18) treatment methods, wherein the DNA methyltransferase inhibitor is a pharmaceutical composition containing azacitidine or decitabine as an active ingredient.

[0035] (21) (18) treatment methods, wherein the BCL-2 inhibitor is a pharmaceutical composition containing Venecra as an active ingredient.

[0036] Invention Effects Combining compound (I), its pharmaceutically acceptable salt or hydrate, with a DNA methyltransferase inhibitor can induce cell death with greater efficiency compared to using compound (I) and the DNA methyltransferase inhibitor alone. CDC7 inhibitors cause genomic instability in cancer cells, thereby inducing DNA damage and apoptosis. On the other hand, DNA methyltransferase inhibitors induce DNA damage and apoptosis by inhibiting abnormal DNA methylation in cancer cells and activating tumor suppressor genes. Therefore, the combination of these compounds (I) with DNA methyltransferase inhibitors is expected to enhance anticancer effects and achieve high efficacy against various hematologic malignancies.

[0037] Furthermore, by combining compound (I), its pharmaceutically acceptable salt or hydrate, with a DNA methyltransferase inhibitor and a BCL-2 inhibitor in a three-dose combination, synergistic effects can be expected, potentially leading to high efficacy against various hematologic malignancies. Attached Figure Description

[0038] [ Figure 1 ] Figure 1 This is a graph showing the cell proliferation inhibition rate of the combination of DNA methyltransferase inhibitor (azacitidine) and compound (I) on human acute monocytic leukemia cell line (THP-1).

[0039] [ Figure 2A ] Figure 2A This figure illustrates the effect of the combination of a DNA methyltransferase inhibitor (azacitidine) and compound (I) on THP-1 cells by studying changes in the expression levels of γH2AX (a marker of DNA damage) and cleaved caspase 3 (a marker of apoptosis).

[0040] [ Figure 2B ] Figure 2B This figure illustrates the effects of a combination of a DNA methyltransferase inhibitor (decitabine) and compound (I) on MV-4-11 cells by studying changes in the expression levels of γH2AX (a marker of DNA damage) and caspase-3 cleavage (a marker of apoptosis).

[0041] [ Figure 3 ] Figure 3 This is a diagram showing the effect of a combination of DNA methyltransferase inhibitor (azacitidine) and compound (I) on apoptosis in THP-1 cells.

[0042] [ Figure 4A ] Figure 4AThis is an equivalent line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (azacitidine) and compound (I) on THP-1 cells.

[0043] [ Figure 4B ] Figure 4B This is an isoelectric line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (azacitidine) and compound (I) on the human acute myeloid leukemia cell line (MV-4-11).

[0044] [ Figure 4C ] Figure 4C This is an isoline diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (azacitidine) and compound (I) on the human acute monocytic leukemia cell line (MOLM-14).

[0045] [ Figure 4D ] Figure 4D This is an isoelectric line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (azacitidine) and compound (I) on the human erythroleukemia cell line (TF-1).

[0046] [ Figure 4E ] Figure 4E This is an equivalent line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (decitabine) and compound (I) on the human acute myeloid leukemia cell line (MV-4-11).

[0047] [ Figure 4F ] Figure 4F This is an isoelectric line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (decitabine) and compound (I) on the human acute monocytic leukemia cell line (MOLM-14).

[0048] [ Figure 4G ] Figure 4G This is an equivalent line diagram (50% inhibition) showing the interaction between the DNA methyltransferase inhibitor (decitabine) and compound (I) on the human erythroleukemia cell line (TF-1).

[0049] [ Figure 5 ] Figure 5 This is a Fa-CI plot representing the interaction of a combination of DNA methyltransferase inhibitor (azacitidine), BCL-2 inhibitor (venecla) and compound (I) on human acute myeloid leukemia cell line (MV-4-11).

[0050] [ Figure 6A ] Figure 6A The results indicate that in a mouse subcutaneous transplantation model of the human acute myeloid leukemia cell line (MV-4-11), the combination of compound (I) and the DNA methyltransferase inhibitor (azacitidine) inhibited tumor proliferation.

[0051] [ Figure 6B ] Figure 6B The results indicate that in a mouse subcutaneous transplantation model of the human acute myeloid leukemia cell line (MV-4-11), the combination of compound (I) and the DNA methyltransferase inhibitor (decitabine) inhibited tumor proliferation.

[0052] [ Figure 7A ] Figure 7A The results indicate that in a mouse subcutaneous transplantation model of human acute myeloid leukemia cell line (MV-4-11), the combination of compound (I), a DNA methyltransferase inhibitor (azacitidine), and a BCL-2 inhibitor (venecla) inhibited tumor proliferation.

[0053] [ Figure 7B ] Figure 7B The results indicate that in a mouse subcutaneous transplantation model of human acute myeloid leukemia cell line (MV-4-11), the combination of compound (I), a DNA methyltransferase inhibitor (decitabine), and a BCL-2 inhibitor (venecla) inhibited tumor proliferation. Detailed Implementation

[0054] (1) DNA methyltransferase inhibitors In this invention, a DNA methyltransferase inhibitor refers to a pharmaceutical composition containing a drug that inhibits DNA methyltransferase activity as an active ingredient. This includes pharmaceutical compositions containing low-molecular-weight compounds, peptides, proteins, nucleic acids (siRNA, miRNA, aptamers, etc.), and other high-molecular-weight compounds that inhibit the physiological function of DNA methyltransferases. Additionally, it also includes agents that are activated in vivo to inhibit the physiological function of DNA methyltransferases, i.e., prodrugs of DNA methyltransferase inhibitors.

[0055] Drugs that inhibit DNA methyltransferase activity include, for example, azacitidine and decitabine, which have the following structures, as well as their pharmaceutically acceptable salts, hydrates and prodrugs.

[0056] (2) BCL-2 inhibitors The BCL-2 inhibitors used in this invention refer to agents that inhibit the physiological function of BCL-2 in cells, including low molecular weight compounds, peptides, proteins, nucleic acids (siRNA, miRNA, aptamers, etc.), and other high molecular weight compounds that inhibit the physiological function of BCL-2.

[0057] BCL-2 is a member of the BCL-2 family that regulates cell death, negatively regulating apoptosis. Besides follicular lymphoma, DLBCL, and CLL, which are lymphoid B-cell tumors, BCL-2 is also activated in multiple myeloma and T-cell tumors. Since BCL-2 inhibitors induce apoptosis in these cancer cells, they exhibit anti-tumor effects. Therefore, further anti-tumor effects can be expected not only through monotherapy but also through combination with other agents.

[0058] Examples of BCL-2 inhibitors include: veneclax, navitoclax, obatoclax, obatoclax mesylate, sabutoclax, APG-1252, AZD-0466, APG-2575, ABBV-167, S-65487, and S-55746.

[0059] (3) Pharmaceutical compositions containing compound (I) In this invention, compound (I) refers to the furanone derivative (I) shown in 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. Pharmaceutically acceptable salts, hydrates, and prodrugs are also included in compound (I).

[0060] 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 strongly inhibits CDC7.

[0061] Furthermore, pharmaceutically acceptable salts of compound (I) used in this invention include: inorganic acid salts with hydrochloric acid, sulfuric acid, carbonic acid, phosphoric acid, etc.; organic acid salts with fumaric acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. Additionally, 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.; and ammonium salts are also included in this invention.

[0062] The compound (I) used in this invention, its pharmaceutically acceptable salt or hydrate, can be formulated into a conventional pharmaceutical preparation (pharmaceutical composition) suitable for oral, parenteral, or topical administration.

[0063] Formulations for oral administration include solid dosage forms such as tablets, granules, powders, and capsules, as well as liquid dosage forms such as syrups. These formulations can be prepared using conventional methods. Solid dosage forms can be prepared using conventional drug carriers such as lactose, starches such as corn starch, crystalline cellulose such as microcrystalline cellulose, hydroxypropyl cellulose, calcium carboxymethyl cellulose, talc, magnesium stearate, etc. Capsules can be prepared by encapsulating such prepared granules or powders in capsules. Syrups can be prepared by dissolving or suspending the compounds of the present invention, their pharmaceutically acceptable salts, or hydrates in an aqueous solution containing sucrose, carboxymethyl cellulose, etc.

[0064] Preparations intended for parenteral administration include injectable formulations such as intravenous infusions. Injectable formulations can also be prepared using conventional methods and may be appropriately added to 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, methylparaben).

[0065] (4) Combination therapy In the combination therapy of the present invention, a pharmaceutical composition comprising compound (I) as an active ingredient is administered to patients with hematologic malignancies in a two-dose combination with a DNA methyltransferase inhibitor or in a three-dose combination with a DNA methyltransferase inhibitor and a BCL-2 inhibitor.

[0066] One aspect of the invention also includes a kit comprising: a pharmaceutical composition comprising compound (I) as an active ingredient, for combination administration with a DNA methyltransferase inhibitor, and a pharmaceutical composition comprising both a DNA methyltransferase inhibitor and compound (I) as active ingredients. The kit may include instructions for use for both pharmaceutical preparations.

[0067] 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.

[0068] 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.

[0069] Another aspect of the invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered prior to the administration of a DNA methyltransferase inhibitor.

[0070] One aspect of the invention also includes a kit comprising: a pharmaceutical composition containing compound (I) as an active ingredient, for combined administration with a DNA methyltransferase inhibitor and a BCL-2 inhibitor; and a pharmaceutical composition containing a DNA methyltransferase inhibitor, a BCL-2 inhibitor, and compound (I) as active ingredients, respectively. The kit may include instructions for use for both pharmaceutical preparations.

[0071] 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.

[0072] Another aspect of the invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered after the administration of a DNA methyltransferase inhibitor and a BCL-2 inhibitor.

[0073] Another aspect of the invention is a pharmaceutical composition comprising compound (I) as an active ingredient, which is administered prior to the administration of a DNA methyltransferase inhibitor and a BCL-2 inhibitor.

[0074] Azacitidine, decitabine, and veneclade are already marketed for the treatment of hematologic malignancies. Those skilled in the art can consider the type of hematologic malignancy, the patient's weight, age, disease severity, etc., and, based on the possibility of administering these drugs as monotherapy, appropriately determine the dosage of azacitidine or decitabine in the combination therapy of this invention.

[0075] In addition, those skilled in the art may consider the type of blood cancer, the patient's weight, age, the severity of the disease, etc., and based on the administration of these drugs as a combination therapy, appropriately determine the dosage of DNA methyltransferase inhibitors such as azacitidine and BCL-2 inhibitors such as veneclade in the three-dose combination therapy of the present invention.

[0076] The dosage of compound (I) used in this invention may vary depending on the severity of the disease, the patient's age and weight, the form of administration (dosage form), etc., but for adults, it is usually in the range of 10 mg to 400 mg per day, which may be administered once, twice or three times depending on the route of administration, whether orally or parenterally. Example

[0077] Experimental Example 1: Cell proliferation inhibition assay using a single drug (Cell Culture) Human acute monocytic leukemia cell lines (THP-1), human acute myeloid leukemia cell lines (MV-4-11), human acute monocytic leukemia cell lines (MOLM-14), and human erythroleukemia cell lines (TF-1) were cultured in a 5% CO2 incubator using RPMI 1640 medium (Roswell Park Memorial Institute medium, NACALAI) containing 10% fetal bovine serum (Thermo Scientific, SIGMA-ALDRICH, or Cytiva) and 1% penicillin-streptomycin (NACALAI).

[0078] (Cell proliferation inhibition assay) THP-1 cells and MV-4-11 cells were fed at a rate of 2 × 10⁻⁶. 4 Cells / well, MOLM-14 cells and TF-1 cells were cultured at 5 × 10⁻⁶ cells / well. 3 Cells / wells were seeded separately in 96-well plates (cell culture plates). The test compound, diluted with culture medium, was added to achieve a final concentration of 3 nM–30 μM (final DMSO concentration 0.3%). After 72 hours of incubation, resazurin reagent (NACALAI) was added. Three hours later, fluorescence was measured at an excitation wavelength of 560 nm and a fluorescence wavelength of 590 nm. Wells without the compound and without cells were set as 100%, and wells without the compound but with cells were set as 0%. The IC50 of the inhibitory activity was then determined. 50 Values. The results are shown in Table 1.

[0079] [Table 1] Example 1: Dose-responsiveness test of DNA methyltransferase inhibitor in the presence of compound (I) The IC50 of the DNA methyltransferase inhibitor (azacitidine) in the presence of compound (I) was investigated. 50 Value change.

[0080] Based on the results of compound (I) alone, a concentration of compound (I) was selected, with the DMSO-added group serving as a control. The compound (I) was added to the cell plate to achieve a concentration of 3 nM–30 μM (final DMSO concentration 0.4%), and the procedure was performed in the same manner as in Experiment 1. The IC50 in the presence of compound (I) was then determined. 50 value.

[0081] The IC50 of azacitidine in the presence of compound (I) when using THP-1 cells 50 Value changes are shown in Figure 1And in Table 2.

[0082] In this experiment, such as Figure 1 As shown in Table 2, in THP-1 cells, a human acute monocytic leukemia cell line, the IC50 of azacitidine in the presence of compound (I) was... 50 The value decreased.

[0083] [Table 2] The results of Example 1 show that the combination of compound (I) of the present invention and DNA methyltransferase inhibitor has a combined effect on hematologic cancer cells.

[0084] Example 2 Evaluation of DNA damage and apoptosis induction The effects of the combination of DNA methyltransferase inhibitors and compound (I) were investigated in terms of changes in the expression levels of γH2AX (a marker of DNA damage) and caspase-3 cleavage (a marker of apoptosis).

[0085] THP-1 cells or MV-4-11 cells were used at a rate of 2 × 10⁻⁶. 5 Cells were seeded in 12-well plates and cultured overnight in a CO2 incubator. The day after seeding, compound (I) and either azacitidine or decitabine were added to the cells, followed by incubation for another 24 hours. Twenty-four hours after drug addition, cells were lysed with RIPA buffer, and proteins were recovered. Protein expression was investigated by Western blot using anti-γH2AX antibody and anti-lysine caspase 3 antibody (Cell Signaling Technologies).

[0086] like Figure 2A , Figure 2B As shown, in the presence of compound (I), the induction of γH2AX induced by the DNA methyltransferase inhibitor and the cleavage of caspase 3 were both promoted. This demonstrates that the combination of compound (I) and the DNA methyltransferase inhibitor promotes DNA damage and cell death in hematologic malignancies.

[0087] Example 3 Evaluation of apoptosis and cell death induction The study investigated apoptosis induction based on a combination of a DNA methyltransferase inhibitor and compound (I).

[0088] THP-1 cells were fed at a rate of 1×10 5Cells were seeded in 24-well plates, and compound (I) and azacitidine were added to the cells, respectively. The cells were incubated in a CO2 incubator for 48 hours and then recovered. The cell suspension was mixed with an equal volume of Guava NexinREAGENT (Millipore) and analyzed using an SA3800 flow cytometer (Sony).

[0089] like Figure 3 As shown, compared with azacitidine monotherapy, the proportion of dead cells caused by apoptosis of annexin V-positive and 7-AAD-positive cells was significantly increased in the combination compound (I) group, indicating a strong synergistic effect between the two agents.

[0090] Example 4: Evaluation of the combined drug effect of DNA methyltransferase inhibitor and compound (I) based on isoline diagram method The isobologram method is one of the methods for evaluating whether a combination of two agents exhibits additive, synergistic, or antagonistic effects (Chou Cancer Res. 70 (2): 440-6 (2010)). Using the isobologram method, the combined effect of a DNA methyltransferase inhibitor and compound (I) on the inhibition of proliferation of hematologic malignancies was analyzed.

[0091] Based on the cell proliferation inhibition assay results of each cell line using single drugs, the IC50 values ​​were determined using compounds (I) or DNA methyltransferase inhibitors as single drugs. 50 ICs were fabricated based on the concentration of the value. 50 A DMSO solution with a concentration multiplied by 10,000. The two agents were mixed at six ratios: 1:0, 5:1, 3:1, 1:1, 1:5, and 0:1. This mixture was added to cell plates containing each cell line to achieve a final concentration of IC50. 50 The concentration was increased by 0.003 to 30 times (final DMSO concentration was 0.3%). After 72 hours of incubation, resazurin reagent was added, and fluorescence at an excitation wavelength of 560 nm and a fluorescence wavelength of 590 nm was measured after 3 hours. The IC50 values ​​for each mixing ratio were then determined. 50 value.

[0092] Based on the IC50 of a single drug 50 The values ​​and mixing ratios of the two agents were calculated to show the concentrations of compound (I) and DNA methyltransferase inhibitor required for 50% proliferation inhibition. The results of plotting the concentration of DNA methyltransferase inhibitor on the vertical axis and the concentration of compound (I) on the horizontal axis are shown in Figure 4.

[0093] (Evaluation based on equivalent line diagram) For drug A and drug B, the dosage that separately displays a certain effect will be designated as D. A DB When the dose-response curves of two drugs are parallel, if the dose at which the combination of the two drugs shows the same effect as when administered alone lies on the straight line D. A D B If the two lines are above each other, it is determined to be an additive effect. A D B If it is located to the lower left, it is considered a synergistic effect, and when it is located on line D... A D B When it is located in the upper right corner, it is considered an antagonistic reaction.

[0094] In this experiment, such as Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F , Figure 4G As shown, the concentrations of each mixture required to exhibit 50% proliferation inhibition are located at the IC50 values ​​of the single-drug conjugates. 50 The value is located to the lower left of the straight line, therefore the results of Example 4 show that the combination of the compound (I) of the present invention with the DNA methyltransferase inhibitor has a strong synergistic effect.

[0095] Example 5: Evaluation of the combined drug effect of DNA methyltransferase inhibitor and compound (I) based on median-effect analysis Median-effect analysis, a theory proposed by Chou and Talalay, is one of the methods for evaluating the additive, synergistic, or antagonistic effects of combined two-drug regimens (Chou Cancer Res., 70(2): 440-6 (2010)). When the drug concentration is set as D, the 50% inhibitory concentration (median-effect dose) as Dm, the inhibited cell rate as Fa (fraction affected), the uninhibited cell rate as Fu (fraction unaffected), and m as a coefficient, the following relationship holds: D = Dm(Fa / Fu) 1 / m .

[0096] The combination index (CI), a quantitative indicator of the combined effect, is used when the point of action of the two-drug combination is exclusive. If the concentration of drug A during combination is set as (D... A+B ) A Let the x% inhibitory concentration of drug A be set as (Dx). ALet the concentration of agent B during combination be set as (D). A+B ) B Let the x% inhibitory concentration of drug B be set as (Dx). B Then the following formula is given: CI=( D A+B ) A / ( Dx ) A +( D A+B ) B / ( Dx ) B .

[0097] Based on the above, CI is represented by a function of Fa, and the plot with Fa as the horizontal axis and CI as the vertical axis is called the Fa-CI plot. A CI less than 1 indicates a synergistic effect, a CI of 1 indicates an additive effect, and a CI greater than 1 indicates an antagonistic effect. Using the experimental data from the isoline plot of Example 4, the combination index CI of the DNA methyltransferase inhibitor and compound (I) in hematological malignancies with Fa=0.5 was determined.

[0098] [Table 3] [Table 4] As shown in Tables 3 and 4, CI is less than 1, indicating that the combination of DNA methyltransferase inhibitor and compound (I) has a strong synergistic effect.

[0099] Example 6 Evaluation of the three-dose combination of compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor The CI for the three-dose combination was determined using the same method as the Median-effect analysis of Chou-Talalay in Example 5.

[0100] If the concentration of drug A during combination is set as (D A+B+C ) A Let the x% inhibitory concentration of drug A be set as (Dx). A Let the concentration of agent B during combination be set as (D). A+B+C ) B Let the x% inhibitory concentration of drug B be set as (Dx). B Let the concentration of agent C during combination be set as (D A+B+C ) C Let the x% inhibitory concentration of drug C be set as (Dx). C Then the following formula is given: CI=(DA+B+C ) A / (Dx) A +(D A+B+C ) B / (Dx) B +(D A+B+C ) C / (Dx) C .

[0101] The combined index CI of DNA methyltransferase inhibitor, BCL-2 inhibitor and compound (I) in hematologic cancer cells with Fa=0.5 was calculated.

[0102] [Table 5] like Figure 5 As shown in Table 5, compared with the two-dose combination of DNA methyltransferase inhibitor and BCL-2 inhibitor, the CI of the three-dose combination of compound (I) with DNA methyltransferase inhibitor and BCL-2 inhibitor was smaller, indicating that the three-dose combination of compound (I) with DNA methyltransferase inhibitor and BCL-2 inhibitor had a stronger synergistic effect.

[0103] Example 7 Antitumor effect of compound (I) in combination with DNA methyltransferase inhibitor The combined effects of compound (I) with DNA methyltransferase inhibitors (azacitidine and decitabine) were studied using a nude mouse subcutaneous transplantation model of MV-4-11 cell line.

[0104] (Preparation of the tumor-bearing model) MV-4-11 cells cultured in the same manner as in Example 1 were conditioned using HBSS (NACALAI TESQUE) containing 50% matrix gel (BD) to achieve a cell density of 2.5 × 10⁻⁶ cells. 7 A cell preparation solution for transplantation was prepared at a concentration of cells per mL. 0.1 mL of this cell preparation solution was injected subcutaneously into the back of BALB / c Slc-nu / nu mice (female, 5 weeks old, SLC Corporation, Japan). On day 20 post-transplantation of cancer cells, mice were grouped (n=8) based on similar average tumor volumes (calculated as shown below).

[0105] (Preparation of sample solution for administration of the analyte) The dihydrochloride salt of compound (I) was dissolved in 0.1M HCl / 0.5% methylcellulose solution (solvent A) to achieve a concentration of 6 mg / mL on a free basis, thus preparing a sample solution of compound (I) for administration.

[0106] Azacitidine was dissolved in a 5% (v / v) DMSO / 30% (w / v) PEG300 aqueous solution (solvent B) to achieve a concentration of 0.5 mg / mL, thus preparing a sample solution for azacitidine administration.

[0107] Decitabine was dissolved in distilled water (solvent C) to a concentration of 0.025 mg / mL to prepare a sample solution for drug administration.

[0108] (Anti-tumor efficacy trial) In mice with transplanted cancer cells (n=7-8), the test substances for azacitidine (Table 6) or decitabine (Table 7) were administered orally (PO) or intraperitoneally (IP) twice daily (BID, at least 5 hours apart) or once daily (QD) according to the dosing schedule for each test substance. The tumor volume of each mouse was calculated using the following formula to evaluate the antitumor effect.

[0109] Tumor volume = major axis × minor axis × minor axis × 0.5 [Table 6] "off" indicates the date when medication is discontinued.

[0110] [Table 7] "off" indicates the date when medication is discontinued.

[0111] In this experiment, such as Figure 6A and Figure 6B As shown, in the combination of compound (I) and DNA methyltransferase inhibitors (azacitidine, decitabine), tumor proliferation was maximally inhibited. The results of Example 7 show that the combination of compound (I) of the present invention and DNA methyltransferase inhibitors also has a strong synergistic effect in vivo.

[0112] Example 8: Antitumor effect of a three-dose combination of compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor. The same procedure was performed as in Example 7, using a nude mouse subcutaneous transplantation model of the MV-4-11 cell line to study the combined effect of the three agents.

[0113] (Preparation of sample solution for administration of the analyte) The dihydrochloride salt of compound (I) was dissolved in 0.1M HCl / 0.5% methylcellulose solution (solvent A) to achieve a concentration of 3 mg / mL on a free basis, thus preparing a sample solution of compound (I) for administration.

[0114] Azacitidine was dissolved in a 5% (v / v) DMSO / 30% (w / v) PEG300 aqueous solution (solvent B) to achieve concentrations of 0.25 mg / mL and 0.125 mg / mL, thus preparing a sample solution for drug administration.

[0115] Decitabine was dissolved in distilled water (solvent C) to a concentration of 0.025 mg / mL to prepare a sample solution for drug administration.

[0116] Venecra was dissolved in a 5% (v / v) DMSO / 50% (w / v) PEG300 / 5% (w / v) Tween 80 aqueous solution (solvent D) to achieve concentrations of 0.5 mg / mL and 0.25 mg / mL, respectively, to prepare a sample solution for administration of venecra.

[0117] (Anti-tumor efficacy trial) On day 19 after cancer cell transplantation, mice were divided into groups. Each mouse with cancer cells (n=6-8) was administered the test substance orally (PO) or intraperitoneally (IP) according to the dosing schedule of each test substance as shown in Table 8 or Table 9, with the dosage determined based on the individual's body weight on that day. The antitumor effect was evaluated.

[0118] [Table 8] "off" indicates the date when medication is discontinued.

[0119] [Table 9] "off" indicates the date when medication is discontinued.

[0120] In this experiment, such as Figure 7A and Figure 7B As shown, in the three-dose combination of compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor, tumor proliferation was maximally inhibited. The results of Example 8 show that the three-dose combination of compound (I) with a DNA methyltransferase inhibitor and a BCL-2 inhibitor of the present invention also has a strong synergistic effect in vivo.

[0121] Industrial applicability According to the present invention, a treatment method with higher efficacy against hematologic malignancies is provided by a two-dose combination of compound (I) and a DNA methyltransferase inhibitor, and a three-dose combination of compound (I), a DNA methyltransferase inhibitor and a BCL-2 inhibitor, as well as a combination or kit of pharmaceutical compositions for performing the treatment method.

Claims

1. A pharmaceutical composition comprising compound (I), a pharmaceutically acceptable salt or hydrate thereof as an active ingredient, administered in combination with a DNA methyltransferase inhibitor to a patient for the treatment of hematologic malignancies. 。 2. The pharmaceutical composition according to claim 1, administered to a patient in combination with a BCL-2 inhibitor in addition to a DNA methyltransferase inhibitor, for the treatment of hematologic malignancies.

3. The pharmaceutical composition according to claim 1 or claim 2, wherein, Blood cancers include myelodysplastic syndromes (MDS) and / or acute myeloid leukemia (AML).

4. The pharmaceutical composition according to claim 1 or claim 2, wherein, Blood cancers are myelodysplastic syndromes (MDS).

5. The pharmaceutical composition according to claim 1 or claim 2, wherein, Blood cancer is acute myeloid leukemia (AML).

6. The pharmaceutical composition according to claim 1 or claim 2, wherein, DNA methyltransferase inhibitors are pharmaceutical compositions comprising azacitidine, a pharmaceutically acceptable salt or hydrate thereof, as an active ingredient, or decitabine, a pharmaceutically acceptable salt or hydrate thereof, as an active ingredient.

7. The pharmaceutical composition according to claim 6, wherein, DNA methyltransferase inhibitors are pharmaceutical compositions containing azacitidine, its pharmaceutically acceptable salt or hydrate, as the active ingredient.

8. The pharmaceutical composition according to claim 6, wherein, DNA methyltransferase inhibitors are pharmaceutical compositions containing decitabine, its pharmaceutically acceptable salt or hydrate, as the active ingredient.

9. The pharmaceutical composition according to claim 7, wherein, A pharmaceutical composition containing azacitidine, its pharmaceutically acceptable salt or hydrate as an active ingredient, and a pharmaceutical composition containing compound (I), its pharmaceutically acceptable salt or hydrate as an active ingredient, are administered simultaneously or sequentially.

10. The pharmaceutical composition according to claim 8, wherein, A pharmaceutical composition containing decitabine, its pharmaceutically acceptable salt or hydrate as an active ingredient, and a pharmaceutical composition containing compound (I), its pharmaceutically acceptable salt or hydrate as an active ingredient, are administered simultaneously or sequentially.

11. The pharmaceutical composition according to claim 2, wherein, BCL-2 inhibitors are pharmaceutical compositions containing Veneclare, its pharmaceutically acceptable salt or hydrate, as the active ingredient.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein, Blood cancers include myelodysplastic syndromes (MDS) and / or acute myeloid leukemia (AML).

13. The pharmaceutical composition according to claim 12, wherein, Blood cancers are myelodysplastic syndromes (MDS).

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

15. A kit comprising: a pharmaceutical composition comprising a DNA methyltransferase inhibitor and compound (I), and a pharmaceutically acceptable salt or hydrate thereof as an active ingredient, for combination administration according to claim 1.

16. A kit comprising: a pharmaceutical composition comprising a DNA methyltransferase inhibitor, a BCL-2 inhibitor, and compound (I), a pharmaceutically acceptable salt or hydrate thereof as an active ingredient, for combination administration according to claim 2.

17. A treatment for blood cancer, wherein, A pharmaceutical composition comprising compound (I) and its pharmaceutically acceptable salt or hydrate as the active ingredient is combined with a DNA methyltransferase inhibitor and administered to the patient.

18. A treatment for blood cancer, wherein, A pharmaceutical composition comprising compound (I) and its pharmaceutically acceptable salt or hydrate as the active ingredient is combined with a DNA methyltransferase inhibitor and a BCL-2 inhibitor and administered to the patient.

19. The treatment method according to claim 17 or claim 18, wherein, Blood cancers include myelodysplastic syndromes (MDS) and / or acute myeloid leukemia (AML).

20. The treatment method according to claim 17 or claim 18, wherein, DNA methyltransferase inhibitors are pharmaceutical compositions comprising either azacitidine, a pharmaceutically acceptable salt or hydrate thereof as an active ingredient, or decitabine, a pharmaceutically acceptable salt or hydrate thereof as an active ingredient.

21. The treatment method according to claim 18, wherein, BCL-2 inhibitors are pharmaceutical compositions containing Veneclare, its pharmaceutically acceptable salt or hydrate, as the active ingredient.

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