Pharmaceutical composition for the prevention or treatment of cancer, comprising EZH2 degrader and BTK inhibitor

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a BTK inhibitor, and it has been confirmed that the therapeutic effect is significantly superior not only in Ebstein-Barr virus (EBV)-associated lymphoma, which has had low therapeutic efficacy until now, but also in other types of cancer.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a BTK inhibitor, and it has been confirmed that the therapeutic effect is significantly superior not only in Ebstein-Barr virus (EBV)-associated lymphoma, which has had low therapeutic efficacy until now, but also in other types of cancer. Background Technology

[0002] Lymphoma is a blood cancer that occurs when B or T lymphocytes become cancerous. Lymphoma can develop in lymph nodes, the spleen, bone marrow, blood, or other organs, eventually becoming cancer. Although lymphoma is a malignant tumor that arises in lymphatic tissue, it can also occur in non-lymphatic areas. Cases occurring in non-lymphatic areas are called extranodal lymphoma, which is known to frequently appear in the junction of the nose and throat, the gastrointestinal tract, and the brain. Lymphoma accounts for 2.1% of all cancer cases in the United States and 2.8% in the United Kingdom, while it accounts for 4% in Korea, making it a relatively common cancer in Asia. Lymphoma is broadly classified into Hodgkin lymphoma and non-Hodgkin lymphoma. According to WHO statistics, there were 627,439 new cases of lymphoma in 2020, with Hodgkin lymphoma and non-Hodgkin lymphoma accounting for 13% and 87% of the total, respectively.

[0003] The Ebstein-Barr virus (EBV), which belongs to the herpes virus family, infects more than 90% of normal people and causes asymptomatic latent infection in most people, but in individuals whose immune system has been weakened for a long time due to bone marrow transplantation or organ transplantation, EBV is known to cause tumors such as lymphoproliferative disease, Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease.

[0004] Lymphocytic diseases associated with EBV include Hodgkin lymphoma, nasal type extranodal NK-T cell lymphoma, and post-transplant lymphoproliferative disease (PTLD).

[0005] Among lymphomas, existing treatments for EBV-associated tumors, such as Burkitt lymphoma, have included dose reduction of immunosuppressants, the use of antivirals, chemotherapy, and administration of rituximab antibodies; however, effective treatments have not yet been established due to differences in treatment response rates. Therefore, there is currently an urgent need to develop new treatments for the treatment and cure of EBV-associated tumors.

[0006] The bones in our body maintain our body shape, enable movement, and play a role in calcium regulation. Inside the bones is a tissue called bone marrow, which is less dense than bone, and it functions to produce blood cells such as white blood cells, red blood cells, and platelets. Leukemia is a type of cancer that develops in these blood cells; it is a general term for blood cancer in which abnormal blood cells (mostly derived from white blood cells, though rarely from the erythrocyte or platelet lineages) proliferate excessively, thereby suppressing the production of normal white blood cells, red blood cells, and platelets.

[0007] A decrease in the normal white blood cell count can lead to immunosuppression and cause sepsis caused by bacterial infection, a decrease in red blood cells can cause symptoms of anemia (dizziness, headache, shortness of breath), and a decrease in platelets can cause a tendency to bleed. In addition, the overproliferated leukemia cells themselves can cause high fever, fatigue, bone pain, diarrhea, decreased consciousness, shortness of breath, and a tendency to bleed.

[0008] Leukemia is classified into acute and chronic types based on the degree of cell differentiation, or the rate of progression, and into myeloid and lymphoid types based on the origin of the cells. It is commonly classified into four forms: acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia.

[0009] The large intestine is divided into the appendix, cecum, colon, rectum, and anal canal. The colon is further divided into the ascending colon, transverse colon, descending colon, and sigmoid colon. Colorectal cancer is a malignant tumor that develops in the cecum, colon, and rectum. Depending on the location of occurrence, it is called colon cancer if it develops in the colon and rectal cancer if it develops in the rectum; collectively, these are referred to as colorectal cancer or colorectal cancer.

[0010] Unlike benign tumors that remain within the breast, breast cancer is a malignant tumor that can spread outside the breast and threaten life. Since the breast contains various types of cells, any of which can transform into cancer cells, there are quite a number of possible types of breast cancer.

[0011] Breast cancer is the most common cancer among women in developed countries such as the United States and Europe, and is the leading cause of death for American women aged 40 to 55. One in nine women develops breast cancer during their lifetime, and the number of breast cancer patients is also increasing by about 15% annually. In Korea, breast cancer accounted for approximately 11.9% of female cancer patients in 1995, becoming the third most common cancer following cervical and stomach cancer, and the fifth leading cause of death after stomach, liver, uterine, and lung cancers, with its frequency increasing every year.

[0012] EZH2 (Enhancer of zeste homolog 2) is a histone-lysine N-methyltransferase enzyme encoded by the EZH2 gene, which participates in histone methylation and ultimately transcriptional repression. EZH2 levels are abnormally elevated in cancer tissues compared to normal tissues, and EZH2 is most highly expressed in late-stage tumors or in cases with a poor prognosis. Tumor growth can be slowed by inhibiting genes that suppress tumor development or by blocking EZH2 activity.

[0013] Bruton's tyrosine kinase (BTK) is a member of the tyrosine kinase subfamily and belongs to the Tec family of kinases. It is primarily expressed in B cells and is distributed in the lymphatic, hematopoietic, and hematological systems. B cell receptors (BCRs) play a critical role in regulating the proliferation and survival of various lymphomas, including chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). As a key protein kinase in the BCR signaling pathway, Bruton's tyrosine kinase (BTK) is highly associated with various B-cell lymphoid tissue diseases. Therefore, small molecule inhibitors targeting BTK may be beneficial for the treatment of B-cell malignancies and autoimmune diseases. The problem to be solved

[0014] Accordingly, the inventors confirmed that by using a PROTAC (Proteolysis targeting chimera)-based EZH2 inhibitor and a BTK inhibitor in combination, the therapeutic effect is excellent not only on Ebstein-Barr virus (EBV)-associated lymphoma, which has previously shown low therapeutic efficacy, but also on other types of cancer, and thus completed the present invention.

[0015] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a BTK inhibitor.

[0016] Another objective of the present invention is to provide a use for cancer treatment of a pharmaceutical composition comprising an EZH2 inhibitor and a BTK inhibitor.

[0017] Another objective of the present invention is to provide a method for treating cancer by administering an EZH2 inhibitor and a BTK inhibitor in combination. means of solving the problem

[0018] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a BTK inhibitor, and it has been confirmed that the therapeutic effect is significantly superior not only in Ebstein-Barr virus (EBV)-associated lymphoma, which has had low therapeutic efficacy until now, but also in other types of cancer.

[0019] The present invention will be described in more detail below.

[0020] One example of the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising an EZH2 inhibitor and a BTK inhibitor.

[0021] In the present invention, the EZH2 inhibitor may be characterized as being based on PROTAC (Proteolysis targeting chimera).

[0022] PROTAC is a heterofunctional small molecule composed of two active domains and a linker, capable of removing specific unwanted proteins by inducing intracellular protein degradation.

[0023] In the present invention, a PROTAC-based EZH2 inhibitor may refer to a substance intended for the degradation of EZH2 protein.

[0024] In the present invention, the EZH2 inhibitor may be MS1943 or MS177, but is not limited thereto.

[0025] In the present invention, the EZH2 inhibitor may be a compound having the structure of the following structural formula 1 (MS1943) or structural formula 2 (MS177), but is not limited thereto.

[0026] [Structural Formula 1]

[0027]

[0028] [Structural Formula 2]

[0029]

[0030] In the present invention, the BTK inhibitor may be one or more selected from the group consisting of ibrutinib, acalabrutinib, and zanubrutinib, but is not limited thereto.

[0031] In the present invention, cancer may be selected from the group consisting of lymphoma, leukemia, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, stomach cancer, brain cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, esophageal cancer, liver cancer, gallbladder cancer, sarcoma, skin cancer, multiple myeloma, thyroid cancer, and head and neck cancer, but is not limited thereto.

[0032] In the present invention, the lymphoma may be a refractory lymphoma.

[0033] In the present invention, the lymphoma may be an Ebstein-Barr virus (EBV)-associated lymphoma.

[0034] In the present invention, lymphoma may be one or more selected from the group consisting of lymphoproliferative disease, Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), Hodgkin's disease, and diffuse large B-cell lymphoma.

[0035] In the present invention, the EZH2 inhibitor and the BTK inhibitor may exist as separate agents, but are not limited thereto.

[0036] In the present invention, the EZH2 inhibitor and the BTK inhibitor may be administered simultaneously, sequentially, or alternately.

[0037] In the present invention, the EZH2 inhibitor and the BTK inhibitor may be administered sequentially at time intervals of about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks, but are not limited thereto.

[0038] In the present invention, the EZH2 inhibitor may be administered at a dose of 0.1 mg / kg to 1000 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 500 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 5 mg / kg to 1000 mg / kg, 5 mg / kg to 500 mg / kg, 5 mg / kg to 100 mg / kg, or 5 mg / kg to 50 mg / kg, but is not limited thereto.

[0039] In the present invention, the BTK inhibitor may be administered at a dose of 0.1 mg / kg to 1000 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 500 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 5 mg / kg to 1000 mg / kg, 5 mg / kg to 500 mg / kg, 5 mg / kg to 100 mg / kg, or 5 mg / kg to 50 mg / kg, but is not limited thereto.

[0040] In the present invention, the pharmaceutical composition may be administered 1 to 6 times, 1 to 5 times, 1 to 4 times, or 1 to 3 times daily as needed, but is not limited thereto.

[0041] In the present invention, the pharmaceutical composition may be administered in the following ways: orally, buccally, inhaled spray, sublingually, rectalally, transdermally, vaginally, via mucosally, topically, nasally, or intestinally; parenterally, e.g., intramuscular injection, subcutaneously, intramedullary injection, as well as intrathecal or direct cerebral injection, in situ, subcutaneously, intraperitoneally, intravenously, intra-articularly, synovially, intrasternally, intrahepatically, intralesionally, intracranially, intra-abdominally, nasally, or ocularly; or other methods of drug delivery.

[0042] In the present invention, the pharmaceutical composition may further include pharmaceutically acceptable carriers, diluents, or excipients.

[0043] In the present invention, the carrier is one that is commonly used in formulations and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto.

[0044] In the present invention, the diluent may be one or more selected from the group consisting of dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dried starch, and powdered sugar, but is not limited thereto.

[0045] In the present invention, the excipient may be one or more selected from the group consisting of anti-adhesion agents, binders, coating agents, coloring agents, dyes, disintegrants, flavoring agents, lubricants, lubricants, preservatives, adsorbents, sweeteners, vehicles, wetting agents, emulsifiers, and pH buffers, but is not limited thereto.

[0046] In the present invention, the pharmaceutical composition may be in the form of tablets, capsules, granules, syrups, powders, lozenges, sachets, case preparations, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, and injections, but is not limited thereto.

[0047] In the present invention, the term "treatment" means reduction, alleviation, or improvement of symptoms of a disease or pathological condition; improvement of symptoms due to potential metabolism; suppression of a disease or symptoms, e.g., prevention of progression of a disease or disorder; improvement of a disease or pathological condition; induction of regression of a disease or pathological condition; alleviation of a condition caused by a disease or pathological condition; or prevention of symptoms of a disease or pathological condition.

[0048] As used herein, the term "about" means ±10%, more preferably ±5%, and most preferably ±2% of the modifying value, so a person skilled in the art can clearly determine the range of the term "about" based on the modified value.

[0049] Another example of the present invention relates to the use of a pharmaceutical composition comprising an EZH2 inhibitor and a BTK inhibitor for the treatment of cancer.

[0050] Another example of the present invention relates to a cancer treatment method comprising the step of administering an effective amount of an EZH2 inhibitor and a BTK inhibitor to a subject requiring treatment.

[0051] As used herein, the term "subject" includes humans (e.g., patients) and animals (e.g., mice, rats, dogs, cats, rabbits, chickens, monkeys, etc.).

[0052] As used herein, the term "effective amount" means an amount (e.g., dose) of a pharmaceutical composition that provides a significant reduction in the clinical symptoms of the disease or condition to be treated without causing excessive toxic side effects.

[0053] As used herein, the term "dose" means the weight of the active substance (e.g., milligrams (mg)) per kilogram (kg) of the subject's body weight.

[0054] In the present invention, the EZH2 inhibitor and the BTK inhibitor may exist as separate agents, but are not limited thereto.

[0055] In the present invention, the EZH2 inhibitor and the BTK inhibitor may be administered simultaneously, sequentially, or alternately. Effects of the invention

[0056] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a BTK inhibitor, and it has been confirmed that the therapeutic effect is significantly superior not only in Ebstein-Barr virus (EBV)-associated lymphoma, which has had low therapeutic efficacy until now, but also in other types of cancer. Brief explanation of the drawing

[0057] Figure 1 is a graph showing the results of measuring absorbance when B-cell lymphoma cell lines (Daudi) were treated with an EZH2 inhibitor or a BTK inhibitor alone, and when treated with an EZH2 inhibitor and a BTK inhibitor in combination. Figure 2 is a graph showing the results of measuring absorbance when T-cell leukemia cell lines (Jurkat) were treated with an EZH2 inhibitor or a BTK inhibitor alone, and when treated with an EZH2 inhibitor and a BTK inhibitor in combination. Figure 3 is a graph showing the results of measuring absorbance when EZH2 inhibitor or BTK inhibitor was treated alone, and when EZH2 inhibitor and BTK inhibitor were treated in combination, on colorectal cancer cell lines (HCT-116). Figure 4 is a graph showing the results of measuring absorbance when breast cancer cell lines (MDA-MB-231) were treated with an EZH2 inhibitor or a BTK inhibitor alone, and when treated with an EZH2 inhibitor and a BTK inhibitor in combination. Specific details for implementing the invention

[0058] The present invention will be explained in more detail below through the following examples. However, these examples are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0060] Experimental Example 1. Measurement of absorbance of cancer cell lines

[0061] B-cell lymphoma cell line (Daudi), T-cell leukemia cell line (Jurkat), colorectal cancer cell line (HCT-116), and breast cancer cell line (MDA-MB-231) were used, and 2×10^4 / 100 μL of cells per well were cultured in RPMI medium (Gibco) containing 10% FBS (Gibco), antibiotics (Gibco), and glutamax (Gibco). After stimulating with the BTK inhibitor (Ibrutinib) and the PROTAC-based EZH2 inhibitor (MS1943, MS177) under each condition, the cells were cultured in a CO2 incubator for 72 hours. Next, the cells were stimulated with 10 μL of Cell Counting Kit-8 (BIOMAX) reagent per well, and the absorbance was measured at 450 nm after 3 hours. The results are presented in Table 1 and Figure 1 (B-cell lymphoma cell line; Daudi), Table 2 and Figure 2 (T-cell leukemia cell line; Jurkat), Table 3 and Figure 3 (colorectal cancer cell line; HCT-116), and Table 4 and Figure 4 (breast cancer cell line; MDA-MB-231). Statistics were analyzed using a t-test, where * < .05, ** < .01, *** < .001, and **** < .0001 represent the values.

[0062] Treatment group Absorbance (OD) control group 1.513 Ibrutinib alone treatment group (5μM) 0.828 Ibrutinib alone treatment group (10μM) 0.390 MS1943 alone treatment group (5μM) 0.998 MS1943 alone treatment group (10μM) 0.539 MS177 alone treatment group (5μM) 0.541 MS177 alone treatment group (10μM) 0.339 Ibrutinib (5μM) + MS1943 (5μM) combination treatment group 0.256 Ibrutinib (5μM) + MS177 (5μM) combination treatment group 0.233

[0064] Treatment group Absorbance (OD) control group 1.433 Ibrutinib alone treatment group (5μM) 1.286 Ibrutinib alone treatment group (10μM) 1.033 MS1943 alone treatment group (5μM) 0.837 MS1943 alone treatment group (10μM) 0.671 MS177 alone treatment group (5μM) 0.706 MS177 alone treatment group (10μM) 0.491 Ibrutinib (5μM) + MS1943 (5μM) combination treatment group 0.323 Ibrutinib (5μM) + MS177 (5μM) combination treatment group 0.193

[0066] Treatment group Absorbance (OD) control group 2.481 Ibrutinib alone treatment group (5μM) 2.247 Ibrutinib alone treatment group (10μM) 2.100 MS1943 alone treatment group (5μM) 1.975 MS1943 alone treatment group (10μM) 1.588 MS177 alone treatment group (5μM) 2.064 MS177 alone treatment group (10μM) 1.63 Ibrutinib (5μM) + MS1943 (5μM) combination treatment group 1.076 Ibrutinib (5μM) + MS177 (5μM) combination treatment group 1.08

[0068] Treatment group Absorbance (OD) control group 1.915 Ibrutinib alone treatment group (5μM) 1.642 Ibrutinib alone treatment group (10μM) 1.342 MS1943 alone treatment group (5μM) 1.497 MS1943 alone treatment group (10μM) 1.038 MS177 alone treatment group (5μM) 1.443 MS177 alone treatment group (10μM) 0.827 Ibrutinib (5μM) + MS1943 (5μM) combination treatment group 0.785 Ibrutinib (5μM) + MS177 (5μM) combination treatment group 0.508

[0070] As can be seen in Table 1 above, in B-cell lymphoma cell line (Daudi), the BTK inhibitor Ibrutinib monotherapy group showed an absorbance value of 0.390 at 10 µM, and the PROTAC-based EZH2 inhibitor MS1943 monotherapy group showed an absorbance value of 0.539 at 10 µM, whereas the combined treatment group of Ibrutinib (5 µM) and MS1943 (5 µM) showed a significantly reduced absorbance value of 0.256. Additionally, another PROTAC-based EZH2 inhibitor, MS177 monotherapy group showed an absorbance value of 0.339 at 10 µM, while the combined treatment group of Ibrutinib (5 µM) and MS177 (5 µM) showed a significantly reduced absorbance value of 0.233.

[0071] As can be seen in Table 2 above, in T-cell leukemia cell lines (Jurkat), the BTK inhibitor Ibrutinib monotherapy group showed an absorbance value of 1.033 at 10 µM, and the PROTAC-based EZH2 inhibitor MS1943 monotherapy group showed an absorbance value of 0.671 at 10 µM, whereas the combined treatment group of Ibrutinib (5 µM) and MS1943 (5 µM) showed a significantly reduced absorbance value of 0.323. Additionally, another PROTAC-based EZH2 inhibitor, MS177 monotherapy group showed an absorbance value of 0.491 at 10 µM, while the combined treatment group of Ibrutinib (5 µM) and MS177 (5 µM) showed a significantly reduced absorbance value of 0.193.

[0072] As can be seen in Table 3 above, in colorectal cancer cell line (HCT-116), the group treated with the BTK inhibitor Ibrutinib alone showed an absorbance value of 2.100 at 10 µM, and the group treated with the PROTAC-based EZH2 inhibitor MS1943 alone showed an absorbance value of 1.588 at 10 µM, whereas the group treated with the combination of Ibrutinib (5 µM) and MS1943 (5 µM) showed a significantly reduced absorbance value of 1.076. In addition, the group treated with MS177, another PROTAC-based EZH2 inhibitor alone, showed an absorbance value of 1.63 at 10 µM, while the group treated with the combination of Ibrutinib (5 µM) and MS177 (5 µM) showed a significantly reduced absorbance value of 1.08.

[0073] As can be seen in Table 4 above, in breast cancer cell line (MDA-MB-231), the group treated with the BTK inhibitor Ibrutinib alone showed an absorbance value of 1.342 at 10 µM, and the group treated with the PROTAC-based EZH2 inhibitor MS1943 alone showed an absorbance value of 1.038 at 10 µM, whereas the group treated with the combination of Ibrutinib (5 µM) and MS1943 (5 µM) showed a significantly reduced absorbance value of 0.785. In addition, the group treated with the other PROTAC-based EZH2 inhibitor MS177 alone showed an absorbance value of 0.827 at 10 µM, while the group treated with the combination of Ibrutinib (5 µM) and MS177 (5 µM) showed a significantly reduced absorbance value of 0.508.

[0074] Through the above results, it was confirmed that when a BTK inhibitor and a PROTAC-based EZH2 inhibitor are used in combination, the growth inhibition effect of cancer cell lines is significantly superior compared to when each is used alone.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and Ibrutinib, wherein the EZH2 inhibitor is a compound having the structure of the following structural formula 1 or structural formula 2, [Structural Formula 1] [Structural Formula 2] A pharmaceutical composition for the prevention or treatment of cancer, wherein the cancer is one or more selected from the group consisting of B-cell lymphoma, T-cell leukemia, colorectal cancer, and breast cancer. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete

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