WO2023014045A1

WO2023014045A1 - Medicinal use of 4'-thio-5-aza-2'-deoxycytidine selected as well-designed multi-target inhibitor

Info

Publication number: WO2023014045A1
Application number: PCT/KR2022/011392
Authority: WO
Inventors: 정두영; 이진수; 조현용
Original assignee: 주식회사 피노바이오
Priority date: 2021-08-02
Filing date: 2022-08-02
Publication date: 2023-02-09
Also published as: KR20230019802A

Abstract

The present invention relates to medicinal use of 4'-thio-5-aza-2'-deoxycytidine (Aza-T-dCyd) selected as a well-designed multi-target inhibitor. Taking advantages as a well-designed multi-target inhibitor, Aza-T-dCyd can be used as an anti-cancer drug for target patient groups with a variety of adjusted administration regimes or doses.

Description

Pharmaceutical use of 4'-thio-5-aza-2'-deoxycytidine selected as a well-designed multi-target inhibitor

In the present invention, 4'-thio-5-aza-2'deoxycytidine, selected as a well-designed multi-target inhibitor, Aza-T-dCyd) for medicinal use. Taking advantage of being a well-designed multi-target inhibitor, Aza-T-dCyd can be used as an anticancer drug in a variety of tailored regimens, doses, or target patient populations.

Genes in our body have the same base sequence for each cell, but the cells in our body express only different genes depending on the specific tissue and do not express other genes.

The mechanism of action that carries out this epigenetic regulation is called epigenetic regulation. ) other components of the chromatin structure.

Double DNA methylation is the most fundamental epigenetic mechanism of action, and among various gene components such as Gene Body, Promoter, and Enhancer, methylation of C base of CpG sequence controls gene expression level. In general, CpG sequences are abundant in the promoter region, and genes with high CpG methylation in the promoter are not well expressed. Conversely, genes with less CpG methylation are actively expressed (FIG. 1).

The DNA double helix has a width of 2 nm, a distance between nucleotides of 0.34 nm, and a length of 3.4 nm when 10 nucleotides are linked in one cycle of the double helix. 146 nucleotides are wrapped around the histone protein, and the histone octamer is wrapped around 1.6 times. The double helix between the nucleosome and the nucleosome consists of about 200 nucleotides, and the state in which the DNA double helix is wound around the histone octamer is called a nucleosome. DNA polymerase, RNA polymerase, and transcriptional regulator bind to the DNA double strand between nucleosomes.

Gene expression regulation is a phenomenon in which the probability of transcription of a gene region present in DNA is changed by a transcriptional regulator. Transcriptional regulators (TFs) change the probability of gene expression from cell to cell. Different cells have different functions because of different levels of expression of transcriptional regulators. Due to DNA methylation, the genes expressed in each cell are different, so there is a difference in function in each cell. Among the base sequences constituting DNA, a methyl group is attached to the 5th carbon of the cytosine hexagonal ring, and the electron regulator protein cannot be attached to that part, so the genetic information of the DNA double helix cannot be transcribed into RNA.

If a methyl group is attached to the 3rd nitrogen atom instead of the 5th carbon of cytosine, a mutation occurs and must be repaired directly. This mutation repair changes the attached methyl group (CH ₃ ) to CH 2 -OH, and the CH ₂ -OH is decomposed into formaldehyde and protons (H ⁺ ₎ of H ₂ C=O to remove it, resulting in the original cytosine molecule. goes through

DNA methylation, histone modification, and RNA interference, key processes of epigenetics, regulate gene expression.

The phenomenon in which the translation process of mRNA is stopped or the mRNA is degraded by the action of miRNA is called RNA interference. miRNAs are transcribed from DNA by the action of RNA polymerase II.

It is known that DNA methylation in our body occurs in two steps: (1) DNA methyltransferase (DNMT) 3A or 3B methylates de novo DNA during tissue differentiation and development to determine the pattern of gene expression in each tissue box; (2) DNMT1 methylates DNA by faithfully replicating the DNA methylation pattern already established by each cell constituting the tissue.

In general cells, in regions where DNA methylation is highly advanced, DNMT1 binds to other proteins, especially histone proteins and other repressor proteins that suppress gene expression, to form structures that make gene expression difficult, and through this, suppress gene expression. Conversely, when DNA methylation has not progressed much, gene expression is promoted by forming a structure that facilitates gene expression. DNA methylation plays an important role in enabling the smooth expression of a set gene combination for each cell so that each cell in our body performs its designated function well. Therefore, abnormalities in these functions lead to the occurrence of various diseases.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are cancers that develop in the bone marrow. Among the hematopoietic stem cells that make up the bone marrow, clones that produce immature leukemia cells (Leukemic Blast) are replaced by clones that normally produce various blood cells. The number is overwhelmed and the blood is filled with leukemic blast cells that cannot function normally. It is a disease that ultimately leads to death due to the failure of blood function.

The normal hematopoiesis process in our body consists of the following processes. (1) When the hematopoietic stem cell is stimulated to produce blood cells, (2) the hematopoietic stem cell rapidly divides in one step and differentiates into progenitor cells capable of producing a sufficient number of blood cells, (3) progenitor cells After dividing and generating a sufficient number of progenitor cells, (4) stop further division/proliferation in the second step, each has its own function, and (5) go through the process of maturation into functional cells that do not divide any more ( Fig. 2).

Master transcription factor (Master TF) plays a major role in this process. When hematopoietic stem cells are stimulated in step 1, master transcription factors that produce progenitor cells such as CEBP/alpha act strongly to produce a sufficient number of progenitor cells. are generated, and when a sufficient number of progenitor cells are generated, the second stage is created, where Master TFs such as CEBP/epsilon are generated. The 2nd stage Master TFs decompose the 1st stage Master TFs so that rapid division/proliferation no longer occurs and induces cell maturation according to the prescribed process.

In MDS and AML, the gene promoter of the 2nd-stage Master TF leading this metastasis process is overmethylated for various reasons, and the expression of this 2nd-stage Master TF is reduced. As a result, the progenitor cells do not pass to the mature stage and continue to divide/proliferate, constantly producing immature cells (FIG. 3). As this process progresses further, multiple mutations (FLT3-ITD, etc.) accumulate and transform into rapidly progressing malignant AML, which leads to death of the patient.

In many cases of MDS/AML, DNMT1 is overactivated, resulting in abnormal methylation patterns, and on the contrary, it is confirmed that these diseases can be treated by normalizing the epigenetic mechanism of action through inhibition of DNMT1 (FIG. 4).

The causes of hypermethylation of the promoter of the Master TF gene include changes in methylation patterns due to abnormalities in DNMT family enzymes (overexpression, overactivation, deletion/mutation, etc.), and abnormalities in IDH1/2 enzymes. There are various causes, such as pattern abnormalities caused by difficulty in removal, pattern abnormalities caused by difficulty in removing methylation patterns due to TET enzyme abnormalities, and accumulation of epigenetic changes due to old age.

However, in any case, inhibiting the activation of DNMT1, a maintenance DNA methylation machinary that maintains methylation patterns during cell growth/division/survival processes, can resolve the hypermethylation pattern, thereby improving the therapeutic efficacy of MDS/AML. It is confirmed what can be brought.

The treatment process and method for AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), CMML (chronic myelomonocytic leukemia) or ALL (acute lymphocytic leukemia) depends on the classification of the disease, the patient's age and health condition, etc. Although it depends on the standard of care (SoC), general standard of care (SoC) is induction therapy through chemotherapy to reduce blasts using anticancer drugs, unlike solid cancers accompanied by surgery to remove carcinoma, complete It mainly consists of consolidation therapy after reaching remission and maintenance therapy to maintain remission. In particular, for severely ill patients who urgently need treatment, hematopoietic cell transplant (HCT) is the most effective. A good method may be performed, but since it is a treatment that can be performed after completely removing the patient's abnormal bone marrow through strong chemotherapy or radiation therapy, its feasibility is very limited in the case of elderly patients with poor physical condition.

Age itself is the most important risk factor for leukemia, especially AML and MDS, because the possibility of genetic mutations that can cause blood cancer, that is, bone marrow disease, increases markedly with aging. Compared to relatively young patients in good condition, it is difficult to apply standard treatment through conventional chemotherapy, which is highly toxic, in most cases for elderly patients whose physical functions are weakened due to various factors such as underlying diseases. Even after treatment, the prognosis is often poor, resulting in frequent recurrence and high early mortality.

Currently, induction therapy through high-intensity chemotherapy or bone marrow transplantation, which are currently used as standard treatments for MDS and AML, are difficult to apply to elderly patients. Since the treatment effect is much lower and the recurrence of the disease is high due to drug resistance, etc., there is an urgent need to develop therapies and drugs tailored to their characteristics, but currently there is no treatment targeting only these elderly patients, especially those over 65 years of age, on the market. It is a state.

Currently, there is a very high unmet need for these two diseases, but despite constant efforts to develop new treatments, most have not been successful, and there has been no major change in standard treatment for decades, and the treatment has not been very successful. In particular, high-risk patients with genetic abnormalities, sAML (secondary AML) patients who relapsed after treatment, t-AML (treatment-related AML) patients due to past treatment history, drug resistance / refractory patients, and elderly patients It is true.

Most of the patients, especially in the case of AML, die prematurely, with a 5-year survival rate of less than 10% for patients aged 65 years or older. The launch of an effective treatment that can lead to prolongation of survival is itself the biggest unmet need.

In the case of elderly patients who do not respond to induction therapy or who relapse after receiving additional treatment, the prognosis is very bleak. The only way to improve their survival time (OS) is bone marrow transplantation. The reality is that strong induction chemotherapy aimed at complete removal of bone marrow in the body cannot be applied to most elderly patients and relapsed/refractory patients.

Since innovative drugs targeting these patients are not currently on the market, the current situation is changing the dose of existing drugs or administering multiple drugs in combination, but the effect is not great, so the goal of treatment is to extend the survival period (OS). However, the survival rate of elderly patients with AML and MDS is extremely low.

When MDS/AML cells (cell lines or patient-derived samples) are treated with Decitabine/Azacytidine, a DNMT1 inhibitor, hypermethylation of CEBP/epsilon promoter, a second-stage master TF, is reduced, CEBP/epsilon is expressed, and CEBP/epsilon's down-stream effector Tumor suppressor genes such as phosphorus p27 are expressed and show anticancer efficacy.

Two existing commercially available DNMT1 inhibitors, Dacogen (Decitabine) and Vidaza (Azacytidine), have been used as standard treatments for the treatment of MDS/AML in the elderly patient group. Several problems were pointed out, such as ease of use and expansion of the DNMT1 inhibitor market through indication expansion.

On the other hand, acute lymphocytic leukemia (ALL) is a disease that mainly occurs at a young age (~80%), but 20% of all patients occur in adults around the age of 50, and pediatric/adolescent ALL has a relatively good treatment prognosis. In contrast, the prognosis of adult ALL is very poor, so the need for the development of new therapies is very great.

The majority of ALL is B-ALL of B-cell origin, but some are T-cell origin of T-cell origin (hereinafter referred to as T-ALL), and in the case of T-ALL, the development of appropriate targeted therapy is limited. Treatment options are limited after first-line chemotherapy.

For the treatment of adult ALL, high-intensity chemotherapy based on Cyclophosphoamide, Daunorubicin, Vincristine, L-asparaginase, and Prednisone is the first-line treatment option.

About 30-40% of patients relapse or acquire resistance despite such high-intensity chemotherapy, and among these relapsed/resistant patients, B-cell ALL patients have various treatment options such as antibody therapy, ADC, and CAR-T In contrast, in the case of T-ALL patients, the treatment is limited to Nelarabine, so there is a high demand for the development of new treatments.

Drug tolerance is a phenomenon in which the efficacy of a drug decreases due to repeated administration of the drug. Cancer cells that survive drug treatment develop drug resistance due to a feedback mechanism for maintaining homeostasis and/or genetic mutation. Combining multiple drugs may reduce resistance.

In the case of T-ALL, many patients show a good response to treatment using intensive chemotherapy that combines various cytotoxic anticancer drugs, but in the case of T-ALL, which has developed resistance despite the use of standard treatment, there is no suitable treatment, so development of a new treatment The demand is very high. In some of these resistant patients, resistance was found to be caused by overexpression of anti-apoptotic genes such as BCl2 (FIG. 52), and treatment with a combination of DNMT1 inhibitors (Decitabine, etc.) and Venetoclax was experimentally attempted, and excellent therapeutic synergy was achieved. effect has been confirmed. However, despite these clinical results reports, additional development efforts for DNMT1 inhibitors in the T-ALL patient group are not in progress.

However, as existing nucleoside anticancer drugs are applied to the treatment of T-cell ALL, there is a high possibility of developing nucleoside metabolism resistance in resistant patients, so there is a high possibility of developing new DNMT1 inhibitors that can overcome various resistance mechanisms including metabolic resistance beyond the existing Decitabine. There is a high demand for

On the other hand, DNA, which has an important function of storing genetic information, is always damaged by UV, ionizing radiation (IR), various chemicals, or reactive oxygen species (ROS) generated by cellular respiration. Although DNA damage accounts for a very small fraction of the entire human genome, when DNA damage occurs to a proto-oncogene or suppressor gene, it ultimately increases the likelihood of cancer. do. To prevent this damage, cells have their own DNA repair system, which is called the DNA damage response. The DNA damage response maintains the genetic integrity of cells. When DNA is damaged, the cell cycle is stopped and DNA damage is repaired. However, when DNA damage exceeds the cell's ability, the cell induces apoptosis on its own.

DNA repair proceeds in different processes depending on the type of damage. Mismatch Repair (MMR), which corrects errors that occur when DNA is copied by polymerases, and Base Excision Repair (BER), which repairs when one base sequence is wrong, to a greater extent than previous damages. Nucleoid Excision Repair (NER) and Double Stand Break Repair (Double Stand Break Repair) repair DNA double helix strands that are damaged due to exposure to strong damage such as IR. Double-strand break repair methods include homologous recombination (HR), which repairs the damaged strand using the intact strand as a template, and non-homologous end linkage (non-homologous end-linkage, which repairs damage by linking non-complementary ends in G1) -homologous end joining: NHEJ). Although there are many DNA repair processes, it is known that the cause of many diseases is caused by DNA damage.

Cancer is a disease characterized by indiscriminate cell proliferation and unlimited division. In normal cells, there is something called a cell cycle checkpoint, which checks for damage to genes and cells during cell division. When DNA damage is detected, repair or, if the damage is severe, apoptosis occurs. However, if DNA continues to divide without being repaired despite this damage, cancer occurs. The most representative example is the BRCA gene, which plays a role in suppressing cancer, and plays an important role in recovering DNA double-strand breaks through homologous recombination. When DNA damage occurs while there is a mutation in the BRCA gene, which plays an important role in DNA repair, correct DNA repair does not occur, which increases mutations in other genes. In fact, mutations in the BRCA gene increase the incidence of female cancers such as breast and ovarian cancer. It is known that women with mutations in the BRCA gene have a 5- to 6-fold increase in the rate of breast cancer and a 10-fold increase in the incidence of ovarian cancer. Through this, it can be seen that DNA damage is not properly repaired and causes genetic instability of cells, thereby causing cancer.

A common feature of cancer is genetic instability. In most cancers, it is not known specifically what defects in the DNA damage response cause cancer cells to develop and develop, but the link between defects in the DNA damage response and cancer is indisputable.

Defects in homologous recombination repair result in sporadic or hereditary tumors.

Defects in homologous recombination repair are mainly due to mutations in the BRCA gene or epigenetic silencing. According to another report, loss-of-function mutations in genes involved in homologous recombination repair, such as BRCA1, BRCA2, ATM, RAD51C, and RAD51D, were found in hereditary breast cancer, uterine cancer, and pancreatic cancer.

As such, cancer cells contain defects in their DNA damage response. That is, cancer cells cannot adequately cope with DNA damage. That is why substances that can damage DNA are used as anticancer agents to induce apoptosis of cancer cells. A first example is the platinum salts carboplatin and cisplatin. Platinum compounds cause DNA damage by creating DNA inter- and intrastrand crosslinks. This DNA damage is repaired through nucleotide excision repair (NER) and homologous recombination repair (HRR).

So, platinum compounds are effective in cervical cancer patients with defects in the process of homologous recombination. About 40% of malignant cervical cancers are due to mutations in BRCA1 or BRCA2, which are key genes for homologous recombination repair at the reproductive or somatic level, or abnormal homologous recombination repair due to epigenetic inactivity.

As shown in FIG. 5 , there is an unmet need for platinum-based anticancer agents for the treatment of ovarian cancer and bladder cancer.

Ovarian cancer is a cancer with a very high incidence worldwide, reaching 300,000 new patients per year. Although many patients benefit from platinum-based anticancer drugs, it is a very lethal cancer that causes 185,000 deaths annually when resistance develops. It is a disease in high demand.

Ovarian cancer, like other solid cancers, can be divided into stages 1 to 4 (terminal stage). Because it is located deep inside the abdominal cavity and is isolated from the outside, ovarian cancer rarely shows any special subjective symptoms even if it is diagnosed in the early stages. More than 75% of patients diagnosed with ovarian cancer in 2009 were identified as

stage

3 or 4, and in most cases, the recurrence rate is high even after treatment, and the survival rate is not high, making it the worst prognosis among gynecological cancers.

For the treatment of ovarian cancer, PARP inhibitors can be used as target anticancer drugs depending on the mutation status of the BRCA 1/2 gene after initial discovery, but most of the treatment processes are in chemotherapy using platinum-based anticancer drugs including cisplatin and carboplatin. are dependent

In the early stage of anticancer treatment, most ovarian cancer patients respond well to chemotherapeutic agents, but in many patients, they quickly acquire resistance to treatment regimes including platinum-based anticancer agents, and among these patients, complete resistance patients are utilized later. It does not respond to the majority of available therapies and leads to rapid death.

Most ovarian cancer patients have very poor treatment prognosis at the time of diagnosis because there are almost no early subjective symptoms of the disease, and most of them, 85%, recur after the first treatment, and then second-line treatment As a result, 25% show drug resistance even after platinum drug is administered, and in most of these patients, cancer recurrence within 6 months and death must occur within 1 year.

Currently, there is no tertiary treatment available on the market for platinum-resistant patients.

In the case of Decitabine and Azacytidine (Fig. 6), the effect of combined administration with platinum-based anticancer drugs was verified in clinical practice, but concerns were raised that the toxicity caused by platinum-based anticancer drugs could be amplified during priming with Decitabine, and also due to the unstable PK profile of Decitabine. Further development has been suspended as it is predicted that it will not be easy to show consistent efficiency in solid cancers where the PK profile is important, such as ovarian cancer.

Bladder cancer is the second most common cancer of the urinary tract, and the main risk factors are genetic, age, and gender (men have a 4-fold higher risk than women), and smoking is estimated to be the biggest carcinogen. As a result, smokers may be up to seven times more likely to develop bladder cancer than non-smokers.

The increasing trend of bladder cancer patients can be attributed to the increase in population and aging worldwide, and the diagnosis rate of bladder cancer is increasing due to the development of cancer diagnosis technology.

Bladder cancer is also a very lethal cancer that causes 200,000 deaths per year when resistance occurs, although many patients benefit from platinum-based anticancer drugs.

Bladder cancer treatment initially uses an approach to suppress the occurrence of cancer infiltrating the bladder tissue by maintenance chemotherapy after resecting the cancer tissue locally present in the bladder, but in about 40% of patients, cancer cells invade muscle tissue of the bladder. Metastasis occurs through penetration, and treatment with immune checkpoint inhibitors is possible in about 15% of these patients, but the majority of patients are treated with gemcitabine/carboplatin-based chemotherapy.

The cause of resistance to platinum-based anticancer drugs is epigenetic silencing of SLFN11 and various tumor suppressor genes such as p15 and p16, which cause resistance to platinum-based anticancer drugs, as in ovarian cancer. this has been proven

However, in the case of Decitabine and Azacytidine, although their clinical efficacy has been verified and the possibility of securing a mechanism of action and biomarkers has been raised, concerns have been raised that the toxicity of platinum-based anticancer drugs may be amplified during priming with these drugs. In addition, due to the unstable PK profile of the decitabine compound, it is predicted that it will not be easy to show consistent efficiency in solid cancers where the PK profile is important, such as bladder cancer, and further development has been discontinued.

Despite these limitations of Decitabine (toxicity and unstable PK profile), there is a very high need and expectation that such a resensitization treatment can be developed by developing a new DNMT1 inhibitor that can overcome these limitations. The cause of this complete resistance is presumed to be epigenetic silencing of various tumor suppressor genes such as p15 and p16, and resistance to such chemotherapy by inducing epigenetic changes through inhibition of DNMT1 from the early development of DNMT1 inhibitor (Decitabine) was thought to be able to overcome this, and in fact, this possibility has been demonstrated in various researcher-led clinical trials using Decitabine. In particular, it has recently been confirmed that the cause of such complete resistance is dependent on the SLFN11 gene, and as a potential biomarker is secured, the need for a new DNMT1 inhibitor is very high.

Recently, immuno-oncology (IO), such as PD-1/PD-L1 immune checkpoint inhibitors, and combination therapy with existing therapies have been introduced. If new concept pipeline treatments such as N-803, Vicinium, and Instiladrin enter the market, they can provide new treatment opportunities to non-muscle-invasive bladder cancer (NMIBC) patients.

However, until recently, immune checkpoint inhibitors, which were expected to fill the unmet needs of drug-resistant patients in metastatic bladder cancer, do not respond to all patients, and relapses are observed in some patients. It has been confirmed that tolerance is also seen.

In the case of muscle invasive bladder cancer (MIBC), which has a relatively good prognosis, the 5-year survival rate reaches 50%, and the earlier stage, non-muscle invasive bladder cancer (NMIBC) ) is 88%, whereas metastatic bladder cancer (mBC) is less than 10% and most of them die within 2 years, so there is a great demand for effective treatment for metastatic bladder cancer.

As with ovarian cancer, if first-line chemotherapy treatment fails in these patients with late-stage bladder cancer, the choice of second-line and third-line treatments becomes very narrow due to drug resistance, and eventually the 5-year survival rate for these patients is It is only 5%.

There are currently no 3rd or 4th line treatments for these platinum-resistant metastatic bladder cancer patients on the market.

The prognosis of patients undergoing chemotherapy is poor, the average survival time is less than 1 year, and many of the patients who die are due to resistance to platinum-based anticancer drugs. situation.

Therefore, like all other solid cancers, there is a market for treatments for ovarian cancer and bladder cancer for various indications depending on stage, biomarker, and drug resistance.

Because rapidly dividing cells, such as cancer cells, are much more sensitive to the effects of cytotoxic substances than slowly dividing normal cells, DNA-modifying agents can be used to eliminate cancer cells. DNA damage is the mechanism of action of many of the most commonly used chemotherapeutic agents, but the therapeutic agents used clinically become difficult to use as therapeutic agents due to the risk of toxicity due to the narrowing of the therapeutic window as the efficacy increases.

Currently, various drugs (injection and oral preparations, used according to each purpose) are released on the market based on Decitabine/Azacytidine, two types of DNMT1 inhibitor active ingredients (FIG. 6).

As shown in FIG. 6, both Decitabine/Azacytidine components of the chemical structure are activated in the form of Decitabine triphosphate in cells and incorporated into DNA, and then trap the DNMT1 enzyme to form a DNA-DNMT1 covalent adduct. It has a mechanism that removes hypermethylation and exhibits various anticancer effects by various intracellular signal transduction pathways that occur during the DNA damage repair process through degradation of the adduct produced thereafter. It is vulnerable to metabolism by Cytidine Deaminase, which has the disadvantage of limited efficacy due to unstable PK profile. There is a problem of damaging normal tissues in our body, so the amount of use is limited, so the anticancer effect is limited. (3) In cancer cells that survived after the initial treatment, the removal process is overactivated after the formation of the DNA-DNMT1 adduct, suppressing the anticancer effect. The development of improved new drugs is strongly demanded as they have problems such as being vulnerable to resistance.

Focusing on the fact that both 5'-OH and 3'-OH groups in the structure of Decitabine/Azacytidine must be exposed for metabolism by Cytidine Deaminase, Astex Pharmaceuticals/Otsuka developed a Decitabine structure in which deoxyguanidine was linked to the 3'-OH group of Decitabine. A prodrug, SGI-110, was developed. When SGI-110 was actually administered to the human body, it was confirmed that it avoids metabolism by Cytidine Deaminase and exhibits an improved PK profile by slowly releasing the Decitabine component. It became.

As a way to solve the metabolic problem caused by cytidine deaminase, an oral medication (ASTX-727) that takes a cytidine deaminase inhibitor together or an oral medication (CC-486) that stabilizes the PK profile by administering an excessive amount of Azacytidine Although the same improved new drugs have been developed, they also do not provide solutions to (2) and (3) of the problems of Decitabine/Azacytidine described above. In addition, it has problems such as deterioration of the active ingredient (Decitabine) due to the combined use of Cytidine Deaminase inhibitors and gastrointestinal side effects due to excessive drug administration.

Surprisingly, the present inventors found that although Aza-T-dCyd is not superior to Decitabine in terms of DNMT1 inhibition, that is, the degree of demethylation, the mechanism of anti-apoptotic gene expression or anti-apoptotic protein down-regulation is superior. In addition, it was found that the IC ₅₀ value was much lower through the apoptotic mechanism.

In addition, unlike Decitabine/Azacytidine in the chemical structure shown in FIG. 6, Aza-T-dCyd in Chemical Formula 1 has a Thio-nucleoside structure, which inhibits DNMT1, the main drug target, and endonuclease, which is an existing resistance development mechanism. It was confirmed that it blocks hydrolysis at the same time, exhibits strong anti-cancer efficacy through apoptosis mechanism, has metabolic resistance by cytidine deaminase, and has an excellent PK profile even when administered orally. Bladder cancer), it was confirmed that it showed excellent efficacy compared to existing commercial DNMT1 inhibitors and competing technologies under development.

Furthermore, deoxycytidine-based compounds used for chemotherapy of cancer or viral infection require phosphorylation by cellular enzymes to be activated. At this time, phosphorylation of deoxycytidine-based compounds by deoxycytidine kinase (dCK) is considered to be the rate-limiting step in the activation of the compound, and further phosphorylation to diphosphate or triphosphate happens. Due to its Thio-nucleoside structure, the activation rate by dCK (deoxycytidine kinase) in normal cells is significantly lowered, allowing the active ingredient of the drug to be selectively delivered to cancer cells, enabling a wide range of treatments with an excellent safety profile. Since it has a therapeutic window range, it is possible to administer at a high dose that prevents the mechanism of resistance development from operating. Based on this, Aza-T-dCyd is used as a well-designed multi-target inhibitor to fill the unmet needs of patients in the vacuum market where there is no current treatment for hematologic cancer and solid cancer, which are not satisfied with existing treatments.

A first aspect of the present invention is to use the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof, which exerts an anticancer effect through an apoptosis mechanism, by BER (Damage repair) method, Base Excision Repair) provides a pharmaceutical composition characterized in that it is administered at a high dose that induces apoptosis so that the drug resistance development mechanism that overactivates does not work.

At this time, the Aza-T-dCyd compound can decrease DNMT1 protein in a concentration-dependent manner. In addition, Aza-T-dCyd compounds can exert anticancer effects in a dose-dependent manner.

As one embodiment of the present invention, (1) a patient group with resistance to DNMT1 inhibitors; (2) a patient group in need of selective drug delivery to cancer cells compared to normal cells as a targeted anti-cancer agent; (3) a patient group in need of adjusting the expression level of one or more markers of drug efficacy; (4) a patient group with accumulated epigenetic DNA methylation pattern changes in cancer cells compared to normal cells; (5) A patient group in which the standard dose of DNMT1 inhibitor, which is a standard treatment, is limited due to the problem of damaging normal tissues as well as cancer cells because DNA damage is caused through the formation of DNA-DNMT1 adducts when DNMT1 inhibitor is administered; (6) a patient group in need of prevention or treatment of hematological malignancies; (7) Compared to normal individuals, lineage commitment master transcription factors selected from the group consisting of CEBP/alpha, Pu.1 and GATA factors are expressed at high levels, while the expression of CEBP/epsilon or transcription factors in the late developmental stage is not the same as that of each gene. a group of patients whose methylation remains low; (8) a group of patients who are progressing or have progressed to bone marrow cancer; (9) a patient group diagnosed with AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), CMML (chronic myelomonocytic leukemia) or ALL (acute lymphocytic leukemia); (10) patient population with health conditions that deviate from standard therapy or standard chemotherapy; (11) a group of patients whose DNMT3B expression level is outside the normal range; (12) a group of patients requiring abnormal bone marrow removal; (13) a patient group in need of blast reduction; (14) a group of patients with impaired blood function or a group of patients with hematopoiesis abnormalities; (15) a group of patients with abnormal methylation patterns due to overactivation of DNMT1 compared to normal subjects; (16) a group of patients diagnosed with T-cell ALL (acute lymphocytic leukemia); (17) Patients who may develop resistance to nucleoside metabolism when administered with nucleoside anticancer drugs; (18) a patient group with a possibility of developing resistance to platinum-based anticancer drugs or who developed resistance; (19) Platinum-based anticancer drug administration target patient group; (20) epigenetically silencing a tumor suppressor gene and/or SLFN11 compared to normal subjects; (21) a patient group in need of prevention or treatment of ovarian or bladder cancer; (22) a platinum-resistant recurrent end-stage ovarian cancer patient group or a platinum-resistant metastatic bladder cancer patient group; (23) metastatic bladder cancer patient group, p53 mutation bladder cancer patient group or hypermethylated SLFN11 bladder cancer patient group, which is a detailed biomarker for treatment; or (24) Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof may be administered to a patient group diagnosed with stage 3 or 4 ovarian cancer.

In this case, the drug effect marker requiring expression level control may be subject to epigenetic silencing through DNA methylation in cancer cells or the transcription factor of the drug effect marker may be subject to epigenetic silencing through DNA methylation.

A drug efficacy marker requiring expression level control may be a differentiation induction gene, a tumor suppressor gene, or a combination thereof. Non-limiting examples of drug efficacy markers requiring expression level control may include p15, p16, SLFN11, CEBP/epsilon, CDKN1B, Myc antagonists (MAD), or a combination thereof.

Aza-T-dCyd compounds can re-express CEBP/epsilon and/or express the p27 tumor suppressor gene, a down-stream effector of CEBP/epsilon, in cancer cells.

The Aza-T-dCyd compound can overcome the resistance development mechanism by lowering the efficiency or speed of the overactivated BER (Base Excision Repair) repair process through strong resistance to nucleic acid intermediate degrading enzyme (Endonuclease).

The patient group that requires selective drug delivery to cancer cells compared to normal cells may be an elderly patient group aged 65 years or older and/or a patient group with weakened physical functions due to an underlying disease or comorbidity.

In addition, the patient group in need of selective drug delivery to cancer cells compared to normal cells is a patient group whose therapeutic effect is limited due to the occurrence of resistance mechanisms due to side effects caused by acting on cells of the bone marrow, digestive tract, mucous membrane and / or skin. .

Aza-T-dCyd compounds induce biased and selective dimethylation to regions of DNA replicated during early replication, promoter regions important for gene expression, DNMT1-binding regions, and/or replication stress regions, but not genomic region-wide dimethylation. can

In addition, the Aza-T-dCyd compound is a targeted anti-cancer agent that selectively attacks only cancer cells by targeting a specific part of cancer cells that differs from normal cells by reducing DNMT1, re-expression of p15 tumor suppressor gene, and/or reducing DNMT3B expression. can act as

In addition, the Aza-T-dCyd compound can simultaneously block endonuclease participating in DNA damage repair along with the DNMT1 inhibitory mechanism.

Aza-T-dCyd compounds can be rapidly activated by triphosphate in cancer cells compared to normal cells.

Aza-T-dCyd compound delays DNA replication by causing base excision repair and/or mismatch repair through DNA insertion of aza-T-dCTP, a triphosphate of Aza-T-dCyd compound, thereby causing replication stress and DNA Damage response can be increased.

The Aza-T-dCyd compound suppresses the expression of RRM1 protein, a ribonucleotide reductase that is important for dNTP de novo synthesis, induces DNA replication stress by reducing the amount of dCTP and dTTP in cells, and can generate a strong DNA damage response.

Aza-T-dCyd compound has a low activation rate by dCK (deoxycytidine kinase) in normal cells compared to cancer cells due to its Thio-nucleoside structure, so Aza-T-dCyd compound is selectively delivered to cancer cells to secure a safety profile can do. In addition, the Aza-T-dCyd compound forms a DNMT1 trapping complex that lasts longer due to its Thio-nucleoside structure, thereby securing strong anticancer efficacy and/or the possibility of overcoming resistance in resistant patients.

According to one embodiment of the present invention, when the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is used in a pharmaceutical composition for treating hematological cancer, the Aza-T-dCyd compound does not damage DNA in normal tissue cells, hematopoietic It may not affect your ability.

In one embodiment of the present invention, the patient group undergoing or progressing to bone marrow cancer may be a patient group having a gene mutation that can cause bone marrow disease.

Patients diagnosed with AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), or ALL (acute lymphocytic leukemia) are particularly high-risk patients with genomic abnormalities, recurrent secondary AML (sAML) patients, and past treatment history. It may be a treatment-related AML (t-AML) patient group or a drug-resistant/refractory patient group.

Patients who need abnormal bone marrow removal may be candidates for bone marrow transplantation (HCT, hematopoietic cell transplant).

In the pharmaceutical composition in which the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered to a patient group in need of blast reduction according to one embodiment of the present invention, the blast cells are leukemic blast cells (Leukemic Blast) can be The Aza-T-dCyd compound may exert an anticancer effect by differentiating blast cells.

According to one embodiment of the present invention, in the anti-cancer pharmaceutical composition in which the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered to a patient group in which DNMT1 is overactivated and the methylation pattern is abnormal compared to normal subjects, Aza- T-dCyd compounds are activated by triphosphate in cells and used instead of some dC (deoxycytidine) during DNA synthesis. Cancer cell death can be induced by activating the silencing mechanism.

According to one embodiment of the present invention T-cell type In the pharmaceutical composition in which the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered to a patient group diagnosed with ALL (acute lymphocytic leukemia), the patient group may be a relapsed and/or resistant patient group, and the anti-apoptotic gene It may be a patient group in which resistance occurs due to overexpression of .

In the pharmaceutical composition in which the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered to a patient group who is likely to develop resistance to a platinum-based anticancer agent or a patient group who develops resistance according to one embodiment of the present invention, the platinum-based anticancer agent The possibility of resistance development can be derived from patient data on epigenetic silencing of tumor suppressor gene and/or SLFN11, which causes resistance to platinum-based anticancer drugs.

In the pharmaceutical composition in which the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered to a patient group to be administered with a platinum-based anticancer drug according to one embodiment of the present invention, the patient group to be administered with a platinum-based anticancer drug has, for example, a poor prognosis. It may be a patient group who received information/diagnosis. Data values for informative poor prognosis could be the likelihood of disease recurrence and/or early mortality (average survival less than 1 year).

According to one embodiment of the present invention, in a pharmaceutical composition for administering the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof to a patient group epigenetically silencing a tumor suppressor gene and/or SLFN11 compared to normal subjects, the tumor The suppressor gene may be p15 or p16.

The second aspect of the present invention is the Aza-T-dCyd compound of Formula 1 or its compound, which exerts an anticancer effect through an apoptosis mechanism in a patient group who has received information on or diagnosed with a poor prognosis or possibility of resistance when administered with a DNMT1 inhibitor. It provides a pharmaceutical composition characterized by administering a pharmaceutically acceptable salt at a high dose that induces apoptosis.

At this time, the provision of information on the prognosis or the possibility of resistance is (i) quantifying the problem of inhibiting anticancer efficacy as the removal process is overactivated after formation of the DNA-DNMT1 adduct, or (ii) classifying the patient group according to the occurrence/degree of the above problem It may have been derived from reconciliation. Data values for providing poor prognosis may be the likelihood of disease recurrence and/or early mortality (average survival less than 1 year).

A third aspect of the present invention provides a pharmaceutical composition for oral administration comprising the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof.

When administered orally, the Aza-T-dCyd compound exhibits metabolism resistance by cytidine deaminase (CDA) in the liver, thereby exhibiting an excellent PK profile.

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

The present invention relates to 4'-thio-5-aza-2'deoxycytidine (4'-thio-5-aza-2'deoxycytidine, Aza-T-dCyd) compound or a pharmaceutically acceptable salt thereof.

[Formula 1]

Recently, the standard therapy for AML treatment has changed from the existing Decitabine/Azacytidine monotherapy to the combination of these drugs with the BCL-2 inhibitor Venetoclax (trade name: Venclexta), so the present inventors use Aza of Formula 1 instead of Decitabine/Azacytidine. - In the case of T-dCyd, the drug effect was compared and evaluated when co-administered with Venetoclax drug.

As a result, surprisingly, in a xenograft model in which MV4-11 AML cell line was subcutaneously implanted into mice, Aza-T-dCyd (NTX-301), even when treated alone at a clinically relevant concentration, strongly activated Survivin through p53 activation. , Mcl-1 and other anti-apoptotic protein expression levels are lowered to show equal or better anticancer efficacy than Azacytidine/Venetoclax combined administration, as well as Azacytidine/Venetoclax combined administration even at very low doses when Aza-T-dCyd and Venetoclax are administered together. It was found to show very strong anticancer efficacy, such as showing strong cancer growth inhibitory effect compared to combined administration (induction of complete tumor regression in the Aza-T-dCyd 0.5mpk administered group) (FIG. 32 and FIG. 33). From this, it can be inferred that both Aza-T-dCyd and Venetoclax inhibit the action of anti-apoptotic protein when co-administered. The present invention was completed based on this. Details in this regard are described in PCT/KR2022/004846 filed on April 5, 2022, the contents of which are incorporated herein.

In short, DNMT1 inhibitors that inhibit the action of anti-apoptotic proteins can be a very promising approach to overcome the existing Decitabine/Azacytidine resistance. Therefore, the present invention is a process of molecular designing a well-designed multi-target inhibitor that exhibits strong anticancer efficacy by simultaneously blocking the strong inhibition of DNMT1, the main drug target, and the hydrolysis by endonuclease, which is an existing resistance development mechanism, through a multi-pharmacological approach selected Aza-T-dCyd of Chemical Formula 1 as a DNMT1 inhibitor and, in some cases, intends to use it for combined treatment with a BCL-2 inhibitor such as venetoclax.

As a DNMT1 inhibitor, in the case of the present invention, in which Aza-T-dCyd of Formula 1 was selected based on the analysis of the mechanism of action of existing nucleoside anticancer drugs and the mechanism of DNA damage repair activation, which is its resistance action mechanism, surprisingly, the BCL-2 inhibitor When used in combination with the compound of Formula 2 (venetoclax), a synergistic effect more than expected was exhibited (FIG. 32 and FIG. 33).

[Formula 2]

Aza-T-dCyd of Chemical Formula 1 is a DNMT1 inhibitor based on a 4-Thio-2-deoxyribose skeleton, and has both a sugar structure change (4'-thiodeoxyribose structure) and an Aza-cytosine group. As a result of molecular modeling, it was confirmed that thio-deoxyribose-based nucleoside compounds did not significantly affect other DNA components after being inserted into DNA.

Aza-T-dCyd is activated as triphosphate in cells and is used instead of some dC (deoxycytidine) during DNA synthesis. Nucleoside induces cancer cell death by trapping DNMT1 after DNA synthesis and activating various epigenetic action mechanisms. It is an anticancer drug. In particular, Aza-T-dCyd is rapidly activated in cancer cells, incorporated into DNA, and can trap and inhibit the DNMT1 enzyme even at low concentrations.

As described below, the present invention is a well-designed multi-target inhibitor that exhibits strong anticancer efficacy by simultaneously blocking the strong inhibition of DNMT1, the main drug target, and the hydrolysis by endonuclease, which is an existing resistance development mechanism, through a multipharmacological approach. During the design process, Aza-T-dCyd was selected as a DNMT1 inhibitor.

The development of next-generation DNMT1 inhibitors is a promising field with very high unmet needs, and overcoming the existing Decitabine/Azacytidine resistance by inhibiting the action of anti-apoptotic proteins can be a very promising approach.

Since nucleoside compounds with 4-Thio-2-deoxyribose skeleton are resistant to DNA strand cleavage by endonuclease and the mechanism corresponds to the first step of the DNA-damage repair pathway, DNMT1 with 4-Thio-2-deoxyribose skeleton It was judged that the inhibitor could be usefully developed as a next-generation DNMT1 inhibitor capable of overcoming drug resistance by anti-apoptotic protein and DNA-damage repair pathway. As expected, NTX-301 (Aza-T-dCyd) has strong anticancer efficacy/resistance potential compared to existing DNMT1 inhibitors due to its 4-Thio-2-deoxyribose backbone.

[ Drug Aza-T-dCyd selected through well-designed multi-target inhibitor-based molecular design platform technology ]

The present inventors have developed a molecular design basis capable of developing next-generation nucleoside-based anticancer drugs that can easily overcome the main problems of existing nucleoside-based anticancer drugs, such as limited therapeutic effects due to various resistance mechanisms and dose-limiting toxicity in bone marrow/digestive tract, mucous membrane/skin, etc. Using technology, the present invention was completed.

Nucleoside derivatives are organic compounds with structures very similar to those of DNA and RNA, which perform the most core functions of cells constituting our body (maintaining life functions through replication/division), and are central to anticancer chemotherapy/antiviral chemotherapy is playing a negative role.

Since the 1960s, various nucleoside anticancer drugs have been commercialized, such as Cytarabine, Clofarabine, Fludarabine, 5-Fluorouracil (including Capecitabine), Gemcitabine, Decitabine, and Azacytidine. there is.

In the case of existing nucleoside anticancer drugs, they act not only on cancer cells but also on rapidly dividing normal cells such as bone marrow/digestive tract, mucous membrane/skin cells, etc. (thrombocytopenia), Vomiting (vomiting), Pyrexia (fever), Leukopenia (leukopenia), Diarrhea (diarrhea), Injection site erythema (erythema), Constipation (constipation), Neutropenia (neutropenia), Ecchymosis (ecchymosis) ), Petechiae (petechiae), Rigors (chills), Weakness (malaise), Hypokalemia (hypokalemia), and (ii) for Decitabine (Dacogen), Neutropenia (neutropenia), Thrombocytopenia (thrombocytopenia), Anemia (anemia) ), Fatigue (fatigue), Pyrexia (fever), Nausea (nausea), Cough (cough), Petechiae (petechiae), Constipation (constipation), Diarrhea (diarrhea), Hyperglycemia (hyperglycemia), which limits the usage. As a result, various resistance mechanisms occur, which has a problem in that the therapeutic effect is limited.

The present inventors proceeded with molecular design based on the analysis of the mechanism of action of existing nucleoside-based anticancer drugs and the mechanism of activation of DNA damage repair, which is its resistance action mechanism.

Nucleoside-based anticancer drugs exert their anticancer efficacy through various mechanisms of action (DNA synthesis/transcription inhibition, DNA damage induction, or expression of pharmacological effects through inhibition of DNA processing-related enzymes such as DNMT1) after DNA incorporation. (FIG. 7)

Nucleoside-based anticancer drugs form base pairs that do not match normal DNA components during DNA incorporation, resulting in abnormal structures in DNA, which are recognized by BER (Base Excision Repair) among DNA damage repair methods, and DNA damage repair proceeds 8).

In the early stage of administration of nucleoside-based anticancer drugs, after the anticancer drugs are incorporated into DNA, sufficient pharmacological effects are exhibited and cancer cells are efficiently killed. tolerance develops.

Therefore, the present inventors focused on the fact that if a new Nucleoside-based compound is developed that is incorporated into DNA with an efficiency equal to or higher than that of existing Nucleoside-based anticancer drugs and exhibits the desired pharmacological efficacy well, but is also resistant to BER, it is expected to show stronger efficacy than existing anticancer drugs. did

In order to secure a new nucleoside-based anticancer drug skeleton with resistance to BER, the following points were considered during molecular design.

The first step of BER is to break the glycosidic bond in Nucleoside/Nucleotide to create a base-free site and to create a site for DNA damage repair through Endonuclease. DNA damage is repaired by filling in base components (FIG. 6).

The speed and efficiency of the BER repair process are determined by the steps of cleaving the C-N bond after recognizing an abnormal base pair and the step of making a vacant nucleotide site by AP-endonuclease, making it difficult to proceed with one or more of these steps. If so, it is possible to suppress the development of resistance due to overactivation of BER by lowering the efficiency of BER.

Since nucleoside compounds with 4-Thio-2-deoxyribose skeleton are resistant to DNA strand cleavage by endonuclease and the mechanism corresponds to the first step of the DNA-damage repair pathway, DNMT1 with 4-Thio-2-deoxyribose skeleton Inhibitors can overcome drug resistance by anti-apoptotic proteins and DNA-damage repair pathways. Therefore, Aza-T-dCyd (NTX-301) has a stronger anticancer efficacy/resistance potential than conventional DNMT1 inhibitors due to its 4-Thio-2-deoxyribose skeleton (Examples 1 and 2).

Therefore, the present inventors tried to lower the efficiency of BER by taking advantage of the strong resistance to endonuclease in the case of Thio-deoxyribose backbone-based nucleoside derivatives.

On the other hand, the mechanism of action of BER acts as a mechanism of resistance development in cancer cells, but on the contrary, in normal tissues (especially hematopoietic tissues such as bone marrow), it works as a safety device to prevent toxicity by anticancer agents, so anticancer agents that inhibit BER are selectively restricted to cancer cells. The need to be communicated is high.

After entering cells, Nucleoside-based anticancer drugs are activated by various nucleoside kinases and accumulate in cells, and nucleoside compounds that are not activated within an appropriate time are quickly removed. If the difference in activation rate between normal cells and cancer cells can be maximized, excellent safety can be secured by accumulating the active form of the drug only in cancer cells.

According to existing literature reports, nucleoside substituted with various heteroatoms can be activated at different rates or eliminated through metabolism. Among them, Thio-deoxyribose is activated relatively slowly in normal cells, whereas normal Deoxyribose-based nucleoside is toxic in cancer cells. It was confirmed that it was activated at a rate equal to or higher than that and could be used during DNA synthesis. This may show a selective drug delivery effect that is relatively more accumulated in cancer cells than in normal cells.

Aza-T-dCyd utilizes a Thio-deoxyribose skeleton that has improved the existing Deoxyribose skeleton. Compared to the existing Deoxyribose-based nucleoside, such as Decitabine, Aza-T-dCyd is activated rapidly in cancer cells and efficiently incorporated into DNA, but is activated slowly in normal organs including normal bone marrow cells, thereby selecting cancer cells. targeted drug delivery is possible; Not only can it secure the possibility of overcoming metabolism-related resistance by being relatively slowly affected by the main metabolic enzyme of nucleoside anticancer drug called Cytidine Deaminase, but also DNA incorporation and DNMT1 trapping through the Azacytosine functional group that can trap DNMT1 enzyme well, A longer lasting DNA-DNMT Adduct was formed to secure a stronger and differentiated effect.

In addition, as a result of molecular modeling, it was confirmed that thio-deoxyribose-based Nucleoside compounds did not significantly affect other DNA components after being inserted into DNA. This supported the validity of the thio-deoxyribose-based nucleoside drug design.

In summary, the compound that converts the deoxyribose backbone of the existing nucleoside to the 4'-thio-deoxyribose backbone and combines the Azacytosine functional group capable of well trapping the DNMT1 enzyme is a new product with differentiated efficacy and safety from existing DNMT1 inhibitors. Under the expectation that development as a nucleoside DNMT1 inhibitor would be possible, the present inventors found that other nucleoside compounds can overcome the BER resistance mechanism through the same type of nucleoside skeleton change (Deoxyribose → 4'-Thio-deoxyribose) in the case of Aza-T-dCyd And it was confirmed that it can have cancer cell-selective drug delivery properties.

As a result, Aza-T-dCyd is rapidly activated in cancer cells and efficiently incorporated into DNA, compared to the existing deoxyribose-based nucleoside such as Decitabine, by utilizing the Thio-deoxyribose skeleton, which is an improved form of the existing deoxyribose skeleton, but is activated slowly in normal organs including normal bone marrow cells. As a result, cancer cell-selective drug delivery is possible; Not only can it secure the possibility of overcoming metabolism-related resistance by being relatively slowly affected by the main metabolic enzyme of nucleoside anticancer drug called Cytidine Deaminase, but also DNA incorporation and DNMT1 trapping through the Azacytosine functional group that can trap DNMT1 enzyme well, A longer lasting DNA-DNMT Adduct was formed to secure a stronger and differentiated effect.

In short, Aza-T-dCyd has both the desired sugar structure change (4'-thiodeoxyribose structure) and an Aza-cytosine group, so the multipharmacological approach described above can be successfully applied to existing commercial DNMT1 inhibitors and competitive technologies under development. that it shows superior efficacy compared to; and a well-designed multi-target inhibitor that exhibits strong anticancer efficacy by simultaneously blocking the strong inhibition of DNMT1, the main drug target, and the hydrolysis by endonuclease, which is an existing resistance development mechanism, and completed the present invention.

In the present invention, by utilizing Aza-T-dCyd, which simultaneously blocks hydrolysis by endonuclease, which is an existing resistance development mechanism and strong inhibition of DNMT1, which is the main drug target, hematological cancer (myelodysplastic syndrome (MDS)/acute myeloid leukemia ( AML), T-cell acute lymphocytic leukemia (T-ALL)), and solid cancer (platinum-based anticancer drug-resistant ovarian cancer/bladder cancer), and its safety and efficacy in humans have been confirmed in the phase 1 clinical trial.

Furthermore, Aza-T-dCyd is a thio-nucleoside compound in which the deoxyribose structure of nucleoside anticancer drugs is changed to a thio-deoxyribose structure. Through this change, Aza-T-dCyd has the following characteristics to overcome the limitations of existing nucleoside anticancer drugs and to meet unmet needs. can

(1) By forming a DNMT1 trapping complex that lasts longer due to the Thio-nucleoside structure, it has differentiated strong anticancer efficacy compared to Decitabine (trade name: Dacogen) and Azacytidine (trade name: Vidaza) and secures the possibility of overcoming resistance in resistant AML patients;

(2) Due to the Thio-nucleoside structure, the activation rate by dCK (deoxycytidine kinase) in normal cells is significantly lowered, thereby selectively delivering the active ingredients of the drug to cancer cells, securing an excellent safety profile;

(3) Compared to existing Decitabine/Azacytidine, when administered orally, the rate of degradation by Cytidine Deaminase is slow, securing the possibility of overcoming resistance due to metabolism in cancer cells and oral administration; and

(4) Securing safety and oral administration PK profile through administration in human body.

Therefore, Aza-T-dCyd can be a best-in-class DNMT1 inhibitor with strong efficacy, improved safety profile, ease of use, and the possibility of overcoming drug resistance compared to existing DNMT1 inhibitors, which are standard treatments.

[ Aza-T-dCyd non-clinical trial ]

Aza-T-dCyd is rapidly activated in cancer cells, incorporated into DNA, and can trap and inhibit the DNMT1 enzyme even at low concentrations.

In addition, even cell lines resistant to Decitabine can maintain excellent cancer cell death-inducing effects. Such efficacy at the cellular level leads to excellent efficacy in animal models. Aza-T-dCyd showed excellent anticancer efficacy in various animal models in which Decitabine/Azacytidine did not show any therapeutic efficacy.

Aza-T-dCyd shows a PK profile that can be administered orally, and in the GLP-preclinical toxicity test, superior safety compared to Decitabine/Azacytidine was confirmed.

In particular, in clinical trials, a high Disease Control Rate was confirmed when administering a dose of 32 mg/day, and in animal tests, animal models for the corresponding disease group (AML, T-ALL, various solid cancers) were tested in the dose group showing similar drug exposure. As a result, it was confirmed that all of them showed excellent efficacy compared to existing competitive drugs.

In particular, in the phase 1a clinical trial of Aza-T-dCyd for solid cancer conducted by the US National Cancer Institute, efficacy and safety in the human body were also secured, such as an excellent PK profile when administered orally and a strong PD marker in circulating tumor cells. Even at a very low dose, the efficacy of slowing down the progression of cancer patients was observed, showing superior efficacy compared to existing drugs.

Therefore, Aza-T-dCyd can be a DNMT1 inhibitor with stronger efficacy, improved safety profile, ease of use, and the possibility of overcoming drug resistance compared to existing DNMT1 inhibitors, which are standard treatments.

Aza-T-dCyd shows a strong anticancer effect from about 0.5 mpk (mg/kg) administration group in animal models, and the drug exposure at that dose is less than 8mg/day when administered to humans (25% of MTD in phase 1 clinical trial). A wide therapeutic window can be secured at this level.

On the other hand, venetoclax can be administered up to 250 mg/day, and when used in combination with venetoclax, the dosage of Aza-T-dCyd is less than 32 mg/day, for example, 8 mg/day or more and less than 32 mg/day, preferably 8 to 8 mg/day. It may be 24 mg/day. 24 mg is equivalent to 1.5 mpk.

When combined with venetoclax, Aza-T-dCyd can be administered at 25% to 75% of the maximum tolerable dose (MTD).

In the phase 1 clinical trial, which was first conducted on humans after preclinical trials to observe toxicity and side effects, the main goal was the maximum tolerated dose (Maximum Tolerated Dose; MTD) is determined.

In particular, when comparing the efficacy of Aza-T-dCyd with that of Azacytidine, the current standard treatment for AML, and Venetoclax (trade name: Venclexta), a BCL-2 inhibitor, Aza-T-dCyd 2.0mpk The monotherapy group (corresponding to the MTD dose of 32 mg of Aza-T-dCyd in phase 1 clinical trial) showed the same level of efficacy as the Azacytidine/Venetoclax administration group, and the Aza-T-dCyd 0.5 or 1.0mpk and Venetoclax combination administration group (MTD in phase 1 clinical trial) 25 - 50% of the dose of 32mg) was confirmed to show a significantly stronger effect than the standard therapy (Azacytidine/Venetoclax combination) administration group (FIGS. 32 and 33).

Decitabine/Azacytidine, an existing drug with the same mechanism, required a 50% dose reduction from the MTD for administration with Venetoclax, and when the dose was reduced to 25% of the MTD, it showed excellent safety, but showed a problem with weak efficacy. In the case of NTX-301, when the dose is reduced to 25% of the MTD and co-administered with Venetoclax, the effect is significantly superior to that of the Azacytidine/Venetoclax administration group (FIG. 49). It is very likely to show superior safety/efficacy compared to existing standard treatment regimens.

[ Excellent efficacy and safety confirmed in preclinical/clinical development ]

In the present invention, compared to Decitabine/Azacytidine, an existing commercial DNMT1 inhibitor that has insufficient therapeutic efficacy and resulting resistance problems (amplification of problems due to the absence of second/tertiary treatments after resistance develops), Aza-T-dCyd is Excellent anti-cancer efficacy and strong efficacy in non-clinical models resistant to existing treatments were confirmed, and superior efficacy compared to existing Decitabine/Azacytidine in AML animal models and Venetoclax (Venetoclaxta), which is used as a combination that is currently becoming the standard treatment, Excellent synergistic efficacy was confirmed, and a safety profile equivalent to or higher than that of Decitabine / Azacytidine confirmed in the GLP-toxicity test and phase 1 clinical test was confirmed.

Aza-T-dCyd shows much stronger anticancer efficacy than the existing DNMT1 inhibitor Decitabine/Azacytidine and therapies based on them in various preclinical models, while securing excellent safety compared to them in preclinical studies, so there is a high possibility of clinical development success.

The MTD was determined to be 32 mg, and no DLT or

grade

3 or 4 adverse reactions were reported at doses of 16 mg or less, indicating good tolerability. A dose-dependent PK profile was confirmed. Compared to the existing Decitabine or Azacitidine, which did not show efficacy as a single administration in patients with late-stage solid cancer, NTX-301 administration induced stable disease that lasted for more than 5 months even in patients with advanced-stage solid cancer. It was expected to be effective in carcinomas that were insensitive to the existing Decitabine and Azacitidine, such as showing the effect of inhibiting the progression of cancer.

[ Competitive Advantage on the Technology Roadmap ]

In the case of Aza-T-dCyd, it enables treatment of terminal cancer patients undergoing relapse, development of an anticancer drug market completely independent of the existing anticancer drug market, and combination treatment.

Aza-T-dCyd induces apoptosis of cancer cells more strongly than Decitabine/Azacytidine due to its selective activation in cancer cells due to the difference in structure compared to Decitabine/Azacytidine, and maintains excellent cancer cell apoptosis inducing effect even in cell lines resistant to Decitabine. In addition, it can show excellent anticancer efficacy in various animal models in which Decitabine/Azacytidine does not show any therapeutic effect. Aza-T-dCyd shows a PK profile that can be administered orally (FIG. 51), and a superior safety profile compared to Decitabine/Azacitidine was confirmed in the GLP-preclinical toxicity test.

In addition, the superiority/safety of Aza-T-dCyd has already been confirmed in clinical trials. In the phase 1a clinical trial for solid cancers led by the US NCI, Aza-T-dCyd demonstrated excellent safety by not showing any obvious side effects even at drug doses higher than the dose of Decitabine, and even at very low doses, it prevented disease progression in cancer patients. The slowing effect was observed, showing superior efficacy compared to existing drugs.

Based on Decitabine and Azacytidine, which are the active ingredients of DNMT1 inhibitors released on the market, various drugs (injection, oral, and used according to each purpose) are on the market.

In the case of Decitabine/Azacytidine, it is vulnerable to metabolism by Cytidine Deaminase present in our body, and its PK profile is unstable, which limits its effectiveness. Surprisingly, compared to Decitabine/Azacytidine, aza-T-dCyd drug and aza-T-dCTP drug are degraded by cytidine deaminase at a slower rate when administered orally, thereby overcoming metabolic resistance by cytidine deaminase in cancer cells as well as having excellent oral administration. It was found to have a PK profile (Example 3).

In addition, due to the Thio-nucleoside structure, aza-T-dCyd significantly lowers the activation rate by dCK (deoxycytidine kinase) in normal cells, thereby selectively delivering the active ingredients of the drug to cancer cells, thereby securing an excellent safety profile. Since it has a wide therapeutic window, it is possible to administer at a high dose that prevents the mechanism of resistance development from operating.

Furthermore, through pharmacokinetic analysis, (1) the anti-cancer effect of aza-T-dCyd is Cmax dependent rather than AUC dependent; and (2) exposure to higher doses of the aza-T-dCyd drug for a short period of time is an effective anti-cancer therapy.

Because it causes DNA damage through the formation of DNA-DNMT1 adducts, there is a problem of damaging not only cancer cells but also normal tissues of the body such as bone marrow/gastrointestinal mucosa. In the cancer cells that survive thereafter, the removal process after the formation of DNA-DNMT1 adducts is overactivated, resulting in suppression of anticancer efficacy, which makes them vulnerable to the development of resistance.

Therefore, there is a strong demand for the development of improved new drugs, and they have the limitations of low efficacy and high toxicity of existing treatments and resistance within 14 months. As such, Aza-T-dCyd can be used as a first-line and second- or third-line treatment.

For AML/MDS patients aged 65 years or older, most of whom are unable to undergo bone marrow transplantation, Aza-T-dCyd has been tested through preclinical and phase 1 clinical trials. It also secures the advantage of oral administration, showing the possibility of a high competitive advantage compared to existing treatments.

In the case of elderly MDS/AML, unlike the development of various targeted anti-cancer drugs/immuno-anticancer drugs/cell therapy drugs in general MDS/AML, applicable development technologies are limited, and recurrent T-ALL, platinum-based anti-cancer drug resistant ovarian cancer/bladder cancer, etc. Even in the field of technology, applicable development technologies are limited.

Aza-T-dCyd's technical superiority is its strong efficacy and differentiated safety profile due to its Thio-nucleoside skeleton, and it has expanded indications compared to existing DNMT1 inhibitors (existing DNMT1 inhibitors (mainly MDS/AML) vs. Aza-T-dCyd (expanded to various hematological and solid cancers)), excellent efficacy in various situations where existing DNMT1 inhibitors are ineffective (strong efficacy in solid cancer patients in phase 1 clinical trials, etc.) and excellent safety (non-clinical and phase 1 clinical results) It has a clear competitive advantage in terms of efficacy and safety.

[ Pharmaceutical composition ]

In the present invention, the compound of Formula 1 and the compound of Formula 2 may each independently exist in a solid or liquid form. It can exist in the solid state, in crystalline or amorphous form, or as mixtures thereof. For crystalline or non-crystalline compounds, pharmaceutically acceptable solvates may be formed. In crystalline solvates, solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve non-aqueous solvents such as but not limited to ethanol, isopropanol, DMSO, acetic acid, ethanolamine or ethyl acetate, or they may involve water as a solvent incorporated into the crystalline lattice. Solvates, in which water is the solvent incorporated into the crystalline lattice, are typically referred to as “hydrates”. Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The present invention includes all such solvates.

Certain compounds of the invention, including their various solvates that exist in crystalline form, may exhibit polymorphism (ie, the ability to occur in different crystalline structures). These different crystalline forms are typically known as “polymorphs”. The present invention includes 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 such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. Different polymorphs can be prepared, for example, by changing or adjusting the reaction conditions or reagents used to prepare the compound. For example, changes in temperature, pressure or solvent can create polymorphs. Additionally, one polymorph may spontaneously convert to another polymorph under certain conditions.

In the present specification, the 4'-thio-5-aza-2'-deoxycytidine (aza-T-dCyd) drug includes not only the compound of Formula 1, but also pharmaceutically acceptable salts thereof, solvates thereof, and prodrugs thereof ( prodrugs).

"Pharmaceutically acceptable salts thereof" refers to pharmaceutically acceptable organic or inorganic salts. Exemplary salts are sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, Tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, genticinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate , glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts Including, but not limited to. A pharmaceutically acceptable salt thereof may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Moreover, pharmaceutically acceptable salts may have more than one charged atom in their structure. It may have multiple counter ions if multiple charged atoms are part of a pharmaceutically acceptable salt. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterion.

The term “prodrug” has the meaning of being used in the relevant technical field. For example, a physiologically active substance or a therapeutically active organic compound is chemically modified, and the parent compound is released or released in vivo under enzymatic or other conditions. means a compound designed to A prodrug is converted into the desired compound in vivo after administration. Although it is a useful drug, it can be used clinically by applying chemical modifications to those that have unsuitable properties in terms of side effects, stability, solubility, absorption, and duration of action.

"Solvate" means a compound of Formula 1 or a salt thereof that further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

As used herein, “cancer” refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, blood-borne tumors (eg, multiple myeloma, lymphoma, and leukemia) and solid tumors. Non-limiting examples of blood cancer include non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myelogenous leukemia, chronic myeloid leukemia, and the like, and non-limiting examples of solid cancer include gastric cancer. , kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.

As used herein, "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 (eg, horse, pig or donkey), chimpanzee or monkey.

In the present specification, the anticancer effect or therapeutic effect of an anticancer agent refers to an action that reduces the severity of cancer, reduces the size of a tumor, or delays or slows down the progression of cancer, which occurs while a patient is suffering from a specific cancer. can be referred to

For example, the anticancer effect of an anticancer drug may be Cell Viability (change in the degree of cytotoxicity or cell number) of cancer cells after treatment with an anticancer drug in vitro and/or in vivo. there is. For example, it can be confirmed indirectly through a drug response test through a cell line or a non-clinical animal model (xenograft). In addition, even in cancer patients, the anticancer effect of the anticancer agent can be directly confirmed, and related data can be derived and used as a database. In addition, when designing an anticancer drug dosage guideline, animal model PK parameters and/or toxicity profile may be considered in parallel.

The anticancer effect of an anticancer agent may be inferred from in-vitro data, % Maximum effect of the anticancer agent, such as IC ₅₀ , IC ₆₀ , IC ₇₀ , IC ₈₀ and IC ₉₀ , and the highest blood level of the drug It can also be confirmed in non-clinical animal models and clinical cancer patients through in-vivo data such as concentration (Cmax) and/or area under the blood drug concentration-time curve (AUC).

Reactivity of an anticancer agent means clinical sensitivity in terms of anticancer effect.

"Sensitivity" and "susceptibility" when referring to treatment with an anti-cancer agent are relative terms that refer to the degree of effectiveness of a compound in alleviating or reducing the progression of the tumor or disease being treated.

An "effective patient's anticancer effect/response" is, for example, 5%, 10%, 15%, 20% of a patient's response, as measured by any suitable means, such as gene expression, cell counts, assays, and the like. , 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more inhibition.

In the present specification, the dose is the dose at which drug efficacy is expected. In the present invention, the medicinal effect may be an anticancer effect. The reactivity (anti-cancer effect) of an anti-cancer agent is the degree of response, and the % maximum effect of the anti-cancer agent, such as IC ₅₀ , IC ₆₀ , IC ₇₀ , IC ₈₀ and IC ₉₀ , the value of exerting toxicity to normal cells (LC ₅₀ ) can be

For example, dosage forms for oral use can be formulated using a variety of formulation techniques known in the art. For example, it may include a biodegradable (hydrolyzable) polymeric carrier used to adhere to the oral mucosa. It is designed to slowly erode over a predetermined period of time, wherein drug delivery is provided essentially entirely.

Drug delivery in an oral dosage form avoids the weaknesses encountered with oral drug administration, such as slow absorption, degradation of the active agent by fluid present in the gastrointestinal tract, and/or first pass and inactivation in the liver. For biodegradable (hydrolyzable) polymeric carriers, virtually any such carrier can be used as long as the desired drug release profile is not compromised, and the carrier is compatible with any other ingredient present in the oral dosage unit. . Generally, polymeric carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the oral mucosa. Examples of polymeric carriers useful herein are acrylic acid polymers (eg, carbomers). In some embodiments, non-limiting examples of other ingredients that can be incorporated into an oral dosage form include disintegrants, diluents, binders, lubricants, flavoring agents, coloring agents, preservatives, and the like. In some embodiments, it may be in the form of a conventionally formulated tablet, lozenge, or gel for buccal or sublingual administration.

In some embodiments, administration of the compound is continued at the physician's discretion when the patient's condition improves; Alternatively, the dose of drug to be administered may be temporarily reduced or temporarily discontinued for some length of time (ie, a "holiday"). The length of the washout can vary between 2 days and 1 year, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days. days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dose reduction during the washout is 10%-100%, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

When improvement of the patient's condition occurs, a maintenance dose is administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced as a function of symptoms, to a level at which improved disease, disorder or condition is maintained. However, patients require intermittent treatment over a long period of time upon any recurrence of symptoms.

The amount of a given agent that will correspond to such amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (eg body weight) of the subject in need of treatment, but nevertheless include, for example, the formulation to be administered, the administration It can be routinely determined in a manner known in the art depending on the route and the particular circumstances surrounding the subject to be treated. In general, however, doses used for adult human treatment will typically range from 0.02-5000 mg/day, or about 1-1500 mg/day.

A single dose herein may be given as a single dose or in divided doses administered simultaneously, for example as 2, 3, 4 or more sub-doses.

In some embodiments, oral formulations are unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. In some embodiments, the unit dose is in the form of patches containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers may be used, in which case it is typical to include a preservative in the composition.

In some embodiments, formulations for parenteral injection are presented in unit dosage form, including but not limited to ampoules, or in multi-dose containers, with an added preservative.

It is typically prepared for parenteral administration, i.e., bolus, intravenous, and intratumoral injection, in unit dose injectable form with a pharmaceutically acceptable parenteral vehicle. It is optionally mixed in the form of a lyophilized preparation or aqueous solution with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

Aza-T-dCyd is equipped with stronger efficacy compared to existing HMA ingredients such as Vidaza (azacytidine) and Dacogen (decitabine), an improved safety profile, convenience of administration in oral form, and the possibility of overcoming drug resistance.

Aza-T-dCyd aims to become a new treatment after 2/3L, which is an unmet need, so it can obtain a status as a standard treatment preferentially in the course of anticancer treatment that must be performed according to strict treatment guidelines.

In particular, Aza-T-dCyd is a thio-nucleoside compound in which the deoxyribose structure of nucleoside anticancer drugs is changed to a thio-deoxyribose structure. Through this change, Aza-T-dCyd has the following characteristics to overcome the limitations of existing nucleoside anticancer drugs and meet the unmet needs. can

In addition, Aza-T-dCyd has a strong efficacy and differentiated safety profile due to the Thio-nucleoside skeleton, and has expanded indications compared to existing DNMT1 inhibitors (existing DNMT1 inhibitors (mainly MDS/AML) vs. Aza-T-dCyd ( Expanded to various hematologic cancers and solid cancers)), excellent efficacy in various situations where existing DNMT1 inhibitors do not show drug efficacy (strong efficacy in solid cancer patients in phase 1 clinical trials, etc.) and excellent safety (non-clinical and phase 1 clinical results) It has a clear competitive advantage in terms of efficacy and safety.

Therefore, according to the present invention, 4'-thio-5-aza-2'deoxycytidine, a multi-target inhibitor including DNMT1 inhibitor, Aza-T-dCyd) can be applied in various ways in terms of administration method, dose, target patient group, or combination therapy with venetoclax, an inhibitor of BCL-2 that mediates apoptosis.

1 is a schematic diagram showing the mechanism of action of epigenetics.

2 is a schematic diagram showing a normal hematopoietic process in our body. The role of the master TF (especially CEBP/epsilon) that leads each step is very important in step 1, which secures cell numbers through rapid cell division, and step 2, which stops cell division and proceeds with cell maturation.

Figure 3 is a schematic diagram showing abnormal functions in the hematopoietic process and occurrence of leukemia due to the delay in conversion from step 1 to step 2 due to decreased expression of Master TF (CEBP/epsilon).

4 is a schematic diagram showing that treatment with a DNMT inhibitor induces normal DNA methylation in hypermethylated MDS/AML hematological cancer cells and induces differentiation into normal cells and exerts anticancer effects through the re-expression of CEBP/epsilon genes.

5 is a schematic diagram of the unmet needs of platinum-based anticancer drugs for the treatment of ovarian cancer and bladder cancer.

6 is the chemical structure of Decitabine and Azacytidine.

7 is a mechanism of action of nucleoside-based anticancer drugs.

8 is a DNA damage repair process of nucleoside-based anticancer drugs by BER action mechanism.

Figure 9 shows the results confirming that DNMT1 is inhibited when NTX-301 and Decitabine are treated at different concentrations in MV4-11 (AML cell line) cells.

10 is a result confirming that DNMT1 is inhibited when KG-1a (AML cell line) cells are treated with NTX-301 and T-dCyd.

11 is a result confirming that DNMT1 is inhibited when NTX-301 and T-dCyd are treated in CCRF-CEM (ALL cell line) cells.

12 is a result confirming that DNMT1 is inhibited when NTX-301 and T-dCyd are treated with NCI-H23 (Lung carcinoma cells), HCT-116 (Colon carcinoma cells), and IGROV-1 (Ovarian carcinoma cells) cell lines.

13 is a result confirming re-expression of the p15 tumor suppressor gene due to DNMT1 inhibition when Aza-T-dCyd was treated in MV4-11 cells.

FIG. 14 compares and analyzes re-expression patterns of the p15 tumor suppressor gene due to DNMT1 inhibition when MV4-11 cells were treated with Aza-T-dCyd and Decitabine.

15 is a result confirming the CEBP/epsilon expression pattern induced when THP-1 cells were treated with NTX-301 (Aza-T-dCyd) at each concentration.

16 shows the results of confirming intracellular DNMT1, CEBP/epsilon, and CDKN1B levels by treating various AML cell lines (MV4-11, HL-60, and KG-1a) with Aza-T-dCyd.

17 is a result of confirming NTX-301 target engagement.

18 is a graph showing the growth inhibitory effect of Aza-T-dCyd on hematological cancer cell lines.

19 is a comparison chart of the growth inhibitory effects of Aza-T-dCyd and Decitabine on hematological cancer cell lines.

20 is the mechanism of action of Aza-T-dCyd's DNA damage response.

21 shows the mechanism of action of Aza-T-dCyd.

22 shows the measurement results of pyrimidine level and RRM1 expression upon NTX-301 treatment.

23 shows the results of confirming the increased expression of DNA replication stress-related genes and the efficacy of drugs for each cell line due to DNA adducts formed during NTX-301 treatment.

24 shows the results confirming the increase in DNA damage response markers due to p53 pathway activation during NTX-301 treatment.

25 shows the selective DNA dimethylation pattern and RNA-seq data analysis results induced by NTX-301 treatment.

26 shows the pharmacokinetic evaluation results of Aza-T-dCyd for Mice.

27 shows the pharmacokinetic evaluation results of Aza-T-dCyd for Mice.

28 shows the pharmacokinetic evaluation results of Aza-T-dCyd in rats.

29 shows the pharmacokinetic evaluation results of Aza-T-dCyd for dogs.

30 shows a comparison of tumor growth and body weight differences for each treatment group in the Molm13 Xenograft model.

31 is a result showing inhibition of DNMT3B expression in cancer tissue after NTX-301 was tested in the Molm13 Xenograft model.

32 is a comparison result of the survival rate and weight difference for each treatment group compared to competing drugs in the MV4-11 systemic AML model.

33 is a comparison result of survival rate and weight difference for each treatment group compared to competing drugs in the MV4-11 systemic AML model.

34 is a comparison result of some CBC values for each treatment group in the MV4-11 systemic AML model.

35 shows the results of comparison of efficacy / weight comparison of MV4-11 Luc cell line Xenograft.

36 shows body luminescence comparison results in MV4-11 Luc cell line Xenograft.

37 is a result of confirming the anticancer efficacy of Aza-T-dCyd in the HL-60 Xenograft model.

38 shows the results of confirming changes in DNMT1, DNMT3A, and DNMT3B after treatment with Aza-T-dCyd in the HL-60 leukemia Xenograft model.

39 shows the drug efficacy of the combined administration of NTX-301 and Venetoclax in the MV4-11 AML cell line xenograft model.

40 shows the results of confirming the efficacy of Aza-T-dCyd and T-dCyd in ALL PDX models.

41 shows results confirming the anticancer efficacy of NTX-301 in a solid cancer Xenograft model.

42 is a result of measuring body weight change for each dose group of Aza-T-dCyd in the Comet assay experiment.

43 shows Comet assay results for each NTX-301 dose group.

44 shows results of cell viability and composition evaluation in in vivo bone marrow cells.

45 is a graph showing repeated dose toxicity (GLP) results of Aza-T-dCyd in rodents and non-rodents.

46 shows the drug blood concentration upon administration of NTX-301 for each patient.

47 shows the highest blood concentration and drug exposure degree (AUC) upon administration of NTX-301 by dose.

48 relates to a preclinical model of AML demonstrating an improved therapeutic index of NTX-301.

A, B: For female NOD/SCID mice bearing MV4-11 tumors (n = 8 per group, 6 groups), NTX-301 [(1.5, 2.0, or 2.5 mg/kg) (p.o.)], DAC[ (2.5 mg/kg) (i.p.)] or AZA [5.0 mg/kg (i.p.)], Kaplan-Meier plot of survival probability (A) and plot showing time to endpoint (moribund state or end of study). day) (B).

C, D: For female NOD/SCID mice bearing MV4-11 tumors (n = 8 per group, 3 groups), NTX-301 [(2.0 mg/kg) (p.o.)] or AZA [(5.0 mg/kg) ) (i.p.), tumor growth measured by quantification of bioluminescence emission (photons/sec) (C) and bioluminescence images at day 42 (D).

E, F: For female NOD/SCID mice bearing luciferase-labeled MV4-11 tumors (n = 6 per group, 4 groups), upon treatment with NTX 301 [(daily at 1.0 or 2.0 mg/kg) or 1.0 twice daily (p.o.) at mg/kg], bioluminescence image (E) and quantification graph of bioluminescence emission (photons/sec) (F).

G-I: female NMRI nude mice (n = 6 per group, 5 groups) bearing subcutaneous MOLM-13 tumors, upon NTX-301 treatment [(0.2, 0.4, 0.8, or 1.5 mg/kg) (i.p.)], Tumor growth (G), number of cells per gram of tumor (H) and anti-CD33 staining intensity (I). The red arrow in G indicates the time point of NTX-301 treatment.

AZA azacytidine, DAC decitabine, NTX NTX-301; mpk mg/kg, p.o. Oral administration, i.p. Intraperitoneal administration. P-values (versus vehicle) are assigned and presented as follows: *p < 0.05; **p < 0.001; ***p < 0.0001.

49 shows the benefits provided by NTX-301 in combination therapy with VCX.

A: A GSEA plot showing that genes controlling sensitivity or resistance to VCX are significantly enriched among transcriptome changes induced by treatment with NTX-301 or DAC in MV4-11 cells for 48 hours.

B: A line plot showing survival (%) of parental cells (Con) and TP53 -knockdown (shp53) MV4-11 cells when treated with NTX-301 + VCX (left) or DAC + VCX (right) for 72 hours.

C: Matrix showing combination therapy index (CI) values in parental (top) and TP53 -knockdown (bottom, shp53) MV4-11 cells treated with NTX-301 + VCX or DAC + VCX at the indicated concentrations for 72 hours. CI values < 1 (blue) indicate synergistic drug combinations. Darker blue correlates with stronger synergy, and CI values > 1 (gray) indicate no synergy.

D: Graph showing the growth of subcutaneously implanted MV4-11 tumors in female BALB/c nude mice (n = 5 mice per group, 8 groups) when treated with NTX-301 or AZA as monotherapy or in combination with VCX. am.

E : Kaplan-Meier curve showing survival probability of female NCG mice (n = 8 mice per group, 6 groups) intravenously injected with MV4-11 cells when treated with NTX-301 or AZA as monotherapy or in combination with VCX am.

AZA azacytidine, DAC decitabine, NTX NTX-301, VCX venetoclax. *p < 0.05; **p < 0.01; ***p < 0.001.

50 is a graph showing long-term disease control lasting 5 months or more when NTX-301 is administered to patients with advanced solid cancer.

51 is the PK profile of Aza-T-dCyd in patients with advanced solid cancer in phase 1 clinical trial.

52 shows the mechanism of action of B-cell lymphoma (BCL)-2 and its inhibitor, venetoclax, which mediate apoptosis.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, but do not limit the protection scope of the present invention.

실시예 1 : In vitro pharmacologyExample 1: In vitro pharmacology

1-1. DNMT1 Inhibition 1-1. DNMT1 Inhibition

Aza-T-dCyd is a second/tertiary treatment for AML-resistant patients whose disease has progressed after Decitabine skeleton-based drug therapy, a treatment for other blood cancers such as T-cell ALL, and an adjuvant treatment for a number of solid cancers. Inhibition of DNMT1, a drug target, and reduction of the intracellular amount of DNMT1 protein can be an important drug efficacy marker (PD marker) of Aza-T-dCyd.

Cytidine-like structural nucleoside DNMT1 inhibitors such as Decitabine are known to be incorporated into DNA to irreversibly trap DNMT1 and induce degradation through proteolytic enzyme complexes. It has been established through various basic and clinical studies, and the utilization of DNMT1 inhibitors in patients.

1-1-1. AML cell line에서의 세포 내 DNMT1 단백질 감소 확인1-1-1. Confirmation of intracellular DNMT1 protein reduction in AML cell line

In order to evaluate the DNMT1 inhibitory efficacy of Aza-T-dCyd in AML cell lines, standard AML cell lines MV4-11 (FIG. 9) and KG-1a (FIG. 10) were treated with Aza-T-dCyd and the positive control substance Decitabine. and Western blotting was performed for DNMT1 protein. As shown in FIGS. 9 and 10 , it was confirmed that Aza-T-dCyd decreased DNMT1 protein in a concentration-dependent manner in both cell lines. In particular, it was confirmed that DNMT1 was completely reduced even at a very low concentration of 20 nM.

1-1-2. ALL cell line/고형암 세포주1-1-2. ALL cell line/solid cancer cell line

The same DNMT1 inhibitory efficacy was evaluated in other hematological cancer cell lines, ALL cell line and solid cancer cell line, and Aza-T-dCyd inhibited DNMT1 in a concentration-dependent manner in CCRF-CEM (ALL) (FIG. 11) and solid cancer cell line (FIG. 12). confirmed that it can be done.

1-2. Cellular Pharmacodynamics1-2. Cellular Pharmacodynamics

1-2-1. p15 Tumor Suppressor Gene의 재발현 확인1-2-1. Confirmation of re-expression of the p15 Tumor Suppressor Gene

In the DNA of hematopoietic stem cells of patients with MDS and AML, many of the cytosine in the expression promoter of a number of tumor suppressor genes are methylated, and their expression is suppressed. As a result, important functions such as DNA damage repair and cell cycle checkpoint are suppressed compared to normal. is caused

Abnormal DNA methylation of multiple tumor suppressor genes has been reported in many types of Leukemia, including AML.

When the methylation pattern is normalized by treatment with an epigenetic control drug 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. At this time, intracellular PD markers Re-expression of phosphorus p15 is induced. Through existing Decitabine/Azacytidine clinical trials and patient treatment, p15 re-expression in p15-silenced cancer cells and cancer patients was established as a PD marker to monitor the pharmacodynamic response of DNMT1 inhibitors.

Therefore, NTX-301 was applied to MV4-11 cells, an AML cell line, in order to confirm the re-expression pattern of tumor suppressor genes through an epigenetic regulatory mechanism caused by DNMT1 inhibition during Aza-T-dCyd treatment. It was treated by concentration. A dose-dependent re-expression of p15 tumor suppressor gene, a PD marker induced by DNMT1 inhibition, was confirmed, and strong re-expression of p15 mRNA was confirmed through RT-PCR (FIG. 13).

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 when Aza-T-dCyd and Decitabine were treated. As a result, Aza-T-dCyd was 60 nM, It was confirmed that the expression level of the p15 tumor suppressor gene was significantly increased in a dose-dependent manner under the condition of treatment at a concentration of 200 nM (FIG. 14 and Table 1). In addition, it was confirmed that p15 upregulation was induced by Aza-T-dCyd more than Decitabine.

Therefore, the results confirming the reduction of DNMT1 and the re-expression of the p15 tumor suppressor gene upon treatment with Aza-T-dCyd suggest that Aza-T-dCyd is highly likely to be developed as a new target anti-cancer drug.

Table 1 below compares the degree of p15 mRNA re-expression after treatment with a DNMT1 inhibitor (Aza-T-dCyd/Decitabine) in the MV4-11 cell line, which is an AML cell line (fold increase based on 0 nM).

In addition, it was confirmed that Aza-T-dCyd induced better p15 upregulation than Decitabine.

Therefore, as a result of confirming the reduction of DNMT1 and the re-expression of the p15 tumor suppressor gene when treated with Aza-T-dCyd, Aza-T-dCyd has high potential as a new target anticancer agent.

1-2-2. 추가 PD 마커 (CEBP/epsilon, CDKN1B)의 변화 확인1-2-2. Confirmation of changes in additional PD markers (CEBP/epsilon, CDKN1B)

In the case of malignant cells from MDS/CMML/AML patients, CEBP/alpha, Pu.1, and GATA factors, which are lineage commitment master TFs, are expressed at high levels, whereas the expression of late developmental stage transcription factors including CEBP/epsilon is low for each gene. The problem is that it is kept low due to hypermethylation.

CEBP/epsilon is a protein that induces maturation of immature hemocytes and suppresses abnormal proliferation. As the expression of this protein is induced, the expression of CDKN1B and Myc antagonists (MAD) is increased. CDKN1B, as a CDK inhibitory protein, is known to be responsible for stopping cell cycle proliferation or inducing differentiation. The expression of CEBP/epsilon and CDKN1B induces differentiation of AML malignant cells and exhibits strong anticancer efficacy.

It has been reported that treatment with decitabine inhibits DNMT1 to induce DNA demethylation and re-expression of CEBP/epsilon, resulting in apoptosis and cell differentiation, thereby exhibiting excellent anticancer efficacy.

CEBP/epsilon and CDKN1B expressions were confirmed and comparatively analyzed to confirm the target engagement pattern induced by DNMT1 inhibition during NTX-301 treatment in various AML cell lines (FIG. 15).

DNMT1 inhibition was induced in a dose-dependent manner, and CEBP/epsilon expression and CDKN1B expression were increased when NTX-301 was treated at different concentrations in THP-1 cells, an AML cell line. Maturation induction and differentiation induction functions were confirmed.

AML cell lines MV4-11, HL-60, and KG-1a cells were treated with Aza-T-dCyd for 72 hours and the intracellular protein expression levels were checked. As a result, the expression of DNMT1 was inhibited and the expression of CEBP/epsilon and CDKN1B was confirmed to increase. In particular, it was confirmed that the expression increased the most in MV4-11 and HL-60 cells (FIG. 16).

1-3. Target engagement1-3. Target engagement

Through previous experiments, MV4-11 cells, which were identified as responsive to Aza-T-dCyd, were treated with DNMT1 knock-out (siRNA) to construct a KO cell line that did not affect cell survival, division, or growth.

When the DNMT1 knock-out cell line produced was treated with NTX-301 at different concentrations, it was confirmed that cytotoxicity was reduced, which is considered to be the result of weakening the pharmacological action on MV4-11 cells. Through this, it was confirmed that Aza-T-dCyd has a strong drug effect through DNMT1 (FIG. 17).

1-4. Cellular cytotoxicity1-4. Cellular cytotoxicity

Growth-inhibitory activity was measured to confirm the inhibitory effect of Aza-T-dCyd, a DNMT1 inhibitor, on cell proliferation in Leukemia cell lines.

1-4-1. AML1-4-1. AML

In various AML cell lines, Aza-T-dCyd (SRI-9639, NTX-301) showed excellent cell survival inhibitory efficacy with IC ₅₀ values as shown in FIG. 18 .

1-4-2. Leukemia 세포주의 세포 증식에 대한 DNMT1 저해제의 효과 비교1-4-2. Comparison of effects of DNMT1 inhibitors on cell proliferation of Leukemia cell lines

Growth-inhibitory activity was measured to confirm the effect of Aza-T-dCyd and Decitabine, which are DNMT1 inhibitors, on cell proliferation in Leukemia cell lines. As a result of treatment with DNMT1 inhibitors for 72 hours each, the GI ₅₀ value (half macimal growth inhibitory concentrations) of Decitabine, which is known to be effective in inhibiting leukemia cell growth, was 0.024 μM to 4.3 μM, and the GI ₅₀ value of Aza-T-dCyd was was measured from 0.014 μM to 0.69 μM. Overall, when comparing the inhibitory efficacy against cell proliferation, it was confirmed that the efficacy of Aza-T-dCyd was significantly superior to that of Decitabine (FIG. 19).

1-4-3. 암종별 세포주에서 Aza-T-dCyd 의 IC50값 결과1-4-3. Results of IC50 values of Aza-T-dCyd in cell lines by carcinoma type

In order to secure a biomarker for Aza-T-dCyd, cytotoxicity was evaluated for 200 cell lines, and drug activity profiling analysis was performed.

As a result of drug responsiveness analysis of Aza-T-dCyd through drug activity profiling and genome analysis on 200 cell lines, it was confirmed that strong efficacy can be secured in various solid cancers (Table 2).

세포주cell line	암 종류cancer type	Aza-T-dCyd IC50 (uM)Aza-T-dCyd IC50 (uM)
NALM-6NALM-6	ALLALL	0.03380.0338
RS4;11RS4;11	ALLALL	0.02030.0203
JurkatJurkat	ALLALL	0.06950.0695
MOLT-16MOLT-16	ALLALL	0.01820.0182
CCRFCEMCCRFCEM	ALLALL	0.1190.119
MOLT-4MOLT-4	ALLALL	0.0350.035
HL-60HL-60	AMLAML	0.5140.514
KG-1KG-1	AMLAML	0.4610.461
MV-4-11MV-4-11	AMLAML	0.1830.183
THP-1THP-1	AMLAML	5.125.12
MC116MC116	B-cell lymphomaB-cell lymphoma	0.02970.0297
JeKo-1JeKo-1	B-cell lymphomaB-cell lymphoma	0.03850.0385
DBDB	B-cell lymphomaB-cell lymphoma	0.1350.135
DOHH-2DOHH-2	B-cell lymphomaB-cell lymphoma	0.6090.609
HTHT	B-cell lymphomaB-cell lymphoma	1.031.03
NU-DUL-1NU-DUL-1	B-cell lymphomaB-cell lymphoma	0.02450.0245
SU-DHL-10SU-DHL-10	B-cell lymphomaB-cell lymphoma	0.03660.0366
SU-DHL-4SU-DHL-4	B-cell lymphomaB-cell lymphoma	0.01750.0175
SU-DHL-8SU-DHL-8	B-cell lymphomaB-cell lymphoma	0.07060.0706
ARH-77ARH-77	B-cell lymphomaB-cell lymphoma	0.4130.413
BC-1BC-1	B-cell lymphomaB-cell lymphoma	0.3380.338
IM-9IM-9	B-cell lymphomaB-cell lymphoma	1.051.05
MHH-PREB-1MHH-PREB-1	B-cell lymphomaB-cell lymphoma	0.03890.0389
RPMI 8226RPMI 8226	B-cell myelomaB-cell myeloma	3.273.27
SKO-007SKO-007	B-cell myelomaB-cell myeloma	4.294.29
CA46CA46	Burkitt's lymphomaBurkitt's lymphoma	0.02550.0255
DaudiDaudi	Burkitt's lymphomaBurkitt's lymphoma	0.4510.451
GA-10GA-10	Burkitt's lymphomaBurkitt's lymphoma	0.03350.0335
NAMALWANAMALWA	Burkitt's lymphomaBurkitt's lymphoma	0.01090.0109
RajiRaji	Burkitt's lymphomaBurkitt's lymphoma	0.07520.0752
EB2EB2	Burkitt's lymphomaBurkitt's lymphoma	8.198.19
MEG01MEG01	CMLCML	1.071.07
BV-173BV-173	CMLCML	0.005320.00532
CML-T1CML-T1	CMLCML	0.1090.109
EM-2EM-2	CMLCML	0.2550.255
TF-1TF-1	ErythroleukemiaErythroleukemia	0.7330.733
L-428L-428	Hodgkin's lymphomaHodgkin's lymphoma	0.3650.365
RPMI 6666RPMI 6666	Hodgkin's lymphomaHodgkin's lymphoma	1.051.05
H9H9	T-cell LymphomaT-cell Lymphoma	0.4680.468
J-RT3-T3-5J-RT3-T3-5	T-cell LymphomaT-cell Lymphoma	0.1060.106
OE33OE33	Head and NeckHead and Neck	0.1610.161
A-673A-673	SarcomaSarcoma	0.6980.698
A375A375	Skin (Melanoma)Skin (Melanoma)	0.06670.0667
C32C32	Skin (Melanoma)Skin (Melanoma)	>10>10
H4H4	CNS (Glioma)CNS (Glioma)	0.08010.0801
U-118 MGU-118 MG	CNS (Glioma)CNS (Glioma)	>10>10
56375637	BladderBladder	0.2960.296
HT-1197HT-1197	BladderBladder	7.137.13
HT1376HT1376	BladderBladder	3.283.28
J82J82	BladderBladder	1.981.98
T24T24	BladderBladder	1.531.53
TCCSUPTCCSUP	BladderBladder	1.181.18
UM-UC-3UM-UC-3	BladderBladder	0.2890.289
SCaBERSCaBER	BladderBladder	0.1870.187
647-V647-V	BladderBladder	0.4080.408
BFTC-905BFTC-905	BladderBladder	0.1190.119
Hs 821.THs 821.T	SarcomaSarcoma	>10>10
AU565AU565	BreastBreast	>10>10
CAMA-1CAMA-1	BreastBreast	>10>10
MDA MB 231MDA MB 231	BreastBreast	0.210.21
MDA MB 415MDA MB 415	BreastBreast	>10>10
MDA MB 453MDA MB 453	BreastBreast	1.891.89
MDA MB 468MDA MB 468	BreastBreast	1.341.34
SK-BR-3SK-BR-3	BreastBreast	9.329.32
EFM-19EFM-19	BreastBreast	3.953.95
ChaGoK1ChaGoK1	Lung (NSCLC)Lung (NSCLC)	3.413.41
LS1034LS1034	ColonColon	1.411.41
LS411NLS411N	ColonColon	0.2650.265
NCI-H508NCI-H508	ColonColon	0.5420.542
NCI-H747NCI-H747	ColonColon	0.3190.319
Caki-1Caki-1	KidneyKidney	0.08560.0856
HCT-15HCT-15	ColonColon	1.021.02
SW1417SW1417	ColonColon	>10>10
SW403SW403	ColonColon	0.6190.619
SW480SW480	ColonColon	1.581.58
SW620SW620	ColonColon	0.2340.234
HCT-116HCT-116	ColonColon	0.10.1
SK-MEL-28SK-MEL-28	Skin (Melanoma)Skin (Melanoma)	2.52.5
DaoyDaoy	CNS (Medulloblastoma)Medulloblastoma (CNS)	3.613.61
A204A204	SarcomaSarcoma	0.1570.157
Hs 729Hs 729	SarcomaSarcoma	>10>10
RL95-2RL95-2	Female GU (Uterus)Female GU (Uterus)	0.7050.705
OE19OE19	Head and NeckHead and Neck	>10>10
OE21OE21	Head and NeckHead and Neck	0.1520.152
HT-1080HT-1080	SarcomaSarcoma	0.120.12
AGSAGS	StomachStomach	0.05840.0584
Hs 746THs 746T	StomachStomach	0.6660.666
A172A172	CNS (Glioma)CNS (Glioma)	>10>10
DBTRG-05MGDBTRG-05MG	CNS (Glioma)CNS (Glioma)	>10>10
DK-MGDK-MG	CNS (Glioma)CNS (Glioma)	>10>10
T98GT98G	CNS (Glioma)CNS (Glioma)	1.391.39
U-87 MGU-87 MG	CNS (Glioma)CNS (Glioma)	1.641.64
HepG2HepG2	LiverLiver	0.8920.892
OVCAR3OVCAR3	Female GU (Ovary)Female GU (Ovary)	3.443.44
FaDuFaDu	Head and NeckHead and Neck	0.2080.208
HuCCT1HuCCT1	LiverLiver	0.1050.105
BT20BT20	BreastBreast	>10>10
BT474BT474	BreastBreast	>10>10
Hs 578THs 578T	BreastBreast	>10>10
MCF7MCF7	BreastBreast	6.476.47
ZR-75-1ZR-75-1	BreastBreast	>10>10
COR-L23COR-L23	Lung (NSCLC)Lung (NSCLC)	0.09440.0944
A549A549	Lung (NSCLC)Lung (NSCLC)	0.190.19
COR-L105COR-L105	Lung (NSCLC)Lung (NSCLC)	2.242.24
D283 MedD283 Med	CNS (Medulloblastoma)Medulloblastoma (CNS)	0.06880.0688
G-361G-361	Skin (Melanoma)Skin (Melanoma)	1.081.08
Hs 688(A).THs 688(A).T	Skin (Melanoma)Skin (Melanoma)	>10>10
Hs 852.THs 852.T	Skin (Melanoma)Skin (Melanoma)	>10>10
MALME3MMALME3M	Skin (Melanoma)Skin (Melanoma)	7.457.45
MeWoMeWo	Skin (Melanoma)Skin (Melanoma)	1.041.04
RPMI-7951RPMI-7951	Skin (Melanoma)Skin (Melanoma)	0.5270.527
SH-4SH-4	Skin (Melanoma)Skin (Melanoma)	0.6980.698
SK-MEL-3SK-MEL-3	Skin (Melanoma)Skin (Melanoma)	0.5220.522
WM-266-4WM-266-4	Skin (Melanoma)Skin (Melanoma)	0.4290.429
TTTT	Endocrine (Thyroid)Endocrine (Thyroid)	>10>10
SK-N-ASSK-N-AS	CNS (Neuroblastoma)Neuroblastoma (CNS)	1.171.17
SK-N-FISK-N-FI	CNS (Neuroblastoma)Neuroblastoma (CNS)	2.712.71
MG-63MG-63	Bone (Osteosarcoma)Bone (Osteosarcoma)	0.990.99
SKOV3SKOV3	Female GU (Ovary)Female GU (Ovary)	2.922.92
HuP-T4HuP-T4	PancreasPancreas	0.3210.321
Hs 766THs 766T	PancreasPancreas	9.49.4
PSN-1PSN-1	PancreasPancreas	0.1910.191
YAPCYAPC	PancreasPancreas	1.051.05
AsPC-1AsPC-1	PancreasPancreas	2.122.12
BxPC-3BxPC-3	PancreasPancreas	0.6990.699
CFPAC-1CFPAC-1	PancreasPancreas	1.751.75
Capan-1Capan-1	PancreasPancreas	3.323.32
HPAF-IIHPAF-II	PancreasPancreas	0.7370.737
Mia PaCa-2Mia PaCa-2	PancreasPancreas	0.8810.881
NCIH441NCIH441	Lung (NSCLC)Lung (NSCLC)	1.31.3
ACHNACHN	KidneyKidney	0.2310.231
Caki-2Caki-2	KidneyKidney	0.3630.363
Detroit 562Detroit 562	Head and NeckHead and Neck	0.08110.0811
22Rv122Rv1	ProstateProstate	2.712.71
DU145DU145	ProstateProstate	0.4320.432
PC-3PC-3	ProstateProstate	0.4410.441
NCI-H292NCI-H292	Lung (NSCLC)Lung (NSCLC)	0.1940.194
SW1463SW1463	ColonColon	0.3030.303
SW837SW837	ColonColon	0.3540.354
HT-29HT-29	ColonColon	0.3230.323
769-P769-P	KidneyKidney	0.06570.0657
786-O786-O	KidneyKidney	0.2490.249
A498A498	KidneyKidney	0.07380.0738
A-704A-704	KidneyKidney	>10>10
A-253A-253	Head and NeckHead and Neck	0.6010.601
KATO IIIKATO III	StomachStomach	0.8070.807
DMS114DMS114	Lung (SCLC)Lung (SCLC)	2.22.2
DMS273DMS273	Lung (SCLC)Lung (SCLC)	5.665.66
DMS53DMS53	Lung (SCLC)Lung (SCLC)	4.224.22
NCIH446NCIH446	Lung (SCLC)Lung (SCLC)	3.043.04
NCI-H69NCI-H69	Lung (SCLC)Lung (SCLC)	>10>10
SKMES1SKMES1	Lung (NSCLC)Lung (NSCLC)	0.3140.314
SW579SW579	Endocrine (Thyroid)Endocrine (Thyroid)	1.051.05
BHT-101BHT-101	Endocrine (Thyroid)Endocrine (Thyroid)	0.2350.235
Cal 27Cal 27	Head and NeckHead and Neck	0.3190.319
SCC-25SCC-25	Head and NeckHead and Neck	0.1260.126
SCC-4SCC-4	Head and NeckHead and Neck	2.862.86
SCC-9SCC-9	Head and NeckHead and Neck	1.831.83
Calu6Calu6	Lung (NSCLC)Lung (NSCLC)	0.2890.289
639-V639-V	BladderBladder	1.571.57
SK-UT-1SK-UT-1	SarcomaSarcoma	0.3430.343
MES-SAMES-SA	SarcomaSarcoma	0.1350.135
A388A388	Skin (Head and Neck)Skin (Head and Neck)	0.190.19
A427A427	Lung (NSCLC)Lung (NSCLC)	0.07750.0775
A431A431	Skin (Head and Neck)Skin (Head and Neck)	0.2560.256
A7A7	Skin (Melanoma)Skin (Melanoma)	1.191.19
BE(2)CBE(2)C	CNS (Neuroblastoma)Neuroblastoma (CNS)	0.3730.373
BM-1604BM-1604	ProstateProstate	4.414.41
C32TGC32TG	Skin (Melanoma)Skin (Melanoma)	0.9530.953
C-33AC-33A	Female GU (Cervix)Female GU (Cervix)	0.4970.497
C-4 IIC-4 II	Female GU (Cervix)Female GU (Cervix)	0.7730.773
CGTH-W-1CGTH-W-1	Endocrine (Thyroid)Endocrine (Thyroid)	0.1430.143
Colo 205Colo 205	ColonColon	0.08110.0811
Colo 320DMColo 320DM	ColonColon	0.09230.0923
DLD-1DLD-1	ColonColon	0.2210.221
HeLaHeLa	Female GU (Cervix)Female GU (Cervix)	1.811.81
Hs 936.T(C1)Hs 936.T(C1)	Skin (Melanoma)Skin (Melanoma)	0.3980.398
HT-3HT-3	Female GU (Cervix)Female GU (Cervix)	0.8180.818
JARJAR	PlacentaPlacenta	0.2960.296
KHOS-240SKHOS-240S	Bone (Osteosarcoma)Bone (Osteosarcoma)	0.2540.254
LS-174TLS-174T	ColonColon	>10>10
MS751MS751	Female GU (Cervix)Female GU (Cervix)	>10>10
NCI-H295RNCI-H295R	Endocrine (Adrenal gland)Endocrine (adrenal gland)	>10>10
NTERA-2 cl.D1NTERA-2 cl.D1	TestisTestis	0.04270.0427
PA-1PA-1	Female GU (Ovary)Female GU (Ovary)	0.03920.0392
PFSK-1PFSK-1	CNS (Glioma)CNS (Glioma)	7.617.61
SJRH30SJRH30	SarcomaSarcoma	0.2910.291
SK-NEP-1SK-NEP-1	KidneyKidney	>10>10
SK-PN-DWSK-PN-DW	SarcomaSarcoma	0.8470.847
SNB-19SNB-19	CNS (Glioma)CNS (Glioma)	0.170.17
SU.86.86SU.86.86	PancreasPancreas	0.5080.508
SW872SW872	SarcomaSarcoma	0.730.73
SW900SW900	Lung (SCLC)Lung (SCLC)	1.981.98
SW962SW962	Female GU (Vulva)Female GU (Vulva)	>10>10
SW982SW982	SarcomaSarcoma	0.1710.171
T47DT47D	BreastBreast	>10>10
U-138MGU-138MG	CNS (Glioma)CNS (Glioma)	>10>10
VA-ES-BJVA-ES-BJ	SarcomaSarcoma	0.2410.241
WiDrWiDr	ColonColon	0.2630.263
D341 MedD341 Med	CNS (Medulloblastoma)Medulloblastoma (CNS)	NANA

실시예 2: Aza-T-dCyd의 작용기전 연구Example 2: A study on the mechanism of action of Aza-T-dCyd

20 and 21 show the DNA damage response mechanism of Aza-T-dCyd and the mechanism of action of Aza-T-dCyd, respectively.

Aza-T-dCyd inhibits the activation of DNA damage responses such as BER, forms a DNA-DNMT1 adduct that lasts longer compared to Decitabine, induces stronger DNA damage, and through this, strongly activates the CHK1-p53 pathway, making it more effective than Decitabine. show stronger anticancer efficacy.

2-1. DNA damage response 유도2-1. Induction of DNA damage response

① DNA incorporation 및 Pyrimidine metabolism 저해① Inhibition of DNA incorporation and Pyrimidine metabolism

Aza-T-dCyd is a pyrimidine analog that is metabolized by various enzymes involved in pyrimidine metabolism within cells.

It was confirmed that when cells were treated with Aza-T-dCyd, the expression of RRM1 protein, a ribonucleotide reductase important for dNTP de novo synthesis, was suppressed, and the amount of dCTP and dTTP in cells was reduced, thereby affecting the intracellular pyrimidine metabolism. (FIG. 22). Reduction of dCTP and dTTP, which act as important precursors when cancer cells replicate DNA, induces replication stress and can generate a strong DNA damage response.

DNA insertion of aza-T-dCTP, a metabolite of Aza-T-dCyd, causes base excision repair and mismatch repair and delays DNA replication, which can cause replication stress and aggravate DNA damage response.

② DNMT1-DNA adduct 형성을 통한 DNA damage response 유도② Induction of DNA damage response through formation of DNMT1-DNA adduct

When treated with Aza-T-dCyd, Aza-T-dCyd inserted into intracellular DNA is trapped through a covalent bond with DNMT1 and forms a DNMT1-DNA adduct.

The formed adduct can disrupt the progress of the DNA replication fork through its bulky structure, thereby collapsing the replication fork and causing a double-strand break, resulting in a strong DNA damage response.

Pyrimidine metabolism, DNA incorporation, and DNMT1 adduct formation result in a strong DNA damage response.

In FIG. 23 , due to the DNA adduct formed during Aza-T-dCyd treatment, the increased expression of DNA replication stress-related genes and the efficacy of drugs for each cell line were confirmed.

③ DDR-p53 pathway 활성화로 인한 DNA damage response 촉진③ Accelerate DNA damage response by activating the DDR-p53 pathway

Aza-T-dCyd treatment increases H2AX phosphorylation, a DNA damage response marker, simultaneously increases phosphorylation of Chk1, a DNA damage sensor, and increases p53 protein expression, which regulates cell cycle/death in DNA damage response, resulting in DNA damage response. As a result, it was confirmed that the tumorigenic activity of hematological malignancies was inhibited (FIG. 24).

2-2.2-2. DNA demethylationDNA demethylation

Treatment with Aza-T-dCyd induces strong DNMT1 depletion and decreases overall DNA methylation levels.

Decitabine induces dimethylation across genomic regions, whereas Aza-T-dCyd induces biased and selective dimethylation in DNA regions that are replicated during early replication, promoter regions important for gene expression, DNMT1-binding regions, and replication stress regions. induce

Through DNA dimethylation caused by Aza-T-dCyd treatment, various tumor suppressor genes that had been suppressed and genes necessary for inducing differentiation of AML cells were reactivated, and through RNA-seq data analysis, expression patterns of the genes were reactivated was confirmed (FIG. 25).

Example 3 : Pharmacokinetic evaluation by concentration - Mice/Rat/Dog PK profile

Aza-T-dCyd was administered orally and intravenously at various concentrations to mice, rats and dogs, and the PK profile was confirmed.

The animal species with the most similar drug distribution in the human body is Mice (CDA (Cytidine Deaminase) is mainly distributed in the liver, showing the most similar metabolic pattern), or rat/dog to confirm that absorption occurs well without differences between species. The PK profile of was confirmed.

Mice, rats, and dogs all show excellent oral administration potential, and as a result of administration in mice, it was confirmed that a Cmax / AUC value at a level that can secure excellent drug efficacy can be easily secured. That is, when administering Aza-T-dCyd at 0.5 mpk or more by oral administration, it is possible to easily secure drug efficacy in vivo.

26 and 27 show the pharmacokinetic evaluation results of Aza-T-dCyd for Mice, FIG. 28 show the pharmacokinetic evaluation results for Aza-T-dCyd for rats, and FIG. 29 show the pharmacokinetic evaluation results for Aza-T-dCyd for dogs. The results of the kinetic evaluation are shown.

These results and the results of basic research to predict drug efficacy (relationship between blood/tissue concentration and drug efficacy - blood dC concentration in Mice is about 1.5uM, blood dC concentration in Human is about 0.01uM, Based on the IC ₅₀ value of NTX-301 at dC concentration, which is more than twice as high as that of human in Mice), the result of predicting the dose showing efficacy in the human body is 32mg/day when administered alone and 8 - 16mg/day when administered in combination. It was predicted at the day level, and the result corresponds to the range below the MTD in the phase 1 clinical trial described below.

실시예 4 : In vivo Pharmacology (PK/PD correlation)Example 4: In vivo Pharmacology (PK/PD correlation)

Surprisingly, despite the fact that Aza-T-dCyd is not superior to Decitabine in terms of DNMT1 inhibition, that is, the degree of demethylation, it is not only superior in expression of anti-apoptotic gene or mechanism of down-regulating anti-apoptotic protein, but also resulted in much lower IC ₅₀ values through an apoptotic mechanism.

Meanwhile, DNA methylation is induced by DNA methyltransferase (DNMT), and there are at least three types of activated DNMTs (DNMT1, DNMT3A, and DNMT3B). Experiments using mice have shown that DNMT3a and DNMT3B are required for initiating and maintaining gene methylation and play an important role in proper development. In cancer tissues, DNMT3B is predominantly overexpressed, whereas DNMT1 and DNMT3a are moderately overexpressed, suggesting that DNMT3B may play a role in cancer development. The DNMT3B gene is located on chromosome 20q11.2 and shows a polymorphism by C→T nucleotide translocation in the new promoter part (C46359T) reversed by 149 base pairs (bp) from the transcription start site. It has been found to increase promoter activity by 30%. Although the mechanism of association is not clear, it is hypothesized that the T mutation increases the expression of DNMT3B, increasing the propensity for epigenetic loss of function of the tumor suppressor gene.

4-1. MDS/AML4-1. MDS/AML

In order to confirm the efficacy of Aza-T-dCyd in an animal model and to confirm whether Aza-T-dCyd exhibits excellent drug efficacy in a dose-dependent manner, a Molm13 Xenograft model experiment (MV4-11 systemic AML model experiment) was performed. (FIG. 30).

After establishing the Molm13 Xenograft model, an AML animal model in which Decitabine/Azacytidine does not show any therapeutic effect, Aza-T-dCyd was orally administered at a dose of 0.2 - 1.5 mg/kg.

It was confirmed that the administration of Aza-T-dCyd showed a strong anticancer effect in a dose-dependent manner without toxic signals (weight change), unlike those described in the literature on Decitabine/Azacytidine.

After administration of Aza-T-dCyd, cancer tissues were collected and analyzed (Western blotting), and as a result (FIG. 31), it was confirmed that Aza-T-dCyd strongly inhibited DNMT1 and DNMT3B in a concentration-dependent manner, and this inhibition of DNMT3B It is judged to contribute greatly to the strong anticancer effect of Aza-T-dCyd.

In the Molm13 AML Xenograft model test, administration of Aza-T-dCyd showed strong anticancer efficacy through inhibition of DNMT1 and strong expression of DNMT3B at the same time (about 90 kDa when treated with Aza-T-dCyd at a concentration of 0.4 mg/kg or higher). A clear reduction of DNMT3B protein in size was observed).

Snap-frozen Molm13-tumors were ground into powder and dissolved in 125 μg/μL of SDS-PAGE sample buffer. For comparison, Molm13 (50,000 cells/μL) was dissolved, separated by SDS-PAGE, and stained with Actin (CIR-fluorescence) and DNMT3B (ECL+). A clear decrease in the DNMT3B band of was observed.

After establishing a systemic MV4-11 engraftment model that can more accurately describe diseases in the actual human body, Aza-T-dCyd was orally administered in an amount of 0.4 - 1.5 mg/kg (FIG. 32).

It was confirmed that the administration of Aza-T-dCyd resulted in a statistically significant level of survival improvement even at the minimum dose of 0.4mg/kg, and in the maximum dose group, more than half of the mice in the entire group survived until the end of the experiment. / In all administrations, a strong dose-dependent anticancer effect was confirmed without significant toxicity signals (weight change).

In order to confirm the superior efficacy of Aza-T-dCyd compared to competing drugs, an MV4-11 systemic AML model experiment was performed to determine whether Aza-T-dCyd showed superior efficacy in a dose-dependent manner.

After establishing the systemic MV4-11 engraftment model, Aza-T-dCyd was administered orally at 1.5, 2.0, and 2.5 mg/kg (for Decitabine/Azacytidine, standard amounts/administration schedules used in preclinical studies were used) ( Figure 33).

While Decitabine showed no therapeutic effect, and Azacytidine showed symptoms that showed no inhibition of leukemia progression as a result of blood test results, Aza-T-dCyd showed very excellent anticancer efficacy against AML, and was excellent in all dose groups. It was confirmed that survival improvement was shown.

After establishment of the whole-body MV4-11 engraftment model, blood was collected before treatment (Day 21) and mid-treatment (

Day

36, 56, 77) in the Aza-T-dCyd and Decitabine/Azacytidine administration groups, and CBC (Complete blood count) numerical values were analyzed (FIG. 34).

In the group administered with Aza-T-dCyd, it was confirmed that there were no changes in major parameters such as white blood cells (WBC), red blood cells (RBC), neutrophils, and platelets. On the other hand, in the case of the Decitabine group, all subjects died before blood samples were obtained, and in the case of the Azacytidine group, severe neutropenia was confirmed.

In order to confirm whether the anticancer efficacy in the whole body MV4-11 engraftment model is directly related to the ability to suppress the number of cancer cells, an experiment was performed to measure body luminescence after whole body engraftment using a Luciferase transfected Mv4-11 cell line to confirm luminescence and In addition, death and weight change were also confirmed (FIG. 35).

It was confirmed that body luminescence was suppressed more strongly in the Aza-T-dCyd-treated group than in the actual Azacytidine-treated group (FIG. 36).

Compared to the strong tumor suppression effect shown when 2.0 mg/kg of NTX-301 (Aza-T-dCyd) was administered to the HL-60 AML Xenograft model, the maximum dose of Decitabine 0.75 mg/kg (if administered above the corresponding dose, the animal died). The experiment could not proceed) did not show any tumor suppression efficacy (FIG. 37).

Since the efficacy of DNMT1 inhibitors is related to the selective profile of DNMT1 and the de novo methyltransferases DNMT3A and DNMT3B targets, the expression of DNMT1, DNMT3A and DNMT3B was confirmed in the group administered with Aza-T-dCyd and Decitabine in the Xenograft model. (FIG. 38).

2mg/kg of NTX-301 (Aza-T-dCyd) or 0.75mg/kg of Decitabine was intraperitoneally administered to the HL-60 Xenograft mice model, and tumor samples were taken on the 11th day to confirm the expression of DNMT1, DNMT3A, and DNMT3B , Aza-T-dCyd and Decitabine-administered groups showed a significant decrease in DNMT1 expression. On the other hand, there was a difference in the expression levels of DNMT3A and DNMT3B in the groups administered with Aza-T-dCyd and Decitabine. DNMT3A expression level was not affected by Aza-T-dCyd, and DNMT3B expression level was significantly decreased by Aza-T-dCyd, but no effect was observed by Deictabine.

It has been reported that high DNMT3B expression is associated with poor prognosis of AML, suggesting that NTX-301 has excellent anticancer effects in a leukiemia model due to the inhibitory effect of DNMT3B by Aza-T-dCyd.

Recently, the standard therapy for AML treatment has changed from single administration of Decitabine/Azacytidine to combining these drugs with Venetoclax, a Bcl2 inhibitor. Accordingly, Aza-T-dCyd evaluated the drug effect when these drugs were administered in combination. . First of all, in the xenograft model in which the MV4-11 AML cell line was subcutaneously implanted into mice, it was confirmed that the Aza-T-dCyd and Venetoclax co-administered group showed very good efficacy.

It was confirmed that complete tumor regression was induced even in the Aza-T-dCyd 0.5mpk administration group, and showed a stronger cancer growth inhibitory effect than the combination administration group of Azacytidine and Venetoclax (FIG. 39).

In the MV4-11 systemic AML model experiment conducted to confirm the superior efficacy of the Aza-T-dCyd / Venetoclax combined administration group compared to competing drugs, when Aza-T-dCyd was administered in combination with the standard dose of Venetoclax, a very low dose of Aza- It can be confirmed that T-dCyd administration alone shows superior efficacy compared to competing drugs.

4-2.4-2. T-ALLT-ALL

In order to confirm the efficacy of Aza-T-dCyd in another indication, T-ALL, the efficacy of Aza-T-dCyd in a patient-derived ALL animal model was evaluated, and as shown in FIG. effect could be confirmed.

Aza-T-dCyd was administered at doses of 0.5, 1, 1.5, and 2 mg/kg in ALL-Patients Derived Xenografts (PDX), and T-dCyd (a DNMT1 inhibitor), a thionucleoside derivative, was used as a comparison group. Aza-T-dCyd is very effective in pediatric ALL PDX at a dose that can be tolerated up to 1.5 mg/kg, and it was also effective in T-ALL including ETP, a subtype of ALL with very high unmet needs. In particular, it was confirmed that a strong tumor regression could be induced even at a low dose compared to the dose in AML and this remission state could be maintained for a long period of time, confirming that the development potential as a future ALL treatment was very high.

4-3. various solid cancers

Unlike existing DNMT1 inhibitors, Aza-T-dCyd showed excellent efficacy in various solid cancer animal models. When Aza-T-dCyd was administered in animal models of colorectal cancer, bladder cancer, ovarian cancer, lung cancer, etc., very excellent effects were confirmed as follows (FIG. 41).

The efficacy of NTX-301 was confirmed through tumor growth curves after administration of Aza-T-dCyd to various solid tumor xenograft models. Excellent anticancer effect was shown in the group where 1 ~ 2mg/kg dose of NTX-301 was repeatedly administered for QD x 5, 2 ~ 6 cycles (test/control (T/C) value ≤ 40%).

Tumor growth inhibition was observed in the group administered with Aza-T-dCyd in Coloretal (HCT-116) xenograft model, and the optimal T/C was 21%. On the other hand, Decitabine's T/C was 45%, which was not very effective.

After administration of Aza-T-dCyd to the Non-Small Cell Lung cancer (NCI-H522) xenograft model, tumor growth inhibition was observed, and the optimal T/C was 31%.

Tumor regression was observed in the group administered with 1mg/kg dose of Aza-T-dCyd in the Ovarian (OVCAR-3) xenograft model, and the optimal T/C was 4%. It was confirmed that the T/C value was 40% at a dose of 0.5 mg/kg, showing a dose-dependent effect.

Tumor regression was observed in the group administered with 1mg/kg dose of NTX-301 to the Bladder Tumor (BL0381PD PDX) xenograft model, and the optimal T/C was 23%. Decitabine showed toxicity after 2 cycles in the 0.75mg/kg dose group.

실시예 5: Safety (GLP-Toxicity)Example 5: Safety (GLP-Toxicity)

As a preliminary toxicity evaluation of the Aza-T-dCyd compound, an experiment to check toxicity symptoms (tissue change) in mice and DNA damage in bone marrow after 2 weeks of systemic administration and evaluation of cell composition in bone marrow of mice after 4 weeks of systemic administration A preliminary toxicity test was performed.

42 shows the results of measuring body weight change for each Aza-T-dCyd dose group during the Comet assay experiment.

During the 2-week systemic administration period, no significant weight change was observed, and it was confirmed that no organ damage was observed during autopsy 2 weeks later.

In addition, when bone marrow was collected after administration for 2 weeks and Comet assay was performed to confirm DNA damage, it was confirmed that there was no change in various indicators indicating DNA damage compared to the control group not treated with the drug at all (FIG. 43).

After 4 weeks of continuous administration, cell composition in the bone marrow of mice was evaluated. As a result of the experiment, there was no change in various cells constituting the bone marrow, confirming that Aza-T-dCyd did not significantly affect hematopoietic ability in normal animals. (FIGS. 44 and 45).

Aza-T-dCyd (NTX-301) was confirmed to have excellent stability and a wide therapeutic window in animal tests as a result of the GLP-toxicity test.

Aza-T-dCyd was confirmed from the above toxicity test results that it is a safe drug that can sufficiently secure a therapeutic concentration based on AUC values within a usable safe range.

In the MDS/AML model, the AUC value of Aza-T-dCyd shows efficacy even at 60h*ng/ml, and shows the best therapeutic efficacy at about 200 - 250h*ng/ml, which is about 50 of the NOAEL value in the most sensitive species. % and about 25% of the HNSTD, which means that Aza-T-dCyd has a very wide therapeutic window.

Other solid cancers and other animal models, such as ALL, showed excellent therapeutic effects even at lower AUC exposures.

GLP-Tox experiments were conducted in rats and dogs, and QD x 5d 2cycles were conducted as PO.

In the GLP rat toxicology study, rats were administered 2.5, 5, and 10 mg/kg of Aza-T-dCyd (15, 30, and 60 mg/m ² ) per day. Target organs of toxicity are bone marrow, thymus, heart, and testes. Testicular toxicity, including reduced sperm count, was recoverable upon discontinuation of administration, and the maximum tolerated dose (MTD) was 10 mg/kg or more (>60 mg/m ² ) per day.

In the GLP dog toxicology study, Aza-T-dCyd was administered at doses of 0.15, 0.5, and 1.0 mg/kg (3, 10, and 20 mg/m ² ) per day. The target organs were bone marrow, thymus, gastrointestinal tract, tonsil, and testis. The maximum tolerated dose (MTD) was between 0.5 - 1mg/kg (10 - 20 mg/m ² ) per day, and the highest non-toxic dose (HNSTD) per day The 0.5 mg/kg (10 mg/m ² ) no observable adverse effect level (NOAEL) was < 0.15 mg/kg (3 mg/m ² ) per day.

실시예 6: 임상개발Example 6: Clinical Development

In patients treated with Aza-T-dCyd, p15 re-expression, a PD marker that inhibits DNMT1, a drug target, was confirmed in circulating tumor cells.

The PK profile of Aza-T-dCyd was confirmed in the patient group administered with Aza-T-dCyd (FIG. 51). We expect high efficacy of Aza-T-dCyd in actual patient population. NTX-310 showed good tolerability in patients with late-stage solid cancer, and showed signs of efficacy, such as inducing stable disease that lasted for more than 5 months in some patients and inhibiting cancer progression.

The PK profile after clinical administration of Aza-T-dCyd agrees well with the predicted results based on the results of existing non-clinical animal PK experiments, especially in the 16 and 32 mg/day administration groups, which are predicted to have the optimal effect range in humans. This is in good agreement with the predicted results from non-clinical studies, so sufficient drug exposure in the human body can be expected.

46 shows the drug blood concentration upon administration of NTX-301 for each patient, and FIG. 47 shows the highest blood concentration and drug exposure degree (AUC) when NTX-301 is administered for each dose.

Aza-T for patients with MDS/AML over the age of 65 who are not eligible for bone marrow transplantation and/or standard chemotherapy and who do not have additional standard therapies due to progression of the disease even after using standard therapies and for T-ALL patients -The safety/possibility of dCyd as a therapeutic agent is being verified (FIG. 51).

In the first human clinical trial (FIH, First in Human), as a result of stepwise dose increase in solid cancer patients, the MTD (maximum tolerated dose) was determined to be 32 mg, and at doses of 16 mg or less, DLT (dose limiting toxicity) , dose-limiting toxicity) and

grade

3 or 4 adverse reactions were not reported. The primary tumor types in the trial were colorectal adenocarcinoma, sarcoma, breast cancer, ovarian cancer, renal cell carcinoma, laryngeal squamous cell carcinoma, uterine cancer, duodenal adenocarcinoma, hepatocellular carcinoma, and paraganglioma.

In a phase 1 clinical trial for solid cancer patients led by another sponsor (Pinobio), the safety/possibility of Aza-T-dCyd as a re-sensitization agent for ovarian cancer/bladder cancer patients resistant to platinum-based anticancer drugs was evaluated in the gradual dose increase and expansion test group. are evaluating The primary tumor types in this clinical trial were endometrial cancer, right tonsillar squamous cell carcinoma, prostate cancer, colorectal cancer, metastatic adencystic carcinoma, pancreatic adenocarcinoma, breast cancer, and adenocarcinoma of the distal bile duct.

Furthermore, we are conducting phase 1/2 clinical trials for patients with MDS, AML, and CMML. For reference, in the case of CMML (Chronic myelomonocytic leukemia), test results in animal models have also come out well, and PDX is currently in progress.

Claims

Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof, which exerts an anticancer effect through an apoptosis mechanism, overactivates BER (Base Excision Repair), one of the DNA damage repair methods A pharmaceutical composition characterized in that it is administered at a high dose that induces apoptosis so that the mechanism of drug resistance is not activated.

[Formula 1]
The pharmaceutical composition according to claim 1, wherein the Aza-T-dCyd compound exhibits an anticancer effect in a dose-dependent manner.
The method according to claim 1, wherein (1) a patient group with resistance to DNMT1 inhibitors; (2) a patient group in need of selective drug delivery to cancer cells compared to normal cells as a targeted anti-cancer agent; (3) a patient group in need of adjusting the expression level of one or more markers of drug efficacy; (4) a patient group with accumulated epigenetic DNA methylation pattern changes in cancer cells compared to normal cells; (5) A patient group in which the standard dose of DNMT1 inhibitor, which is a standard treatment, is limited due to the problem of damaging normal tissues as well as cancer cells because DNA damage is caused through the formation of DNA-DNMT1 adducts when DNMT1 inhibitor is administered; (6) a patient group in need of prevention or treatment of hematological malignancies; (7) Compared to normal individuals, lineage commitment master transcription factors selected from the group consisting of CEBP/alpha, Pu.1 and GATA factors are expressed at high levels, while the expression of CEBP/epsilon or transcription factors in the late developmental stage is not the same as that of each gene. a group of patients whose methylation remains low; (8) a group of patients who are progressing or have progressed to bone marrow cancer; (9) a patient group diagnosed with AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), CMML (chronic myelomonocytic leukemia) or ALL (acute lymphocytic leukemia); (10) patient population with health conditions that deviate from standard therapy or standard chemotherapy; (11) a group of patients whose DNMT3B expression level is outside the normal range; (12) a group of patients requiring abnormal bone marrow removal; (13) a patient group in need of blast reduction; (14) a group of patients with impaired blood function or a group of patients with hematopoiesis abnormalities; (15) a group of patients with abnormal methylation patterns due to overactivation of DNMT1 compared to normal subjects; (16) a group of patients diagnosed with T-cell ALL (acute lymphocytic leukemia); (17) Patients who may develop resistance to nucleoside metabolism when administered with nucleoside anticancer drugs; (18) a patient group with a possibility of developing resistance to platinum-based anticancer drugs or who developed resistance; (19) Platinum-based anticancer drug administration target patient group; (20) epigenetically silencing a tumor suppressor gene and/or SLFN11 compared to normal subjects; (21) a patient group in need of prevention or treatment of ovarian or bladder cancer; (22) a platinum-resistant recurrent end-stage ovarian cancer patient group or a platinum-resistant metastatic bladder cancer patient group; (23) metastatic bladder cancer patient group, p53 mutation bladder cancer patient group or hypermethylated SLFN11 bladder cancer patient group, which is a detailed biomarker for treatment; or (24) A pharmaceutical composition characterized by administering the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof to a patient group diagnosed with stage 3 or 4 ovarian cancer.
The method of claim 3, wherein the Aza-T-dCyd compound has strong resistance to nucleic acid intermediate degrading enzyme (Endonuclease), resistance development mechanism by lowering the efficiency or speed of the overactivated BER (Base Excision Repair) repair process. A pharmaceutical composition characterized by overcoming.
According to claim 3, the patient group in need of selective drug delivery to cancer cells compared to normal cells is an elderly patient group aged 65 years or older and/or a patient group with weakened physical function due to an underlying disease or comorbidity; Or a pharmaceutical composition characterized in that the patient group whose therapeutic effect is limited due to the occurrence of a resistance mechanism is limited due to side effects that also appear by acting on cells of the bone marrow, digestive tract, mucous membrane and / or skin.
The method of claim 3, wherein the Aza-T-dCyd compound targets a specific part of cancer cells that differs from normal cells by reducing DNMT1, re-expressing p15 tumor suppressor gene, and/or decreasing DNMT3B expression, thereby selectively targeting only cancer cells. A pharmaceutical composition characterized by acting as an attacking target anticancer agent.
The pharmaceutical composition according to claim 1, wherein the Aza-T-dCyd compound is rapidly activated by triphosphate in cancer cells compared to normal cells.
The method of claim 7, wherein the Aza-T-dCyd compound delays DNA replication by causing base excision repair and/or mismatch repair through DNA insertion of aza-T-dCTP, a triphosphate of the Aza-T-dCyd compound, An anti-cancer pharmaceutical composition characterized by inducing replication stress and aggravating DNA damage response.
The method of claim 1, wherein the Aza-T-dCyd compound inhibits the expression of RRM1 protein, which is an important ribonucleotide reductase for dNTP de novo synthesis, induces DNA replication stress by reducing the amount of dCTP and dTTP in cells, and induces a strong DNA damage response A pharmaceutical composition characterized in that it can generate.
The method of claim 1, wherein the Aza-T-dCyd compound has a low activation rate by dCK (deoxycytidine kinase) in normal cells compared to cancer cells due to its Thio-nucleoside structure, so the Aza-T-dCyd compound is selectively delivered to cancer cells. A pharmaceutical composition characterized by securing a safety profile.
The method of claim 3, wherein the drug effect marker requiring expression level control is subject to epigenetic silencing through DNA methylation in cancer cells, or the transcription factor of the drug effect marker is subject to epigenetic silencing through DNA methylation. A pharmaceutical composition characterized in that
The anti-cancer pharmaceutical composition according to claim 11, wherein the drug efficacy marker requiring expression level control is a differentiation induction gene, a tumor suppressor gene, or a combination thereof.
[Claim 12] The anti-cancer pharmaceutical composition according to claim 11, wherein the pharmacological markers requiring expression level control are p15, p16, SLFN11, CEBP/epsilon, CDKN1B, Myc antagonists (MAD), or a combination thereof.
The pharmaceutical composition according to claim 1, wherein the Aza-T-dCyd compound re-expresses CEBP/epsilon in cancer cells and/or expresses the p27 tumor suppressor gene, which is a down-stream effector of CEBP/epsilon.
The pharmaceutical composition according to claim 3, wherein the Aza-T-dCyd compound does not affect the hematopoietic ability without DNA damage in normal tissue cells.
The method of claim 1, wherein the Aza-T-dCyd compound is activated by triphosphate in the cell and used instead of some dC (deoxycytidine) during DNA synthesis, and after DNA synthesis, DNMT1 is irreversibly trapped to form DNA- A pharmaceutical composition characterized by inducing cancer cell death by forming a DNMT1 adduct and activating the epigenetic silencing mechanism.
Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof, which exerts an anticancer effect through an apoptosis mechanism, to a patient group who has received information on or diagnosed with poor prognosis or the possibility of resistance when DNMT1 inhibitor is administered to apoptosis A pharmaceutical composition characterized in that it is administered in a high dose that induces.

[Formula 1]
The method of claim 17, wherein the provision of information on prognosis or the possibility of resistance is (i) quantifying the problem of inhibition of anticancer efficacy as the removal process is overactivated after formation of the DNA-DNMT1 adduct, or (ii) whether or not the problem occurs / degree A pharmaceutical composition characterized in that it is derived from classifying the patient group according to.
18. The pharmaceutical composition according to claim 17, characterized in that the data value of providing information of poor prognosis is the possibility of disease recurrence and/or early mortality (mean survival less than 1 year).
A pharmaceutical composition for oral administration comprising the Aza-T-dCyd compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]
21. The pharmaceutical composition for oral administration according to claim 20, wherein the Aza-T-dCyd compound exhibits metabolic resistance by cytidine deaminase (CDA) in the liver when administered orally.