Combination therapy of 4'-thio-5-aza-2'-deoxycytidine and venetoclax

Abstract

The present invention relates to a combination therapy of 4'-thio-5-aza-2'-deoxycytidine (Aza-T-dCyd), a multi-targeted inhibitor including a DNMT1 inhibitor, and venetoclax, an inhibitor of BCL-2, which mediates apoptosis.

Description

[Technical Field]

[0001] The present invention relates to a combination therapy of 4'-thio-5-aza-2'-deoxycytidine (Aza-T-dCyd), a multitarget inhibitor including a DNMT1 inhibitor, and venetoclax, an inhibitor of BCL-2 that mediates apoptosis. [Background technology]

[0002] Nucleoside derivatives are organic compounds with structures very similar to the components of DNA and RNA, which perform the most core functions of cells that make up our bodies (maintaining life functions through replication and division), and they play a central role in anti-cancer chemotherapy and antiviral chemotherapy.

[0003] Since the 1960s, various nucleoside-based anticancer drugs such as cytarabine, clofarabine, fludarabine, 5-fluorouracil (including capecitabine), gemcitabine, decitabine, and azacytidine have been commercialized (Figure 4). Of these, many are classified as major anticancer drugs and are included in the WHO's Essential Medication List.

[0004] Decitabine (also known as Dacogen® or 5-Aza-2'-deoxycytidin) is a pyrimidine nucleotide analog of cytidine that inhibits DNA methyltransferase (DNMT) and induces DNA hypomethylation. Specifically, decitabine functions by being incorporated into the DNA strand during replication. When a DNA methyltransferase such as DNMT1 binds to DNA and replicates methylation to the daughter strand, the DNMT becomes irreversibly bound to decitabine and cannot be separated. Therefore, the action of decitabine is cell division-dependent. The drug requires cells to divide to act. Thus, cells that divide much faster than most other cells in the body (e.g., cancer cells) are more severely affected by decitabine. In other words, decitabine is used to treat cancers such as leukemia, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), in which DNA hypermethylation is important for development.

[0005] Existing nucleoside-based anticancer drugs act not only on cancer cells but also on rapidly dividing normal cells such as bone marrow / gastrointestinal tract and mucosal / skin cells, and therefore, as side effects, (i) in the case of azacitidine (VidAza), may include nausea, anemia, thrombocytopenia, vomiting, fever, leukopenia, diarrhea, and injection site (ii) In the case of decitabine (Dacogen), erythema (injection site erythema), constipation, neutropenia (neutropenia), ecchymosis (petechiae), rigors (chills), weakness (fatigue), and hypokalemia (hypokalemia) may occur.

[0006] Although the genes in our bodies have the same base sequence in every cell, the cells in our bodies are very precisely regulated so that only genes different from each other are expressed by specific tissues, while other genes are not expressed.

[0007] The mechanism by which such epigenetic regulation occurs is called epigenetic regulation, and this mechanism consists of (1) changes in DNA, (2) changes in histones that physically bind to DNA and form the chromatin structure, and (3) changes in other components that make up the chromatin structure.

[0008] Of these, DNA methylation, as the most fundamental epigenetic mechanism, regulates gene expression by methylating the carbon base of CpG sequences in various gene components such as Gene Body, Promoter, and Enhancer. Generally, CpG sequences are abundant in promoter regions, and genes with significant CpG methylation in the promoter region are less likely to be expressed. Conversely, genes with less CpG methylation are actively expressed (Figure 1).

[0009] In our bodies, DNA methylation is known to consist of two stages: (1) DNA methyltransferase (DNMT) 3A or 3B methylates de novo DNA during tissue differentiation and development, determining the gene expression pattern for each tissue; (2) DNMT1 methylates DNA by faithfully replicating the DNA methylation pattern already established in each cell that makes up the tissue.

[0010] In typical cells, in regions where DNA methylation is highly advanced, DNMT1 binds to other proteins, particularly histone proteins and other repressor proteins that suppress gene expression, forming a structure that discourages gene expression. Conversely, where DNA methylation is not advanced, it forms a structure that facilitates gene expression, thereby promoting gene expression. DNA methylation thus plays a crucial role in ensuring that each cell in our body can perform its specific function effectively by smoothly expressing the predetermined combination of genes. Therefore, abnormalities in these functions can lead to the development of various diseases.

[0011] Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are cancers that develop in the bone marrow. In these diseases, clones of hematopoietic stem cells that produce immature leukemic cells (Leukemic Blasts) outnumber clones that produce a variety of normal blood cells, resulting in the blood being filled with leukemic blasts that do not function properly. Ultimately, these diseases lead to death due to blood dysfunction.

[0012] The normal hematopoietic process in our bodies consists of the following steps: (1) When hematopoietic stem cells are stimulated to produce blood cells, (2) the hematopoietic stem cells rapidly divide in one step and differentiate into progenitor cells that can produce a sufficient number of blood cells, (3) the progenitor cells divide and a sufficient number of progenitor cells are generated, (4) further division / proliferation stops in two steps, each with its own function, and (5) they mature into functional cells that no longer divide (Figure 2).

[0013] The main role in this process is played by master transcription factors (Master TFs). In the first stage, when hematopoietic stem cells are stimulated, master transcription factors such as CEBP / alpha, which generate progenitor cells, act strongly to ensure that a sufficient number of progenitor cells are produced. Once a sufficient number of progenitor cells have been produced, the process moves to the second stage, where Master TFs such as CEBP / epsilon are produced. The Master TFs in the second stage degrade the Master TFs from the first stage, preventing further rapid division / proliferation and inducing cell maturation according to a predetermined process.

[0014] In MDS and AML, the gene promoter of Master TF in the two stages that drive this metastasis process is hypermethylated for various reasons, resulting in decreased expression of Master TF in these two stages. As a result, progenitor cells cannot progress to the maturation stage and continue to divide / proliferate, constantly producing immature cells. If this process progresses further, several mutations (such as FLT3-ITD) accumulate, leading to rapidly progressing malignant AML and ultimately death of the patient.

[0015] A significant number of MDS / AML cases were found to be caused by hyperactivation of DNMT1, resulting in abnormal methylation patterns. Conversely, it was confirmed that these diseases can be treated by normalizing the epigenetic mechanism through inhibition of DNMT1 (Figure 3).

[0016] Promoter hypermethylation of the Master TF gene can be caused by a variety of factors, including changes in the methylation pattern due to abnormalities in DNMT enzymes (overexpression, overactivation, deletion / mutation, etc.), abnormalities in the pattern resulting from difficulty in removing the methylation pattern due to abnormalities in IDH1 / 2 enzymes, abnormalities in the pattern resulting from difficulty in removing the methylation pattern due to abnormalities in TET enzymes, and the accumulation of epigenetic changes due to aging. In any case, it has been confirmed that inhibiting the activation of DNMT1, a Maintenance DNA methylation Machinery that maintains the methylation pattern during cell growth / division / survival processes, can resolve the hypermethylation pattern, thereby providing therapeutic efficacy for MDS / AML.

[0017] The treatment process and methods for AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), or ALL (acute lymphoblastic leukemia) vary depending on the disease classification, the patient's age, and health condition. However, unlike solid tumors which require surgery to remove the cancer, the general standard of care (SoC) mainly consists of induction therapy using anticancer drugs to reduce blasts, consolidation therapy performed after achieving complete remission, and maintenance therapy to maintain remission. In severe cases where treatment is urgent, hematoietic cell transplantation (HCT) can be performed, which is the most effective method. However, this treatment requires the complete removal of the patient's abnormal bone marrow through intensive chemotherapy or radiation therapy, so its feasibility is very limited for elderly patients in poor health.

[0018] The likelihood of gene mutations that can cause blood cancers, i.e., bone marrow diseases, increases significantly with age. Therefore, age itself can be considered the most important risk factor for leukemia, especially AML and MDS. However, compared to relatively young patients in good physical condition, elderly patients whose physical function is weakened due to various factors such as underlying diseases are often unable to receive standard treatment with highly toxic existing chemotherapy. Even if they undergo treatment through chemotherapy, the prognosis is often poor, with frequent relapses and a very high early mortality rate.

[0019] When MDS / AML cells (cell lines or patient-derived samples) are treated with the DNMT1 inhibitor decitabine / azacitidine, the permethylation of the CEBP / epsilon promoter, a Master TF, decreases in two steps, while CEBP / epsilon is expressed. This leads to the expression of tumor suppressor genes such as p27, which are downstream effectors of CEBP / epsilon, thus demonstrating anticancer efficacy.

[0020] Two existing commercialized DNMT1 inhibitors, Dacogen (decitabine) and VidAza (azacitidine), are used as standard therapeutic agents for the treatment of MDS / AML in the elderly patient group. However, several problems have been pointed out, including insufficient efficacy, high toxicity, problems with usability due to the administration route (IV or subcutaneous injection), and the expansion of the DNMT1 inhibitor market due to the expansion of indications.

[0021] On the other hand, most acute lymphoblastic leukemia (ALL) is B-cell origin B-ALL, but some are T-cell origin T-cell ALL (hereinafter T-ALL). In the case of T-ALL, the development of appropriate targeted therapy is restricted, so the treatment options are limited after primary chemotherapy.

[0022] The treatment of adult ALL is high-intensity chemotherapy based on Cyclophosphoamide, Daunorubicin, Vincristine, L-asparaginase, Prednisone, etc. as the primary treatment option.

[0023] Approximately 30 - 40% of patients relapse or acquire resistance despite such high-intensity chemotherapy. Among such relapsed / resistant patients, in the case of B-cell ALL patients, there are various treatment options such as antibody therapeutics, ADCs, and CAR-Ts. In contrast, in the case of T-ALL patients, the therapeutic agent is limited to nelarabine, so there is a high demand for the development of new therapeutic agents.

[0024] In the case of T-ALL, many patients respond well to intensive chemotherapy using a combination of various cytotoxic anticancer drugs. However, in cases of T-ALL where resistance develops despite the use of standard treatment, there is no appropriate treatment, and there is a very high demand for the development of new therapies. In some of these resistant patients, resistance is understood to develop due to the overexpression of anti-apoptotic genes such as BCL-2. In such cases, treatment with a combination of DNMT1 inhibitors (such as decitabine) and venetoclax has been experimentally attempted, and excellent therapeutic synergy effects have been confirmed.

[0025] However, since existing nucleoside-based anticancer drugs are used to treat T-cell ALL, nucleoside metabolic resistance is likely to develop in resistant patients. Therefore, there is a high need for the development of new DNMT1 inhibitors that can overcome various resistance mechanisms, including metabolic resistance, beyond existing decitabine.

[0026] Based on decitabine and azacitidine, which are DNMT1 inhibitor active ingredients that have been released to the market, various drugs (injectable, oral, and used according to their respective applications) are available on the market.

[0027] Both components of decitabine / azacytidine, with the chemical structures shown in Figure 4, are activated in cells in the form of decitabine triphosphate, then incorporated into DNA, and subsequently trap the DNMT1 enzyme to form a DNA-DNMT1 covalent adduct. Subsequently, the process of DNA damage repair through the degradation of the generated adduct has a mechanism that exhibits permethylation removal and various anticancer effects through various intracellular signaling pathways. However, (1) both decitabine and azacitidine components are vulnerable to metabolism by cytidine deaminase, which is present in our bodies, and have the disadvantage of limited efficacy due to an unstable PK profile. (2) Because it causes DNA damage through the formation of DNA-DNMT1 adducts, it damages not only cancer cells but also normal tissues in our bodies such as bone marrow / gastrointestinal mucosa, and its anticancer efficacy is limited as the amount used is restricted. (3) In cancer cells that survive after initial treatment, the anticancer efficacy is suppressed due to hyperactivation of the removal process after DNA-DNMT1 adduct formation, and it shows vulnerabilities to the development of resistance. Therefore, there is a strong need for the development of improved new drugs.

[0028] Decitabine / azacitidine has the disadvantage of being vulnerable to metabolism by cytidine deaminase present in our bodies, and its potency is limited due to an unstable PK profile.

[0029] Focusing on the fact that both the 5'-OH and 3'-OH groups in the decitabine / azacitidine structure must be fully exposed for metabolism by cytidine deaminase, Astex Pharmaceuticals / Otsuka developed SGI-110, a decitabine prodrug with a structure in which deoxyguanidine is linked to the 3'-OH group of decitabine. When administered to humans, SGI-110 was confirmed to avoid metabolism by cytidine deaminase and exhibit an improved PK profile by slow-releasing the decitabine component. However, it was found that this improved PK profile did not translate into an improvement in actual pharmacological efficacy.

[0030] To address the metabolic problems caused by cytidine deaminase, improved new drugs have been developed, such as oral formulations that include a cytidine deaminase inhibitor (ASTX-727) or oral formulations that stabilize the PK profile by administering an excessive amount of azacytidine (CC-486). However, these drugs also fail to provide solutions to problems (2) and (3) of the decitabine / azacitidine problem described above. Furthermore, there are problems such as reduced efficacy of the active ingredient (decitabine) due to concomitant use of cytidine deaminase inhibitors and gastrointestinal side effects due to drug overdose. Therefore, there is a need to develop a novel DNMT1 inhibitor that can solve these problems.

[0031] Currently, decitabine / azacitidine or combination therapy with these agents and venetoclax is the standard treatment for elderly MDS / AML patients. However, there are problems such as a limited therapeutic response in only about 40% of patients when the drugs are administered alone, and a therapeutic response in only about 65% of patients when administered in combination, as well as rapid tolerance issues and cross-resistance between the two drugs, decitabine and azacitidine.

[0032] Furthermore, in the case of elderly MDS / AML, while a variety of targeted anticancer drugs, immunosuppressants, and cell therapies have been developed for general MDS / AML, the applicable development technologies are limited. Similarly, in the fields of recurrent T-ALL and platinum-resistant ovarian / bladder cancer, the applicable development technologies are limited. [Overview of the project] [Problems that the invention aims to solve]

[0033] To address the aforementioned problems related to DNMT1 inhibitors, the present invention provides a combination therapy comprising 4'-thio-5-aza-2'-deoxycytidine (Aza-T-dCyd), a multitarget inhibitor including DNMT1 inhibition, and venetoclax, an inhibitor of BCL-2 that mediates apoptosis. [Means for solving the problem]

[0034] A first aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug, wherein both drugs are administered in combination in the same or different dosage forms.

[0035] A second aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug, wherein both drugs are intended to be administered to a subject via the same or different routes.

[0036] A third aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug, wherein both drugs are intended to be administered to a subject parenterally or orally.

[0037] A fourth aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug administered simultaneously or sequentially.

[0038] A fifth aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug, which is administered simultaneously with a venetoclax drug.

[0039] A sixth aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug, which is administered sequentially with a venetoclax drug.

[0040] A seventh aspect of the present invention provides a kit comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug.

[0041] In the first to seventh embodiments, the 4'-thio-5-aza-2'-deoxycytidine drug can be administered at a dose of 8 mg / day or more and less than 32 mg / day.

[0042] In the first to seventh embodiments, the 4'-thio-5-aza-2'-deoxycytidine drug can be administered at a dose of 75% or less of the maximum tolerable dose (MTD), preferably 25% to 75%, and more preferably 25% to 50%.

[0043] In the first to seventh embodiments, the 4'-thio-5-aza-2'-deoxycytidine drug can reduce or inhibit the DNMT1 protein in a concentration-dependent manner.

[0044] In the first to seventh embodiments, the 4'-thio-5-aza-2'-deoxycytidine drug can be administered at a high dose that prevents the resistance mechanism that overactivates BER (Base Excision Repair), one of the DNA damage repair methods, from activating. In this case, the 4'-thio-5-aza-2'-deoxycytidine drug can overcome the resistance mechanism by reducing the efficiency or rate of the BER repair process that has been overactivated in the resistance mechanism through its strong resistance to endonuclease.

[0045] The pharmaceutical compositions or drugs in the kits of the first to seventh embodiments can be administered to patients who have developed resistance to DNMT1 inhibitors; patients in whom epigenetic genetic DNA methylation pattern changes have accumulated in cancer cells compared to normal cells; patients who have been informed of or diagnosed with a poor prognosis or the possibility of developing resistance when administered DNMT1 inhibitors; patients who express a lineage commitment Master transcription factor selected from the group consisting of CEBP / alpha, Pu.1, and GATA factors at a high level compared to healthy individuals, while the expression of CEBP / epsilon or late-stage developmental transcription factors is maintained at a low level by hypermethylation of each gene; patients who may develop nucleoside metabolic resistance when administered nucleoside anticancer drugs; patients who are candidates for administration of platinum-based anticancer drugs; patients who may develop resistance to or have developed resistance to platinum-based anticancer drugs; and / or patients who have been epigenetically silenced to tumor suppressor genes and / or SLFN11 compared to healthy individuals.

[0046] In this case, information regarding prognosis or the possibility of resistance development may be derived from (i) quantifying the problem of suppression of anticancer efficacy due to hyperactivation of the removal process after DNA-DNMT1 adduct formation, or (ii) classifying patient groups based on the presence / degree of the above problem. For example, data values ​​for providing information on poor prognosis may be the likelihood of symptom recurrence and / or early mortality (less than one year of average survival).

[0047] In the first to seventh embodiments, when DNMT1 inhibitors are administered, DNA damage is caused through DNA-DNMT1 adduct formation, resulting in damage not only to cancer cells but also to normal tissues. This problem limits the standard dose of DNMT1 inhibitors, which are standard therapeutic agents. In such patient groups, a 4'-thio-5-aza-2'-deoxycytidine drug can be administered as a targeted anticancer agent at a dose greater than or equal to the aforementioned limited standard dose.

[0048] The pharmaceutical compositions of the first to sixth embodiments can be administered to a group of patients diagnosed with AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), or ALL (acute lymphoblastic leukemia), a group of patients with platinum-resistant recurrent end-stage ovarian cancer, a group of patients with platinum-resistant metastatic bladder cancer, a group of patients with metastatic bladder cancer, a group of patients with p53-mutated bladder cancer or hypermethylated SLFN11 bladder cancer, which are target biomarkers for treatment, or a group of patients diagnosed with ovarian cancer as stage 3 or 4. In particular, it can be administered to patients with chronic myelomonocytic leukemia, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia, high-risk patients with genomic abnormalities, patients with relapsed secondary AML (sAML), patients with treatment-related AML (t-AML) due to past treatment history, and drug-resistant / refractory patients.

[0049] The present invention will be described below.

[0050] In this specification, "drug" refers to all substances used for the diagnosis, cure, alleviation, treatment, or prevention of disease (excluding food or equipment), or for affecting the structure or function of the body. For example, all chemical or biological substances that affect the body and its metabolism. The chemical name of a drug represents its atomic or molecular structure.

[0051] For a drug to work effectively in vivo, its concentration in the body must be maintained within the therapeutic range for a certain period of time. If the drug is present in excess, toxicity will occur; if the amount is too low, no therapeutic effect will be observed.

[0052] Drug efficacy refers to the period during which a drug remains in the body without being broken down, thus providing the expected effect for its target indication. A slower metabolic rate results in a longer duration of blood concentration, thus extending the duration of efficacy.

[0053] Cells, the basic units that make up the body, regulate cell division and death to maintain tissue homeostasis. Of these, active cell death is called apoptosis or programmed cell death. In apoptosis, a pre-existing suicide mechanism within the cell is activated by internal and external stimuli, causing the cell to die as planned. Unlike cell necrosis, the contents of the dying cell are not released outside the cell, and it does not damage other cells. Morphologically, it refers to a process involving a decrease in cell density, destruction of the cell membrane, and condensation of chromosomes, along with the formation of an apoptotic body, and the action of phagocytic cells. Biochemically, it refers to DNA fragmentation, where chromosomal DNA separates from large fragments into smaller fragments.

[0054] Generally, the apoptosis mechanism is a series of processes in which intracellular proteins are degraded by proteolytic enzymes called caspases, while signals are transmitted simultaneously. Several types of caspases are involved in apoptosis. Caspase-8 is activated by apoptosis-inducing substances such as TNF-α or Fas ligand, activating a series of other caspases and inducing apoptosis. Meanwhile, during apoptosis, cytochrome c is released through a pathway in the mitochondrial membrane and is regulated by BCL-2 system proteins that constitute the pathway. It has been reported that released cytochrome c binds to Apaf-1, caspase-9, and dATP, activating caspase-9, which in turn activates caspase-3, thereby inducing apoptosis (Figure 9).

[0055] There are two factors that determine tumor growth: first, cell proliferation, and second, cell death. If the cell cycle of tumor cells is stopped by cytotoxic substances, the tumor cells will die through apoptosis.

[0056] As shown in Figure 9, B-cell lymphoma (BCL)-2 mediates apoptosis.

[0057] While cancer treatments induce multiple types of apoptosis, the activation of the apoptotic pathway regulated by BCL-2 is the most crucial factor in the therapeutic efficacy of oncogenic kinase inhibitors and cytotoxic agents. However, defects in the mitochondrial apoptotic pathway allow various cancers to develop resistance to cytotoxic drugs.

[0058] BCL-2 overexpression has been demonstrated in chronic lymphocytic leukemia (CLL) cells, mediating tumor cell survival and associated with resistance to chemotherapy agents.

[0059] Venetoclax is a potent and selective small molecule inhibitor of the anti-apoptotic protein BCL-2. In other words, venetoclax is an apoptosis-inducing anticancer agent. Venetoclax directly binds to the BH3-binding groove of BCL-2, replacing BH3 motif-containing proapoptotic proteins such as BIM. This causes BIM, which does not bind to BCL-2, to initiate mitochondrial membrane permeabilization (MOMP), caspase activity, and apoptosis (Figure 9). In non-clinical studies, this drug showed cytotoxicity against tumor cells overexpressing BCL-2.

[0060] In recent years, the standard therapy for AML has shifted from the existing monotherapy of decitabine / azacitidine to the combination of these drugs with venetoclax (trade name: Venclexta), a BCL-2 inhibitor. Therefore, the inventors compared and evaluated the efficacy of Aza-T-dCyd (chemical formula 1 below) when administered in combination with venetoclax, instead of decitabine / azacitidine.

[0061] [ka]

[0062] Surprisingly, in a xenograft model in which the MV4-11 AML cell line was subcutaneously implanted into mice, Aza-T-dCyd (NTX-301), even when administered alone at a clinically relevant concentration, not only reduced the expression levels of anti-apoptotic proteins such as Survivin and Mcl-1 through potent p53 activation, demonstrating anticancer efficacy equivalent to or better than that of azacitidine / venetoclax combination therapy, but also showed extremely potent anticancer efficacy even at very low drug doses compared to azacitidine / venetoclax combination therapy (inducing complete tumor regression in the Aza-T-dCyd 0.5mpk dose group) (Figures 28-33). From this, it can be inferred that when both Aza-T-dCyd and venetoclax are administered in combination, the action of anti-apoptotic proteins is suppressed. The present invention was completed based on this.

[0063] In the present invention, the drug administered in combination with the Aza-T-dCyd drug is not limited to the compound of chemical formula 2 below (venetoclax), as long as it is a BCL-2 inhibitor, and falls within the scope of the present invention.

[0064] [ka]

[0065] The present invention is a DNMT1 inhibitor for combination therapy with venetoclax drugs, characterized by the selection of Aza-T-dCyd of chemical formula 1 based on an analysis of the mechanism of action of existing nucleoside anticancer agents and the DNA damage repair activation mechanism which is the mechanism of resistance to them.

[0066] DNMT1 inhibitors, which inhibit the action of anti-apoptotic proteins, could be a very promising approach to overcome the resistance of existing decitabine / azacitidine. Therefore, the present invention aims to design a well-designed multi-target inhibitor that exhibits potent anticancer efficacy by simultaneously inhibiting DNMT1, the primary drug target, and blocking hydrolysis by endonucleases, the mechanism by which resistance develops, through a multi-pharmacological approach. In the process of designing this well-designed multi-target inhibitor, Aza-T-dCyd (chemical formula 1) is selected as the DNMT1 inhibitor and is intended for use in combination therapy with venetoclax drugs.

[0067] Aza-T-dCyd, represented by chemical formula 1, is a DNMT1 inhibitor based on a 4-thio-2-deoxyribose skeleton, possessing both a sugar structure change (4'-thiodeoxyribose structure) and an Aza-cytosine group. Molecular modeling confirmed that the thiodeoxyribose-based nucleoside compound, after insertion into DNA, does not significantly affect other DNA components.

[0068] Aza-T-dCyd is a nucleoside-based anticancer agent that is activated by triphosphate within cells, replaces some deoxycytidine (dC) during DNA synthesis, and induces cancer apoptosis by trapping DNMT1 after DNA synthesis and activating various epigenetic mechanisms of action. In particular, Aza-T-dCyd is rapidly activated within cancer cells, integrated into DNA, and can effectively inhibit the DNMT1 enzyme even at low concentrations.

[0069] As shown in Figure 5, nucleoside anticancer agents exert their anticancer effects through a variety of mechanisms after DNA incorporation (inhibition of DNA synthesis / transcription, induction of DNA damage, or inhibition of DNA processing-related enzymes such as DNMT1).

[0070] As shown in Figure 6, nucleoside anticancer drugs form base pairs that do not match the normal DNA components during DNA uptake, resulting in abnormal structures in the DNA. This is recognized by BER (base excision repair), one of the DNA damage repair methods, and DNA damage repair proceeds.

[0071] In the initial stages of administration of nucleoside-based anticancer drugs, the drug is incorporated into the DNA and exerts its full pharmacological effect, efficiently killing cancer cells. However, as administration of anticancer drugs continues, BER becomes overactivated, and the drug is removed before it can exert its full effect, leading to the development of resistance to the anticancer drug.

[0072] Therefore, focusing on the fact that if a new nucleoside compound is developed that can be incorporated into DNA with an efficiency equal to or greater than that of existing nucleoside anticancer drugs, expresses the desired pharmacological efficacy well, and is resistant to BER, it is expected to exhibit stronger efficacy than existing anticancer drugs, the present invention has selected Aza-T-dCyd of chemical formula 1 as a DNMT1 inhibitor that can be used in combination therapy with venetoclax drugs.

[0073] The first step in BER is to cleave the glycosidic bond in the nucleoside / nucleotide, creating a base-deficient site where DNA damage can be repaired via endonucleases. Subsequently, various enzymes use the base information from the opposite side to replenish the other complementary base components, thereby repairing the DNA damage (Figure 6).

[0074] The rate and efficiency of the BER repair process are determined by the steps of cleaving the CN bond after recognizing the abnormal base pair and creating a gap in the nucleotide position by AP-endonuclease. By making one or more of these steps difficult, the efficiency of BER can be reduced, thereby suppressing the development of resistance due to BER overactivation.

[0075] Nucleoside compounds with a 4-thio-2-deoxyribose skeleton are resistant to DNA strand breaks by endonucleases, and since this mechanism corresponds to the first step of the DNA damage repair pathway, DNMT1 inhibitors with a 4-thio-2-deoxyribose skeleton can overcome drug resistance mediated by anti-apoptotic proteins and the DNA damage repair pathway. Therefore, Aza-T-dCyd (NTX-301) has the potential to overcome potent anticancer efficacy / resistance compared to existing DNMT1 inhibitors with a 4-thio-2-deoxyribose skeleton (Examples 1 and 2).

[0076] On the other hand, while the BER mechanism acts as a mechanism for developing resistance in cancer cells, it conversely functions as a safety mechanism to prevent toxicity from anticancer drugs in normal tissues (especially hematopoietic tissues such as bone marrow). Therefore, there is a growing need for anticancer drugs that inhibit BER to be selectively delivered only to cancer cells.

[0077] Nucleoside-based anticancer drugs, after entering cells, are activated by various nucleoside kinases and accumulate within the cells. Nucleoside compounds that are not activated within a suitable time are rapidly removed. By maximizing the difference in activation rates between normal cells and cancer cells, excellent safety can be ensured by accumulating the active drug only in cancer cells.

[0078] According to previous literature, nucleosides substituted with various heteroatoms can be activated or removed by metabolism at different rates. However, in the case of thiodeoxyribose, while it is activated relatively slowly in normal cells, it is activated at a rate equal to or greater than that of common deoxyribose-based nucleosides in cancer cells, and it has been confirmed that it can be used in DNA synthesis. This can lead to a selective drug delivery effect, where it accumulates relatively more in cancer cells than in normal cells.

[0079] Aza-T-dCyd utilizes a modified thiodeoxyribose skeleton, an improved version of the conventional deoxyribose skeleton. Compared to conventional deoxyribose-based nucleosides such as decitabine, it is rapidly activated in cancer cells and efficiently incorporated into DNA, while being slowly activated in normal organs, including normal bone marrow cells, enabling selective drug delivery to cancer cells. Furthermore, it not only ensures the possibility of overcoming metabolism-related resistance by being relatively slowly affected by cytidine deaminase, the major metabolic enzyme of nucleoside anticancer drugs, but also ensures a more potent and differentiated effect by forming a longer-lasting DNA-DNMT adduct after DNA incorporation and DNMT1 trapping through an azacytidine functional group that effectively traps the DNMT1 enzyme, thereby ensuring a more potent and differentiated effect.

[0080] Unlike decitabine / azacitidine with the chemical structure shown in Figure 4, Aza-T-dCyd of chemical formula 1, through its thio-nucleoside structure, not only exhibits potent anticancer efficacy but also demonstrates excellent PK profile even with oral administration, as well as resistance to metabolic resistance by cytidine deaminase. This confirms its applicability to hematological malignancies and solid tumors (ovarian cancer / bladder cancer) (Examples 1-4).

[0081] Furthermore, deoxycytidine compounds used in chemotherapy for cancer or viral infections require phosphorylation by cellular enzymes for activation. In this case, phosphorylation of deoxycytidine compounds by deoxycytidine kinase (dCK) is considered a rate-limiting step in the activation of the compound, leading to further phosphorylation to diphosphate or triphosphate. Aza-T-dCyd, due to its thio-nucleoside structure, significantly reduces the activation rate by dCK (deoxycytidine kinase) in normal cells, allowing for selective delivery of the drug's active ingredient to cancer cells, ensuring an excellent safety profile and a wide therapeutic window, enabling administration at high doses that do not trigger resistance mechanisms.

[0082] Aza-T-dCyd compounds can be activated by rapid triphosphate formation in cancer cells compared to normal cells. DNA insertion of Aza-T-dCTP, the triphosphate of Aza-T-dCyd compounds, induces base excision repair and / or mismatch repair, delaying DNA replication and thereby inducing replication stress, which can accelerate the DNA damage response.

[0083] The Aza-T-dCyd compound can induce DNA replication stress and generate a strong DNA damage response by suppressing the expression of the RRM1 protein, a ribonucleotide reductase important for dNTP de novo synthesis, thereby reducing intracellular dCTP and dTTP levels.

[0084] Unlike decitabine / azacitidine, Aza-T-dCyd exhibits potent anticancer efficacy by simultaneously inhibiting the primary drug target DNMT1 through its thio-nucleoside structure and blocking endonucleases involved in DNA damage repair, a pre-existing resistance mechanism (Figure 6).

[0085] Decitabine / azacitidine has the disadvantage of being vulnerable to metabolism by cytidine deaminase present in our bodies, and its efficacy is limited due to an unstable PK profile. Surprisingly, when Aza-T-dCyd and Aza-T-dCTP drugs were orally administered compared to decitabine / azacitidine, they were found to be degraded more slowly by cytidine deaminase, overcoming metabolic resistance to cytidine deaminase in cancer cells, and possessing a superior PK profile even when administered orally (Example 3).

[0086] Furthermore, Aza-T-dCyd's thio-nucleoside structure significantly reduces the activation rate of dCK (deoxycytidine kinase) in normal cells, allowing for selective delivery of the drug's active ingredient to cancer cells and ensuring an excellent safety profile. Therefore, it provides a broad therapeutic window, enabling administration at high doses that prevent the development of resistance.

[0087] Furthermore, pharmacokinetic characterization revealed that (1) the anticancer effect of Aza-T-dCyd is Cmax-dependent rather than AUC-dependent; and (2) exposure to larger amounts of Aza-T-dCyd over a short period of time is an effective anticancer treatment.

[0088] At the cellular level, Aza-T-dCyd induces significantly improved changes in drug efficacy markers (PD markers) compared to decitabine / azacitidine, thereby demonstrating a more potent cancer apoptosis-inducing effect than decitabine / azacitidine (Example 1).

[0089] Furthermore, these improved changes in PD markers allow for the maintenance of superior cancer apoptosis-inducing efficacy even in cell lines resistant to decitabine. This efficacy at the cellular level leads to superior efficacy in animal models, and Aza-T-dCyd demonstrated superior anticancer efficacy in a variety of animal models where decitabine / azacitidine showed no therapeutic effect whatsoever.

[0090] Aza-T-dCyd exhibits an orally administered PK profile and demonstrated superior safety compared to conventional decitabine / azacitidine in GLP-preclinical toxicity studies.

[0091] In particular, clinical trials confirmed a high disease control rate at a dose of 32 mg / day, and animal studies using similar dose groups to test animal models of the disease group (AML, T-ALL, and various solid tumors) showed superior efficacy compared to existing competing drugs.

[0092] In particular, in a Phase 1a clinical trial of Aza-T-dCyd for solid tumors conducted by the National Cancer Institute, no major side effects were observed even when Aza-T-dCyd was administered at approximately twice the dose of existing decitabine. The drug demonstrated efficacy and safety in humans, including an excellent PK profile when administered orally and confirmation of a potent PD marker in circulating tumor cells. Even at very low doses, it was observed to slow disease progression in cancer patients, demonstrating superior efficacy compared to existing drugs.

[0093] Therefore, Aza-T-dCyd can be a DNMT1 inhibitor that offers potent efficacy, an improved safety profile, ease of use, and the potential to overcome drug resistance compared to existing DNMT1 inhibitors that are the standard of treatment.

[0094] On the other hand, venetoclax can be administered up to 250 mg / day, and when used in combination with venetoclax, the dose of Aza-T-dCyd can be less than 32 mg / day, for example, 8 mg / day or more but less than 32 mg / day, preferably 8 to 24 mg / day. 24 mg corresponds to 1.5 mpk.

[0095] When used in combination with venetoclax, Aza-T-dCyd can be administered at 25% to 75% of the maximum tolerable dose (MTD).

[0096] The primary goal of Phase 1 clinical trials, the first to be conducted in humans after preclinical studies to observe toxic and adverse drug reactions, is to determine the maximum tolerated dose, which is the maximum dose that can be tolerated by the subject without causing adverse reactions.

[0097] In animal models, Aza-T-dCyd showed strong anticancer efficacy in the group administered approximately 0.5 mpk (mg / kg), and the drug exposure at that dose, when administered to humans, is at a level of 8 mg / day or less (25% of the MTD in Phase 1 clinical trials), ensuring a wide therapeutic window.

[0098] When comparing the efficacy of the current standard AML treatment, which is azacitidine in combination with the BCL-2 inhibitor venetoclax (trade name: Venclexta), with the efficacy of Aza-T-dCyd alone, the Aza-T-dCyd 2.0mpk monotherapy group (equivalent to the MTD dose of 32 mg of Aza-T-dCyd in Phase 1 clinical trials) showed efficacy at a similar level to the azacitidine / venetoclax group. The Aza-T-dCyd 0.5 or 1.0mpk in combination with venetoclax (25-50% of the MTD dose of 32 mg in Phase 1 clinical trials) showed significantly stronger efficacy compared to the standard therapy (azacitidine / venetoclax combination) group (Figures 28-33).

[0099] In the case of decitabine / azacitidine, an existing drug with the same mechanism of action, a 50% dose reduction from the MTD was necessary for co-administration with venetoclax. While a dose reduction to 25% of the MTD showed excellent safety, it revealed a problem of weak efficacy. In the case of Aza-T-dCyd, when the MTD dose was reduced to 25% and administered in co-administration with venetoclax, it showed significantly better efficacy compared to the azacitidine / venetoclax administration group. Therefore, when compared with the results of existing drugs, there is a very high possibility that Aza-T-dCyd will show superior safety / efficacy to the human body compared to conventional standard therapy (Example 5).

[0100] In the present invention, the compounds of chemical formula 1 and chemical formula 2 can each exist independently in solid or liquid form. In the solid state, they can exist in crystalline or amorphous form, or as mixtures thereof. In the case of crystalline or amorphous compounds, pharmaceutically acceptable solvates can be formed. In crystalline solvates, solvent molecules are incorporated into the crystal lattice during crystallization. Solvates may contain non-aqueous solvents, such as, without limitation, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or these may contain water as the solvent incorporated into the crystal lattice. Solvates in which water is the solvent incorporated into the crystal lattice are usually called “hydrates.” Hydrates include not only stoichiometric hydrates but also compositions containing a variable amount of water. The present invention includes all such solvates.

[0101] Certain compounds of the present invention, including their diverse solvates existing in crystalline form, can exhibit polymorphism (i.e., the ability to exist in different crystalline structures). These different crystalline forms are commonly known as "polymorphs." The present invention encompasses all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometric arrangement, and other descriptive properties of the crystalline solid state. Thus, polymorphs can have different physical properties, so to speak, such as shape, density, hardness, deformability, stability, and solubility. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. For example, different polymorphs can be produced by changing or adjusting the reaction conditions or reagents used in the production of the compound. For example, changes in temperature, pressure, or solvent can produce polymorphs. Furthermore, one polymorph can spontaneously transform into another polymorph under specific conditions.

[0102] In this specification, 4'-thio-5-aza-2'-deoxycytidine (Aza-T-dCyd) drugs include not only the compound of chemical formula 1 described above, but also its pharmaceutically acceptable salts, solvates, and prodrugs.

[0103] "Pharmacologically acceptable salts" refer to pharmaceutically acceptable organic or inorganic salts. Exemplary salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, azid phosphates, isonicotinates, lactates, salicylates, acid citrates, tallates, oleates, tannates, pantothenates, vitallates, ascorbates, succinates, maleates, gentisinates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1, This includes, but is not limited to, 1'-methylene-bis-(2-hydroxy-3-naphthoate) salts. A pharmaceutically acceptable salt may contain other molecules, e.g., acetate ions, succinate ions, or other counterions. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have one or more extra charged atoms in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0104] The term "prodrug" has its meaning as used in the relevant technical field. For example, it refers to a compound that has been chemically modified from a physiologically active substance or a therapeutically active organic compound, and is designed to release or liberate the parent compound enzymatically or under other conditions in vivo. Prodrugs are converted into the target compound in vivo after administration. They are useful drugs that have unsuitable properties in terms of side effects, stability, solubility, absorption, or duration of action, and are made clinically usable through chemical modifications.

[0105] A "solvate" refers to a compound of chemical formula 1 or a salt thereof that further contains a stoichiometric or non-stoichiometric amount of solvent bonded by non-covalent forces. When the solvent is water, the solvate is a hydrate.

[0106] In this specification, “cancer” typically refers to a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematological malignancies (e.g., multiple myeloma, lymphoma, and leukemia) and solid tumors. Non-limited examples of hematological malignancies include non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia. Non-limited examples of solid tumors include gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.

[0107] In this specification, “patient,” “subject,” and “subject” refer to animals such as mammals. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal such as a dog, cat, livestock (e.g., a horse, pig, or donkey), chimpanzee, or monkey.

[0108] In this specification, the anticancer effect or therapeutic effect of an anticancer agent refers to an effect that occurs while a patient has a particular type of cancer, reducing the severity of the cancer, reducing the size of the tumor, or slowing or slowing the progression of the cancer.

[0109] For example, the anticancer effect of anticancer drugs can be measured in the cell viability (degree of cytotoxicity or change in cell number) of cancer cells after treatment with the anticancer drug in vitro and / or in vivo. This can be indirectly confirmed, for example, through drug response tests using cell lines or nonclinical animal models (xenografts). Furthermore, the anticancer effect of anticancer drugs has been directly confirmed from cancer patients, and related data can be derived and used as a database. In addition, when designing anticancer drug administration guidelines, PK parameters and / or toxicity profiles of animal models can be considered in parallel.

[0110] The anticancer effect of an anticancer drug is expressed as the in vitro data of the drug's maximum effect (%), for example, IC. 50 ,I C 60 ,I C 70 ,I C 80 , and IC 90 This can also be inferred from other data, and can be confirmed from non-clinical animal models and clinical cancer patients through in-vivo data such as the maximum blood concentration (Cmax) and / or area under the blood drug concentration-time curve (AUC).

[0111] The responsiveness of anticancer drugs refers to their clinical sensitivity in terms of their anticancer effect.

[0112] When used in relation to the treatment of cancer, "sensitivity" and "sensitive" are relative terms referring to the degree of a compound's effect in mitigating or reducing the progression of the tumor or disease being treated.

[0113] "Effective anti-cancer effect / response of a patient" may be, for example, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more inhibition in the patient's response, as measured by any suitable means, such as so-called gene expression, cell counting, analysis results, etc.

[0114] The dosage administered herein is the dosage expected to have a medicinal effect. The medicinal effect in the present invention can be an anti-cancer effect. The reactivity (anti-cancer effect) of an anti-cancer agent is the degree of reaction, which is the % maximum effect of the anti-cancer agent, for example, IC 50 IC 60 IC 70 IC 80 and IC 90 It may also be a value that exhibits toxicity to normal cells (LC 50 ), )

[0115] For example, an oral dosage form can be formulated using various dosage form technologies known in the art. For example, it can include a biodegradable (hydrolyzable) polymer carrier used for adhesion to the oral mucosa. It is made to erode gradually over a预定 period, where drug delivery is essentially provided entirely.

[0116] Drug delivery in oral formulations avoids weaknesses encountered in oral drug administration, such as slow absorption, degradation of the activator by fluids present in the gastrointestinal tract, and / or first-pass inactivation in the liver. For biodegradable (hydrolyzable) polymer carriers, virtually any such carrier can be used as long as the desired drug release profile is not impaired, and the carrier is compatible with any other components present in the oral dose unit. Generally, polymer carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the moist surface of the oral mucosa. In this specification, an example of a useful polymer carrier is acrylic acid polymer (e.g., carbomer). In some embodiments, non-limiting examples of other components that can be incorporated into oral formulations include disintegrants, diluents, binders, lubricants, flavorings, colorants, and preservatives. In some embodiments, for oral or sublingual administration, the formulation may be in the form of conventionally shaped tablets, lozenges, or gels.

[0117] In some embodiments, administration of the compound may continue at the physician's discretion if the patient's condition improves; the dose of the drug to be administered as an alternative may be temporarily reduced or temporarily interrupted for a certain length of time (i.e., “drug-free period”). The length of the drug-free period may vary between 2 days and 1 year, and may include, as mere examples, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. In some embodiments, the dose reduction during drug discontinuation is 10% to 100%, including, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0118] Once the patient's condition improves, the maintenance dose is administered as needed. Thereafter, the dosage, frequency, or both may be reduced as a function of the symptoms to a level where the improved disease, disorder, or condition is maintained. However, if the patient experiences a spontaneous relapse of symptoms, intermittent treatment over a long period may be required.

[0119] The amount of a given formulation corresponding to such a quantity varies depending on factors of the subject requiring treatment, such as the specific compound, the severity of the disease, and identity (e.g., body weight). Nevertheless, it can be routinely determined in methods known in the art, for example, by the formulation to be administered, the route of administration, and the specific circumstances surrounding the subject to be treated. However, generally, doses used for treating adults will typically range from 0.02 to 5000 mg / day, or about 1 to 1500 mg / day.

[0120] The single doses described herein may be provided as a single dose or as divided doses administered simultaneously, for example, as two, three, four or more subdoses.

[0121] In some embodiments, the oral dosage form is a unit dose form suitable for single-dose administration of a precise amount. In the unit dose form, the dosage form is divided into unit doses containing an appropriate amount of one or more compounds. In some embodiments, the unit dose is in the form of packaging containing separate amounts of dosage forms. Non-limiting examples include packaged tablets or capsules, and powder vials or ampoules. The aqueous suspension composition can be packaged in single-dose, non-resealable containers. Alternatively, multi-dose, resealable containers can be used, in which case the composition typically contains a preservative.

[0122] In some embodiments, parenteral injection dosage forms are provided in unit dose forms, or in multi-dose containers, that include ampoules, with added preservatives.

[0123] Typically, it is prepared in a unit-dose injectable form with a pharmaceutically acceptable parenteral vehicle for parenteral administration, i.e., bolus, intravenous, and intratumoral injection. It is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized formulation or aqueous solution. [Effects of the Invention]

[0124] Aza-T-dCyd is a thio-nucleoside compound in which the deoxyribose structure of a nucleoside anticancer drug is changed to a thiodeoxyribose structure. Through this change, it possesses the following characteristics, overcoming the limitations of existing nucleoside anticancer drugs and meeting unmet needs. (1) By forming a DNMT1 trapping complex that is sustained for a longer period due to the thio-nucleoside structure, differentiated potent anticancer efficacy compared to decitabine (trade name: Dacogen) and azacitidine (trade name: VidAza) and ensure the possibility of overcoming resistance in patients with resistant AML; (2) The thio-nucleoside structure significantly reduces the activation rate by dCK (deoxycytidine kinase) in normal cells, thereby selectively delivering the active ingredient of the drug to cancer cells and ensuring a superior safety profile; (3) Compared to existing decitabine / azacitidine, it is broken down more slowly by cytidine deaminase when administered orally, thus potentially overcoming resistance due to metabolism within cancer cells and ensuring the possibility of oral administration; and (4) Ensure safety and oral PK profile through administration to humans.

[0125] Furthermore, Aza-T-dCyd exhibits a potent efficacy and differentiated safety profile due to its thio-nucleoside skeleton, demonstrating a clear competitive advantage in terms of efficacy and safety compared to existing DNMT1 inhibitors. This includes expanded indications (existing DNMT1 inhibitors (primarily MDS / AML) vs. Aza-T-dCyd (extended to various hematological and solid tumors)), superior efficacy in various situations where existing DNMT1 inhibitors do not show efficacy (such as potent efficacy in solid tumor patients in Phase 1 clinical trials), and superior safety (non-clinical and Phase 1 clinical trial results).

[0126] Therefore, the combination therapy of 4'-thio-5-Aza-2'deoxycytidine (Aza-T-dCyd), a multitarget inhibitor containing a DNMT1 inhibitor according to the present invention, and venetoclax, an inhibitor of BCL-2 that mediates apoptosis, is applicable in diverse ways in terms of administration method, dosage, or target patient group. [Brief explanation of the drawing]

[0127] [Figure 1] This is a schematic diagram illustrating the mechanism of action of epigenetics. [Figure 2] This is a schematic diagram illustrating the normal hematopoietic process in our bodies. In stage 1, where cell numbers are secured through rapid cell division, and in stage 2, where cell division stops and cell maturation progresses, the role of Master TFs (especially CEBP / epsilon) that lead each stage is extremely important. [Figure 3] This schematic diagram illustrates how DNMT inhibitor treatment induces normal DNA methylation in hypermethylated MDS / AML hematological cancer cells, leading to differentiation into normal cells through the re-expression of the CEBP / epsilon gene and thus exerting an anticancer effect. [Figure 4] These are the chemical structures of decitabine and azacitidine. [Figure 5] This is the mechanism of action of nucleoside-based anticancer drugs. [Figure 6] This is a DNA damage repair process using nucleoside-based anticancer drugs via the BER mechanism of action. [Figure 7] The mechanism of action of Aza-T-dCyd is to mediate apoptosis and cell cycle arrest through the DNA damage response. [Figure 8] This is the mechanism of action of Aza-T-dCyd. [Figure 9] This describes the mechanism of action of B-cell lymphoma (BCL)-2, which mediates apoptosis, and its inhibitor, venetoclax. [Figure 10] The results show that DNMT1 is inhibited when MV4-11 (AML cell line) cells are treated with NTX-301 (SRI-9639) and decitabine at different concentrations. [Figure 11] This study confirmed DNMT1 inhibition in KG-1a (AML cell line) cells after treatment with NTX-301 (Aza-T-dCyd) and T-dCyd. [Figure 12] This study confirmed DNMT1 inhibition in CCRF-CEM (ALL cell line) cells after treatment with NTX-301 and T-dCyd. [Figure 13] The results show that DNMT1 inhibition was confirmed in NCI-H23 (Lung carcinoma cells), HCT-116 (Colon carcinoma cells), and IGROV-1 (Ovarian carcinoma cells) cell lines upon treatment with NTX-301 and T-dCyd. [Figure 14] This result shows that when MV4-11 cells were treated with Aza-T-dCyd, re-expression of the p15 tumor suppressor gene was confirmed by inhibition of DNMT1. [Figure 15] This study compared and analyzed the re-expression patterns of the p15 tumor suppressor gene induced by DNMT1 inhibition in MV4-11 cells treated with Aza-T-dCyd and decitabine. [Figure 16] This study confirmed the induced CEBP / epsilon expression patterns when THP-1 cells were treated with NTX-301 (Aza-T-dCyd) at different concentrations. [Figure 17]This study describes the results of treating various AML cell lines (MV4-11, HL-60, KG-1a) with Aza-T-dCyd and examining the levels of intracellular DNMT1, CEBP / epsilon, and CDKN1B. [Figure 18] This is the result of confirming NTX-301 target engagement. [Figure 19] This figure shows the growth inhibitory effect of Aza-T-dCyd on hematological cancer cell lines. [Figure 20] This chart compares the growth inhibitory effects of Aza-T-dCyd and decitabine on hematological cancer cell lines. [Figure 21] These are the measurement results for pyrimidine level and RRM1 expression after NTX-301 treatment. [Figure 22] This study confirmed the increased expression of DNA replication stress-related genes due to DNA adducts formed during NTX-301 treatment, as well as the efficacy of the drug in different cell lines. [Figure 23] This result confirms an increase in DNA damage response markers due to p53 pathway activation during NTX-301 treatment. [Figure 24] This shows the results of selective DNA dimethylation induced in MV4-11 and MOLM-13 cell lines upon treatment with NTX-301, as well as the analysis of RNA-seq data. [Figure 25] This shows a comparison of tumor growth and weight differences among different treatment groups in the Molm13 leukemia cell line Xenograft model. [Figure 26] This result shows that NTX-301 suppressed DNMT3B expression in cancer tissue after being tested in a Molm13 leukemia cell line Xenograft model. [Figure 27] This report describes the results of examining changes in DNMT1, DNMT3A, and DNMT3B after treatment with Aza-T-dCyd and decitabine in the HL-60 leukemia cell line Xenograft model. [Figure 28]These are the CRL results showing the efficacy of co-administration of NTX-301 (0.5, 1, 1.5 mg / kg) and venetoclax (100 mg / kg) in the MV4-11 AML cell line xenograft model. [Figure 29] These are the CRL results showing the efficacy of co-administration of NTX-301 (0.5, 1, 1.5 mg / kg) and venetoclax (100 mg / kg) in the MV4-11 AML cell line xenograft model. [Figure 30] This is a pharmaron result showing the efficacy of co-administration of NTX-301 (0.5, 1, 2 mg / kg) and venetoclax (50 mg / kg) in an MV4-11 AML cell line xenograft model. [Figure 31] This is a pharmaron result showing the efficacy of co-administration of NTX-301 (0.5, 1, 2 mg / kg) and venetoclax (50 mg / kg) in an MV4-11 AML cell line xenograft model. [Figure 32] This is a pharmaron result showing the efficacy of co-administration of NTX-301 (0.5, 1, 2 mg / kg) and venetoclax (50 mg / kg) in an MV4-11 AML cell line xenograft model. [Figure 33] This is a pharmaron result showing the efficacy of co-administration of NTX-301 (0.5, 1, 2 mg / kg) and venetoclax (50 mg / kg) in an MV4-11 AML cell line xenograft model. [Modes for carrying out the invention]

[0128] The present invention will be described in more detail below through examples. However, the following examples are merely for the purpose of clearly illustrating the technical features of the present invention and do not limit the scope of protection of the present invention.

[0129] Example 1: In vitro pharmacology

[0130] 1-1. DNMT1 Inhibition

[0131] The key pharmacological markers (PD markers) for Aza-T-dCyd are inhibition of the drug target DNMT1 and a decrease in the amount of DNMT1 protein in cells.

[0132] Cytidine-like nucleoside inhibitors such as decitabine are known to integrate into DNA, irreversibly trap DNMT1, and induce its degradation via proteolytic enzyme complexes. Furthermore, it has been established through various basic and clinical studies, as well as the use of DNMT1 inhibitors in patients, that such loss of DNMT1 protein promotes the expression of differentiation-inducing / anti-oncogenes.

[0133] 1-1-1. Confirmation of decreased intracellular DNMT1 protein in AML cell lines.

[0134] To evaluate the DNMT1 inhibitory efficacy of Aza-T-dCyd in AML cell lines, standard AML cell lines MV4-11 (Figure 10) and KG-1a (Figure 11) were treated with Aza-T-dCyd and the positive control substance decitabine, and Western blotting was performed on the DNMT1 protein. As can be seen from Figures 10 and 11, Aza-T-dCyd was confirmed to reduce DNMT1 protein in a concentration-dependent manner in all cell lines. In particular, it was confirmed that DNMT1 was completely reduced even at a very low concentration of 20 nM.

[0135] 1-1-2.ALL cell line / solid tumor cell line

[0136] The same DNMT1 inhibitory efficacy evaluation was performed on other hematological cancer cell lines (ALL cell lines) and solid tumor cell lines, and it was confirmed that Aza-T-dCyd (NTX-301) can inhibit DNMT1 in a concentration-dependent manner in CCRF-CEM(ALL) (Figure 12) and solid tumor cell lines (Figure 13).

[0137] 1-2. Cellular Pharmacodynamics

[0138] 1-2-1. Confirmed the re-expression of the p15 tumor suppressor gene.

[0139] In hematopoietic stem cells of MDS and AML patients, numerous cytosines in the promoter regions of many tumor suppressor genes are methylated, suppressing their expression. This leads to problems where important functions such as DNA damage repair and cell cycle checkpoints are suppressed compared to normal.

[0140] Abnormal DNA methylation of numerous tumor suppressor genes has been reported in many types of leukemia, including AML, and in particular, inactivation of the p15 tumor suppressor gene by CpG island hypermethylation induces abnormal cell proliferation.

[0141] When methylation patterns are normalized by treatment with epigenetic regulatory drugs that can normalize DNA methylation, such as decitabine, these hematopoietic stem cells differentiate or die and can be usefully used in the treatment of MDS / AML patients, in which case re-expression of the intracellular PD marker p15 is induced. Through existing decitabine / azacitidine clinical trials and patient treatments, p15 re-expression in cancer cells and cancer patients with silencing p15 has been established as a PD marker to monitor the pharmacodynamics of DNMT1 inhibitors.

[0142] Therefore, in order to confirm the mode of tumor suppressor gene re-expression through the epigenetic regulatory mechanism mediated by DNMT1 inhibition during Aza-T-dCyd (NTX-301) treatment, MV4-11 cells, an AML cell line targeting AML cell lines, were treated with Aza-T-dCyd at different concentrations. Re-expression of the p15 tumor suppressor gene, a PD marker induced by DNMT1 inhibition, was confirmed, and strong re-expression of p15 mRNA was confirmed by RT-PCR (Figure 14).

[0143] To compare the efficacy of DNMT1 inhibitors in MV4-11 cells, quantitative RT-PCR was performed to confirm the expression of the p15 tumor suppressor gene after treatment with Aza-T-dCyd (NTX-301) and decitabine. The results showed that the expression level of the p15 tumor suppressor gene was significantly increased under conditions of treatment with Aza-T-dCyd at concentrations of 60 nM and 200 nM (Figure 15 and Table 1).

[0144] Table 1 below compares the degree of p15 mRNA re-expression in the MV4-11 cell line, which is an AML cell line, after treatment with the DNMT1 inhibitor (Aza-T-dCyd / decitabine) (showing the multiplier of increase relative to 0 nM).

[0145] [Table 1]

[0146] Furthermore, it was confirmed that Aza-T-dCyd induces superior p15 upregulation compared to decitabine.

[0147] Therefore, based on the results of confirming a decrease in DNMT1 and re-expression of the p15 tumor suppressor gene upon treatment with Aza-T-dCyd, Aza-T-dCyd has high potential as a novel targeted anticancer agent.

[0148] 1-2-2. Check for changes in additional PD markers (CEBP / epsilon, CDKN1B).

[0149] In malignant cells from MDS / CMML / AML patients, there is a problem in that while lineage commitment master TFs such as CEBP / alpha, Pu.1, and GATA factors are expressed at high levels, the expression of late-stage developmental transcription factors, including CEBP / epsilon, is kept low due to hypermethylation of each gene.

[0150] CEBP / epsilon is a protein that induces maturation of immature blood cells and suppresses abnormal proliferation. The expression of this protein induces increased expression of CDKN1B and Myc antagonists (MAD). CDKN1B is a CDK inhibitory protein that plays a role in halting cell cycle proliferation and inducing differentiation. The expression of CEB / epsilon and CDKN1B thus induces differentiation of AML malignant cells and exhibits potent anticancer efficacy.

[0151] It has been reported that decitabine drug treatment inhibits DNMT1, inducing DNA demethylation and CEBP / epsilon re-expression, leading to apoptosis and cell differentiation, thereby demonstrating excellent anticancer efficacy.

[0152] To confirm the target engagement patterns induced by DNMT1 inhibition during Aza-T-dCyd (NTX-301) treatment in various AML cell lines, CEBP / epsilon and CDKN1B expression were examined and compared (Figure 16).

[0153] When THP-1 cells, an AML cell line, were treated with Aza-T-dCyd at different concentrations, dose-dependent inhibition of DNMT1 was induced, and a correlation between this and increased CEBP / epsilon expression and CDKN1B expression was confirmed. This demonstrated the function of Aza-T-dCyd in inducing maturation and differentiation of immature hematopoiesis.

[0154] After treating AML cell lines MV4-11, HL-60, and KG-1a cells with Aza-T-dCyd (NTX-301) for 72 hours, intracellular protein expression levels were examined. The results showed that DNMT1 expression was inhibited, while CEBP / epsilon and CDKN1B expression increased. The greatest increase in expression was observed in MV4-11 and HL-60 cells (Figure 17).

[0155] 1-3. Target Retention

[0156] When MV4-11 cells, which had been identified as a responsive cell line to Aza-T-dCyd through existing experiments, were treated with DNMT1 knock-out (siRNA), a knockout cell line was created that did not affect cell survival, division, or proliferation.

[0157] When the prepared DNMT1 knock-out cell line was treated with Aza-T-dCyd (NTX-301) at different concentrations, a decrease in cytotoxicity was observed, which is judged to be a result of weakened pharmacological effects on MV4-11 cells. Through this, it was confirmed that Aza-T-dCyd has potent pharmacological activity via DNMT1 (Figure 18).

[0158] 1-4. Cellular cytotoxicity

[0159] To confirm the inhibitory effect of the DNMT1 inhibitor Aza-T-dCyd on cell proliferation in Leukemia cell lines, growth-inhibitory activity was measured.

[0160] 1-4-1.AML

[0161] In various AML cell lines, Aza-T-dCyd (NTX-301) exhibits IC as shown in Figure 19. 50 It possessed a value and showed excellent cell survival inhibitory effects.

[0162] 1-4-2. Comparison of the effects of DNMT1 inhibitors on cell proliferation in Leukemia cell lines

[0163] To confirm the effects of the DNMT1 inhibitors Aza-T-dCyd and decitabine on cell proliferation in Leukemia cell lines, growth-inhibitory activity was measured. Treatment with each DNMT1 inhibitor for 72 hours resulted in a decrease in the GI of decitabine, which is known to be effective in suppressing Leukemia cell growth.50 The values ​​(half maximal growth inhibitory concentrations) range from 0.024 μM to 4.3 μM, and the GI of Aza-T-dCyd 50 The values ​​were measured from 0.014 μM to 0.69 μM. When comparing the overall inhibitory efficacy against cell proliferation, it was confirmed that Aza-T-dCyd was significantly more effective than decitabine (Figure 20).

[0164] 1-4-3. IC of Aza-T-dCyd in cancer-specific cell lines 50 Value result

[0165] To secure a biomarker for Aza-T-dCyd, drug activity profiling analysis was performed, which involved evaluating cytotoxicity in 200 cell lines.

[0166] Drug activity profiling and genetic analysis of 200 cell lines revealed that Aza-T-dCyd exhibits potent efficacy in various solid tumors (Table 2).

[0167] [Table 2] JPEG0007898763000005.jpg210161 JPEG0007898763000006.jpg216161 JPEG0007898763000007.jpg213161 JPEG0007898763000008.jpg207161 JPEG0007898763000009.jpg203161 JPEG0007898763000010.jpg111161

[0168] Example 2: Study of the mechanism of action of Aza-T-dCyd

[0169] Figures 7 and 8 illustrate the mechanisms of action of Aza-T-dCyd in response to DNA damage, and the mechanism of action of Aza-T-dCyd, respectively.

[0170] Aza-T-dCyd exhibits stronger anticancer efficacy compared to decitabine by forming a longer-lasting DNA-DNMT1 adduct and inducing stronger DNA damage, thereby strongly activating the CHK1-p53 pathway, through DNA incorporation and DNMT1 trapping via an azacytosine functional group that can effectively trap the DNMT1 enzyme.

[0171] 2-1. Induction of DNA damage response

[0172] (1) Inhibition of DNA uptake and pyrimidine metabolism

[0173] Aza-T-dCyd is a pyrimidine analog whose metabolism is carried out within cells by various pyrimidine metabolism-related enzymes.

[0174] When cells were treated with Aza-T-dCyd (NTX-301), the expression of RRM1 protein, a ribonucleotide reductase important for dNTP de novo synthesis, was suppressed, and the levels of dCTP and dTTP in the cells decreased, which may affect the pyrimidine metabolic process in cells (Figure 21). A decrease in dCTP and dTTP, which act as important precursors when cancer cells replicate DNA, can induce replication stress and generate a strong DNA damage response.

[0175] DNA insertion of Aza-T-dCTP, a metabolite of Aza-T-dCyd, can induce replication stress and increase the momentum of the DNA damage response by causing base excision repair and mismatch repair, thereby slowing down DNA replication.

[0176] (2) Induction of DNA damage response by DNA-DNMT1 adduct formation

[0177] Upon treatment with Aza-T-dCyd, Aza-T-dCyd inserted into intracellular DNA is trapped through covalent binding with DNMT1, forming a DNA-DNMT1 adduct.

[0178] The formed adductor can disrupt the DNA replication fork by hindering its progression through its bulky structure, causing a double-strand break and triggering a strong DNA damage response.

[0179] Pyrimidine metabolism, DNA uptake, and DNMT1 adduct formation ultimately trigger a strong DNA damage response.

[0180] In Figure 22, we confirmed the increased expression of DNA replication stress-related genes and the drug's efficacy in different cell lines by DNA adducts formed during Aza-T-dCyd treatment.

[0181] (3) Enhancement of the DNA damage response by activating the DDR-p53 pathway

[0182] Treatment with Aza-T-dCyd (NTX-301) increased H2AX phosphorylation, a DNA damage response marker, and simultaneously increased phosphorylation of Chk1, a DNA damage sensor. It also increased the expression of p53 protein, which regulates the cell cycle / death in the DNA damage response, thus promoting the DNA damage response and consequently suppressing tumorigenic activity in hematological cancers (Figure 23).

[0183] 2-2. DNA demethylation

[0184] Treatment with Aza-T-dCyd induces strong DNMT1 depletion and leads to a decrease in overall DNA methylation levels.

[0185] Decitabine induces dimethylation of entire genomic regions, whereas Aza-T-dCyd selectively induces dimethylation by targeting DNA regions replicated during early replication, promoter regions important for gene expression, DNMT1-binding regions, and replication stress regions.

[0186] DNA dimethylation by Aza-T-dCyd treatment reactivates various suppressed tumor suppressor genes and genes necessary for differentiation induction of AML cells, and the reactivated expression patterns of these genes were confirmed through RNA-seq data analysis (Figure 24).

[0187] Example 3: Pharmacokinetic evaluation by concentration - Mice / Rat / Dog PK profiles

[0188] The pharmacokinetic (PK) profile of Aza-T-dCyd was investigated while administering it orally and intravenously to mice, rats, and dogs at various concentrations.

[0189] The animal species with the most similar drug distribution to that of humans is the Mice (which, like humans, have CDA (cytidine deaminase) primarily distributed in the liver and exhibit the most similar metabolic pattern). To confirm whether absorption occurs well across species, the PK profiles were examined in rats and dogs. All three species—Mice, Rat, and Dog—showed excellent potential for oral administration, and administration to Mice resulted in a level of C25 that ensured excellent efficacy. max We confirmed that a / AUC value can be easily achieved. In other words, when Aza-T-dCyd is administered orally at a dose of 0.5 mpk or higher, drug efficacy can be easily ensured in vivo.

[0190] Tables 3 and 4 show the pharmacokinetic evaluation results of Aza-T-dCyd for mice, Table 5 shows the pharmacokinetic evaluation results of Aza-T-dCyd for rats, and Table 6 shows the pharmacokinetic evaluation results of Aza-T-dCyd for dogs.

[0191] [Table 3]

[0192] [Table 4]

[0193] [Table 5]

[0194] [Table 6]

[0195] Based on these results, the predicted dosage for efficacy in humans is 32 mg / day or less when administered alone, and 8-16 mg / day when administered in combination. These results fall within the range below the MTD (Mean Time Tolerance) in Phase 1 clinical trials, as described later.

[0196] Example 4: In vivo Pharmacology (PK / PD correlation)

[0197] 4-1. MDS / AML

[0198] To confirm the efficacy of Aza-T-dCyd in animal models and to verify that Aza-T-dCyd exhibits excellent dose-dependent efficacy, we conducted experiments using the Molm13 Xenograft model (Figure 25).

[0199] After establishing the Molm13 Xenograft model, an animal model of AML that showed no therapeutic effect from decitabine / azacitidine, Aza-T-dCyd (NTX-301) was orally administered at a dose of 0.2–1.5 mg / kg.

[0200] Unlike existing literature on decitabine / azacitidine, administration of Aza-T-dCyd demonstrated a dose-dependent, potent anticancer effect without toxic signals (weight changes).

[0201] Analysis of cancer tissue collected after administration of Aza-T-dCyd (Western blotting) (Figure 26) confirmed that Aza-T-dCyd strongly inhibits DNMT1 and DNMT3B in a concentration-dependent manner. This inhibition of DNMT3B is considered to contribute significantly to the potent anticancer effect of Aza-T-dCyd.

[0202] In a Molm13 AML Xenograft model study, administration of Aza-T-dCyd demonstrated strong anticancer efficacy through inhibition of DNMT1 and simultaneous potent suppression of DNMT3B expression (a clear decrease in the approximately 90 kDa DNMT3B protein was observed when treated with Aza-T-dCyd at concentrations of 0.4 mg / kg or higher).

[0203] After establishing a whole-body MV4-11 engraftment model that more accurately depicts the disease in the actual human body, Aza-T-dCyd was administered orally at doses of 0.4 to 1.5 mg / kg. It was confirmed that Aza-T-dCyd administration resulted in a statistically significant level of survival improvement even at the minimum dose of 0.4 mg / kg. In the highest dose group, more than half of the MCIEs in the overall group survived until the end of the experiment, and a dose-dependent, strong anticancer effect was observed from all doses without significant toxicity signals (weight changes).

[0204] To confirm the superior efficacy of Aza-T-dCyd compared to competing drugs, an MV4-11 whole-body AML model experiment was conducted to determine if Aza-T-dCyd exhibits dose-dependent superior efficacy. After establishing the whole-body MV4-11 engraftment model, Aza-T-dCyd was administered orally at doses of 1.5, 2.0, and 2.5 mg / kg (for decitabine / azacitidine, the standard dose / dosing schedule used in preclinical trials was used). Compared to decitabine, which showed no therapeutic effect, and azacitidine, which showed no suppression of leukemia progression based on blood cell counts, Aza-T-dCyd demonstrated significantly superior anticancer efficacy against AML, and excellent survival improvement was observed in all dose groups.

[0205] After establishing a whole-body MV4-11 engraftment model, blood samples were collected from the Aza-T-dCyd and decitabine / azacitidine administration groups before treatment (Day 21) and at mid-treatment points (Days 36, 56, and 77), and the Complete Blood Count (CBC) values ​​were analyzed. In the Aza-T-dCyd administration group, no changes were observed in major parameters such as white blood cells (WBC), red blood cells (RBC), neutrophils, and platelets. On the other hand, in the decitabine group, all individuals died before blood samples could be obtained, and in the azacitidine group, severe neutropenia was observed.

[0206] To confirm whether the anticancer efficacy in a whole-body MV4-11 engraftment model is directly related to the ability to suppress the number of cancer cells, we conducted an experiment using a luciferase-transfected Mv4-11 cell line to measure body luminescence after whole-body engraftment. We confirmed that the Aza-T-dCyd treatment group suppressed body luminescence more strongly than the azacitidine treatment group. Compared to the strong tumor-suppressing efficacy shown when NTX-301 (Aza-T-dCyd) was administered at 2.0 mg / kg in the HL-60 AML Xenograft model, the maximum dose of decitabine (0.75 mg / kg) (experiments could not be continued due to animal death at doses above this level) showed no tumor-suppressing effect at all.

[0207] The efficacy of DNMT1 inhibitors is related to their selective profile against DNMT1 and the de novo methyltransferases DNMT3A and DNMT3B targets. Therefore, in this Xenograft model, the expression of DNMT1, DNMT3A, and DNMT3B was confirmed in the groups treated with Aza-T-dCyd and decitabine (Figure 27).

[0208] In HL-60 Xenograft mouse models, 2 mg / kg of NTX-301 (Aza-T-dCyd) or 0.75 mg / kg of decitabine was administered intraperitoneally. Tumor samples were collected on day 11, and the expression of DNMT1, DNMT3A, and DNMT3B was examined. It was confirmed that the expression of all DNMT1 molecules was significantly reduced in both the Aza-T-dCyd and decitabine groups. On the other hand, there were differences in the expression levels of DNMT3A and DNMT3B between the Aza-T-dCyd and decitabine groups. The degree of DNMT3A expression was not affected by Aza-T-dCyd, and the expression level of DNMT3B was significantly reduced by Aza-T-dCyd, but no effect was observed with decitabine.

[0209] High DNMT3B expression has been reported to be associated with a poor prognosis in AML, suggesting that NTX-301 has excellent anticancer effects in leukiemia models due to the inhibitory effect of Aza-T-dCyd on DNMT3B.

[0210] 4-2. T-ALL

[0211] To confirm the efficacy of Aza-T-dCyd in another indication, T-ALL, we evaluated its efficacy in patient-derived animal models of ALL.

[0212] Aza-T-dCyd was administered at doses of 0.5, 1, 1.5, and 2 mg / kg in ALL-patient-derived xenografts (PDX), with T-dCyd (DNMT1 inhibitor) used as a comparison group. Aza-T-dCyd was tolerable up to 1.5 mg / kg and was highly effective in pediatric ALL PDX. Its efficacy was also confirmed in T-ALL, including ETP, a subtype of ALL with very high unmet needs. In particular, compared to the doses administered in AML, it was confirmed that even at low doses, it induced strong tumor regression and could maintain this remission state for a long period of time, indicating very high potential for future development as an ALL treatment.

[0213] 4-3. Various Solid Cancers

[0214] Unlike existing DNMT1 inhibitors, Aza-T-dCyd demonstrated superior efficacy in a variety of solid tumor animal models. In animal models of colorectal cancer, bladder cancer, ovarian cancer, and lung cancer, Aza-T-dCyd administration showed remarkably superior efficacy, as shown in Table 7 (cell lines, animals, dosage, and duration of administration for solid tumor animal models).

[0215] [Table 7]

[0216] The efficacy of Aza-T-dCyd was confirmed by examining tumor growth curves after administration to various solid tumor xenograft models. In groups repeatedly administered 1-2 mg / kg doses of Aza-T-dCyd over 2-6 cycles with 5 cycle durations, superior anticancer effects were observed (test / control (T / C) values ​​were proven to be ≤40%).

[0217] In the colorectal (HCT-116) xenograft model, tumor growth suppression was observed in the group treated with Aza-T-dCyd, with an optimal T / C ratio of 21%. In contrast, decitabine showed little effect, with a T / C ratio of 45%.

[0218] In a non-small cell lung cancer (NCI-H522) xenograft model, tumor growth suppression was observed after administration of Aza-T-dCyd, with an optimal T / C ratio of 31%.

[0219] In the Ovarian (OVCAR-3) xenograft model, tumor regression was observed in the group treated with 1 mg / kg dose of Aza-T-dCyd, with an optimal T / C ratio of 4%. At a dose of 0.5 mg / kg, the T / C ratio was 40%, confirming a dose-dependent effect.

[0220] In the Bladder Tumor (BL0382PD PDX) xenograft model, tumor regression was observed in the group treated with 1 mg / kg dose of NTX-301, with an optimal T / C ratio of 23%. In the case of decitabine, toxicity appeared after 2 cycles in the group treated with 0.75 mg / kg dose.

[0221] Example 5: Safety (GLP-Toxicity)

[0222] Preliminary toxicity assessments of the Aza-T-dCyd compound included experiments to check for toxic symptoms (tissue changes) and DNA damage in the bone marrow of mice after 2 weeks of systemic administration, and preliminary toxicity experiments to evaluate the cellular composition of the bone marrow of mice after 4 weeks of systemic administration.

[0223] During the comet assay experiment, weight changes were measured in each group administered Aza-T-dCyd. The results showed no significant weight changes during the two-week systemic administration period, and no organ damage was observed during the subsequent dissection after two weeks.

[0224] Furthermore, when bone marrow was collected two weeks after administration and a Comet assay was performed to check for DNA damage, it was confirmed that no changes in various indicators of DNA damage occurred compared to the control group, which was not treated with any drug.

[0225] After four weeks of continuous administration, the cellular composition of the bone marrow of mice was evaluated. The results of this experiment showed no changes in the various cells constituting the bone marrow, confirming that Aza-T-dCyd does not have a significant effect on hematopoietic capacity in normal animals.

[0226] Aza-T-dCyd (NTX-301) demonstrated excellent safety and a broad therapeutic window in animal studies, as confirmed by GLP toxicity testing.

[0227] Based on the results of the aforementioned toxicity tests, Aza-T-dCyd was confirmed to be a safe drug that can achieve sufficient therapeutic concentrations, as evidenced by its usable AUC value within a safe range.

[0228] In the MDS / AML model, Aza-T-dCyd showed efficacy even at an AUC of 60 h*ng / ml, and exhibited the best therapeutic effect at approximately 200-250 h*ng / ml. This indicates that in the most sensitive species, the NOAEL value was approximately 50%, which is about 25% of that for HNSTD, suggesting that Aza-T-dCyd has a very broad therapeutic window.

[0229] Other solid tumors and other animal models such as ALL showed superior therapeutic effects even with lower AUC exposures.

[0230] GLP-Tox experiments were performed on rats and dogs, using PO with 5 days of QD for 2 cycles.

[0231] In the GLP rat toxicology study, rats were given 2.5, 5, and 10 mg / kg of Aza-T-dCyd (15, 30, and 60 mg / m³) daily. 2 The drug was administered. The toxic organs were the bone marrow, thymus, heart, and testes. Testicular toxicity, including decreased sperm count, was reversible upon discontinuation of administration, and the maximum tolerated dose (MTD) was 10 mg / kg or more per day (>60 mg / m²). 2 ) was.

[0232] In the GLP dog toxicology study, Aza-T-dCyd was administered at 0.15, 0.5, and 1.0 mg / kg per day (3, 10, and 20 mg / m³). 2 The drug was administered to the bone marrow, thymus, gastrointestinal tract, tonsils, and testes. The maximum tolerated dose (MTD) was 0.5-1 mg / kg (10-20 mg / m²) per day. 2 ) is between , and the highest non-toxic dose (HNSTD) is 0.5 mg / kg per day (10 mg / m²). 2 The observable irreversible potency level (NOAEL) was <0.15 mg / kg (3 mg / m³) per day. 2 ) was.

[0233] Example 6: Combination therapy with Aza-T-dCyd and venetoclax

[0234] Recently, the standard therapy for AML has shifted from the existing decitabine / azacitidine monotherapy to the combination of these drugs with venetoclax, a BCL-2 inhibitor. Therefore, after establishing an animal model of AML disease using the MV4-11 cell line, we compared and evaluated the drug effects of co-administration of these drugs with the in vivo drug effects of co-administration of Aza-T-dCyd and venetoclax.

[0235] 6-1.NTX-301 Mouse effective dose

[0236] One of the major metabolic pathways for 5-azacitidine derivatives is deamination mediated by cytidine deaminosease (CDA), which can inactivate Aza-T-dCyd(NTX-301).

[0237] The human dose of NTX-301 needs to be divided by 3 to 4 to adjust the exposure level for mice.

[0238] In human PK simulation studies, the effective human dose of NTX-301 monoform was calculated to be 96 mg / day, which is equivalent to 369 ng of the mouse dose of 2.0 mg / kg. * This is based on the AUC value of h / mL. Since 96 mg / day must be divided by 3-4, the result is 24-32 mg / day.

[0239] For reference, in the NCI's Phase 1 trial, the AUC value for a 16 mg / day dose was approximately 50 ng*h / mL, which, when converted to a mouse AUC value (3-4 times), becomes 150-200 ng*h / mL. This was a safe and effective dose in mice.

[0240] Furthermore, based on the results of combination therapy with venetoclax in a mouse model, 0.5–1.0 mg / kg was determined to be an effective dose, which translates to 8–16 mg + venetoclax in human doses.

[0241] 6-2. Mouse

[0242] Female NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22 / NjuCrl, Charles River) were 9 weeks old on day 1 of the study, with a body weight (BW) range of 19.8–25.3 g. The animals were supplied with any water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed in Enrich-o'Cobs® laboratory animal bedding irradiated in static microisolators with a 12-hour photoperiod at 20–22°C (68–72°F) and 40–60% humidity. The "Guidelines for the Management and Use of Laboratory Animals" were followed in relation to restraint, rearing, surgical procedures, feed and water control, and veterinary management. The animal care and use program is accredited by the International Association of Accredited Laboratory Animal Care (AAALAC), which ensures compliance with standards for the care and use of laboratory animals.

[0243] 6-3. Tumor cell culture

[0244] The MV-4-11 human dual-phenotype B-myelomonocytic leukemia cell line was obtained from the American Type Culture Collection (ATCC® CRL-9591®) and maintained in suspension culture in Iscove's Modified Dulbecco medium containing 100 units / mL penicillin G sodium, 100 μg / mL streptomycin sulfate, and 25 / mL gentamicin.

[0245] The culture medium was supplemented with 10% fetal bovine serum and 2 mM glutamine. Leukemia cells were cultured in tissue culture flasks in a humidified incubator at 37°C under an atmosphere of 5% CO2 and 95% air.

[0246] 6-4. In vivo transplantation

[0247] On the day of transplantation, MV411 cells were harvested during the exponential growth phase and resuspended in phosphate-buffered saline (PBS). 1×10 7 cells (0.2 mL suspension) were injected intravenously (i.v.) into the tail vein of each mouse.

[0248] Fourteen days after tumor cell transplantation designated on study day 1, the animals were randomized into 7 groups (n = 8) according to body weight, and the average body weight was 21.9 - 22.9 g.

[0249] 6 - 5. Therapeutic agent

[0250] The formulation was stored at -20°C protected from light before being formulated.

[0251] On each dosing day, a dosing solution of Aza-T-dCyd (NTX-301) was prepared. Specifically, the required amount of the compound was mixed with N-methyl-2-pyrrolidone (NMP) while vortexing, then polyethylene glycol (PEG) 400 was mixed in, and ultrasonic treatment was performed to obtain a clear solution. Then saline was added, and further vortexing and ultrasonic treatment were performed to obtain a clear solution of 20% NMP / 40% PEG400 / 40% saline. When administered at 10 mL / kg (0.2 mL / 20 g animal) adjusted according to the body weight of each animal, the resulting dosing solutions of 0.05, 0.1, 0.15 or 0.2 mg / mL provided dosing amounts of 0.5, 1, 1.5 or 2 mg / kg, respectively.

[0252] Venetoclax (ABT-199) and azacitidine (VIDAZA) were purchased. Venetoclax powder was newly formulated in doses of PEG 400 and ethanol following phosal 50 propylene glycol (PG). A 10 mg / mL administration solution prepared in 60% Phosal 50 PG / 30% PEG 400 / 10% EtOH was adjusted to the body weight of each animal when administered at a dose volume of 10 mL / kg (0.200 mL / 20 g mouse). Azacitidine powder was dissolved in PBS on each administration day and adjusted according to the body weight of each animal to obtain a 0.25 mg / mL administration solution that delivered 2.5 mg / kg when administered at a dose volume of 10 mL / kg (0.200 mL / 20 g mouse).

[0253] 6-6.Treatment

[0254] In a previous experimental design in which venetoclax was administered at a dose of 100 mg / kg, the dosage and administration schedule were as follows, and the treatment results are shown in Figures 28 and 29.

[0255] Treatment with the vehicle (20% NMP / 40% PEG400 / 40% saline), Aza-T-dCyd (NTX-301), or venetoclax was administered orally (po), while treatment with azacitidine was administered intraperitoneally (ip). All formulations were administered in a volume of 10 mL / kg (0.2 mL / 20 g mouse) proportional to the body weight of each animal.

[0256] Group 1 was used as the control group, and the vehicle was administered in three cycles: once daily for 5 days, followed by a 2-day rest period, then once daily for 5 days, followed by a 9-day rest period (qd×5, 2 off, qd×5, 9 off - 3 cycles).

[0257] Group 2 was administered 2 mg / kg of Aza-T-dCyd (NTX-301) (qd×5, 2 off, qd×5, 9 off - 3 cycles).

[0258] Group 3 received 0.5 mg / kg Aza-T-dCyd (NTX-301) (qd×5, 2 off, qd×5, 9 off - 3 cycles) together with 100 mg / kg venetoclax (5 days once daily, followed by 2 consecutive days of rest, for a total of 9 cycles, 5 / 2×9).

[0259] Group 4 received 1 mg / kg of Aza-T-dCyd (NTX-301) (qd×5, 2 off, qd×5, 9 off - 3 cycles) along with 100 mg / kg of venetoclax (5 / 2×9).

[0260] Group 5 was scheduled to receive 1.5 mg / kg of Aza-T-dCyd (NTX-301) (qd×5, 2 off, qd×5, 9 off - 3 cycles) along with 100 mg / kg of venetoclax (5 / 2×9). Two animals in this group died treatment-related (TR), and treatment was discontinued on day 8. Treatment was restarted on day 10 with 0.5 mg / kg Aza-T-dCyd (NTX-301) and 100 mg / kg venetoclax.

[0261] Group 6 received 2.5 mg / kg azacitidine (biwk x 8) twice weekly for 8 consecutive weeks, along with 100 mg / kg venetoclax (5 / 2 x 9).

[0262] Group 7 was administered 100 mg / kg of venetoclax (5 / 2 x 9).

[0263] Subsequently, the results of an animal model in which venetoclax was administered in combination at a dose reduced by half to 50 mg / kg are shown in Figures 30, 31, 32, and 33.

[0264] During this study, the mice were classified into seven groups (n=8) on the first day. The actual treatments used are summarized in Table 8.

[0265] [Table 8]

[0266] Biw: Twice a week

[0267] 6 - 7. Endpoint for Survival Study

[0268] Treatment outcomes were evaluated based on the time to endpoint (TTE) and the increase / decrease in lifespan (ILS). Individual animals were euthanized at the endpoint when they reached the moribund endpoint or the end of the study (63 days). Signs of impending death due to tumor progression, including hind limb dysfunction, exophthalmos, weight loss, or neurological signs (protrusions, changes in behavior, etc.), necessitated euthanasia, and all deaths due to tumor progression were classified as deaths in the survival study (DSS). TTE (in days) was recorded for each mouse that died of the disease or was euthanized due to extensive tumor progression.

[0269] The median TTE of the treated mice was expressed as a percentage of the median TTE of the control group mice (%T / C), and the increase in lifespan (ILS) was calculated as follows. ILS = %T / C - 100% Here, T = median TTE of treatment, C = median TTE of control. Thus, if T = C, then ILS = 0%. (For reference, in the L1210 model, the National Cancer Institute's threshold for the anti - acute myelomonocytic leukemia activity of a drug is 25% ILS.)

[0270] 6 - 8. Toxicity

[0271] Animal body weight was measured from days 1 to 5 until the end of the study (day 63), with measurements taken three times weekly. Animals frequently exhibited obvious signs of adverse treatment-related (TR) side effects, and noteworthy clinical observations were recorded. Individual body weight was monitored according to the protocol, and all animals with a weight loss exceeding 30% in a single measurement or 25% over three measurements were euthanized and considered TR deaths. The mean body weight loss of the group was also monitored according to the CR Discovery Services protocol. Acceptable toxicity was defined as a mean body weight loss of less than 20% of the group during the study period and no more than one TR death per 10 treated animals (10%). In groups where mean body weight loss exceeded the acceptable limit, administration was discontinued. Once the mean body weight of the group was restored to an acceptable level, administration was resumed at a lower level and / or at a reduced frequency. Deaths were classified as TRs if they were due to therapeutic side effects documented by clinical signs and / or autopsy. TR classification can also be assigned to deaths due to unknown causes during the treatment period or within 14 days after the last dose. If there is evidence that the death is related to the tumor model rather than the treatment, the death is classified as non-treatment related (NTR). NTR deaths are further classified into NTR (accidental or due to human error), NTRm (due to tumor propagation by invasion or metastasis confirmed by autopsy), and NTRu (etiological cause unknown).

[0272] 6-9. Results of a combined drug experiment using Aza-T-dCyd (NTX-301) and venetoclax.

[0273] Tumor Volume

[0274] As shown in Figures 30 and 31, after establishing a xenograft AML model in which MV4-11AML cells were transplanted subcutaneously into mice, administration of azacitidine alone failed to reduce tumor volume, and administration of venetoclax alone showed only a limited level of tumor regression. In contrast, when venetoclax and Aza-T-dCyd were administered in combination, even using 0.5 mpk (15 mg), it showed excellent anticancer effects similar to those of Aza-T-dCyd 2 mpk monotherapy.

[0275] In other words, we confirmed that NTX-301 alone was sufficient for walking (tumor regression), and that the combination of NTX-301 (0.5 mpk) and venetoclax also produced an effect equivalent to that of monotherapy (complete tumor regression).

[0276] Figure 28 (MV4-11 Systemic model survival) also confirmed that when NTX-301 is administered in combination with the standard dose of venetoclax (100 mg / kg), even very low doses of NTX-301 show superior efficacy compared to competing drugs.

[0277] Body Weight

[0278] After establishing a xenograft systemic AML model in which MV4-11 AML cells were transplanted subcutaneously into mice, the anticancer efficacy was confirmed in disease models administered either 2.0 mpk of Aza-T-dCyd alone in one cycle (5 days oral administration, 2 days rest, 5 days oral administration, 9 days rest) (PO) or in combination with 0.5, 1.0, or 2.0 mpk of Aza-T-dCyd and 50 mpk or 100 mpk of venetoclax in one cycle (PO) (venetoclax was administered orally for 3 weeks, with 5 days oral administration followed by 2 days rest). In the control group, azacitidine 5.0 mpk was administered intraperitoneally (IP) alone or in combination with the optimal dose of azacitidine (2.5 mpk) and 50 mpk or 100 mpk of venetoclax.

[0279] As a result, as shown in Figures 28, 29, 32, and 33, the survival rate was greater in the group administered venetoclax and Aza-T-dCyd (0.5mpk) than in the group administered azacitidine and venetoclax in combination or venetoclax alone, thus confirming the superior anticancer effect of the combination administration of venetoclax and Aza-T-dCyd.

[0280] conclusion

[0281] In a xenograft-based AML disease model using MV4-11 cell lines transplanted subcutaneously, NTX-301 alone demonstrated sufficient anticancer efficacy. Furthermore, when NTX-301 and venetoclax were administered in combination, a corresponding drug effect was observed even with a dose one-quarter less than that of NTX-301 alone.

[0282] In the group administered Aza-T-dCyd 0.5mpk, complete tumor regression was induced, confirming that it exhibited a stronger cancer growth inhibitory effect than the group administered azacitidine and venetoclax in combination (Figure 28).

[0283] As shown in Figure 28, with the existing drug decitabine / azacitidine, which operates on the same mechanism, a 50% dose reduction from the MTD was necessary for co-administration with venetoclax. Furthermore, while a 25% dose reduction from the MTD showed excellent safety, it resulted in reduced efficacy. Conversely, with NTX-301, a 25% dose reduction from the MTD and co-administration with venetoclax demonstrated significantly higher efficacy compared to the azacitidine / venetoclax group, suggesting that NTX-301 exhibits superior safety and high efficacy in humans compared to existing standard treatments.

Claims

1. A pharmaceutical composition for the treatment or prevention of cancer comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug, wherein both drugs are administered in combination in the same or different dosage forms.

2. A pharmaceutical composition for the treatment or prevention of cancer comprising the drug 4'-thio-5-aza-2'-deoxycytidine and the drug venetoclax, wherein both drugs are intended to be administered to a subject via the same or different routes.

3. A pharmaceutical composition for the treatment or prevention of cancer comprising the drug 4'-thio-5-aza-2'-deoxycytidine and the drug venetoclax, wherein both drugs are intended to be administered to a subject parenterally or orally.

4. A pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug administered simultaneously or sequentially.

5. A pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug, to be administered simultaneously with the drug venetoclax.

6. A pharmaceutical composition for the treatment or prevention of cancer, comprising a 4'-thio-5-aza-2'-deoxycytidine drug, which is administered sequentially with a venetoclax drug.

7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the 4'-thio-5-aza-2'-deoxycytidine drug is administered at a dose of 8 mg / day or more and less than 32 mg / day.

8. The pharmaceutical composition according to claim 7, characterized in that the 4'-thio-5-aza-2'-deoxycytidine drug is administered at a dose of 25% to 75% of the maximum tolerable dose.

9. The pharmaceutically active ingredient in the 4'-thio-5-aza-2'-deoxycytidine drug is characterized by reducing or inhibiting the DNMT1 protein in a concentration-dependent manner, as described in any one of claims 1 to 6.

10. Patient groups that developed resistance to DNMT1 inhibitors; patients in whom epigenetic genetic DNA methylation pattern changes accumulated in cancer cells compared to normal cells; patients who received information or a diagnosis regarding the possibility of poor prognosis or resistance development when administered DNMT1 inhibitors; CEBP / alpha and Pu compared to healthy individuals. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that it is administered to a group of patients in which a lineage commitment Master transcription factor selected from the group consisting of 1 and GATA factors is expressed at a high level, while the expression of CEBP / epsilon or late-stage developmental transcription factors is maintained at a low level by hypermethylation of each gene; a group of patients in which nucleoside metabolic resistance may develop when administered with nucleoside anticancer agents; a group of patients to whom platinum-based anticancer agents are administered; a group of patients in which resistance to platinum-based anticancer agents may develop or has developed resistance; and / or a group of patients in which tumor suppressor genes and / or SLFN11 have been epigenetically silenced compared to healthy individuals.

11. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that it is administered to a group of patients diagnosed with AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), or ALL (acute lymphoblastic leukemia), a group of patients with platinum-resistant recurrent end-stage ovarian cancer, a group of patients with platinum-resistant metastatic bladder cancer, a group of patients with metastatic bladder cancer, a group of patients with p53 mutation bladder cancer, a group of patients with hypermethylated SLFN11 bladder cancer, or a group of patients diagnosed with ovarian cancer as stage 3 or 4.

12. The pharmaceutical composition according to claim 11, characterized in that it is administered to a group of patients with chronic myelocellular leukemia, a group of patients with T-cell acute lymphoblastic leukemia (T-ALL), a group of patients with chronic lymphocytic leukemia, a high-risk group of patients with genomic abnormalities, a group of patients with relapsed secondary AML (sAML), a group of patients with t-AML (treatment-related AML) due to past treatment history, and a group of drug-resistant / refractory patients.

13. A kit comprising a 4'-thio-5-aza-2'-deoxycytidine drug and a venetoclax drug for the treatment or prevention of cancer.

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