RAS mutant cancer therapies, including ixazomib citrate analogs.

Abstract

To provide a therapeutic agent for RAS gene mutant cancer. [Solution] The present invention provides a cancer treatment for RAS gene mutants comprising an ixazomib citrate ester analog represented by the following formula (1). The cancer treatment of the present invention can inhibit the differentiation, proliferation, and migration of cancer cells in RAS gene mutant cancers, and can selectively suppress the proliferation of cancer cells. In particular, it is effective in treating cancers including KRAS gene mutations. [Formula 1] JPEG2026105758000006.jpg4287

Description

[Technical Field]

[0001] This invention relates to a therapeutic agent for RAS gene mutant cancers, comprising an ixazomib citrate analog. [Background technology]

[0002] RAS is a type of protein involved in cell proliferation. RAS gene mutations are one of the cancer driver factor mutations in the field of cancer, and it is thought that when they occur, abnormal RAS proteins are produced or cell proliferation is significantly promoted, making cancer more likely to develop. For this reason, RAS gene mutations are attracting attention as a target molecule for cancer treatment.

[0003] There are three types of RAS proteins: KRAS, NRAS, and HRAS. These are highly frequent oncogenes, with mutations detected in approximately 30% of cancer patients. For example, KRAS gene mutations are found in about 70-90% of pancreatic cancers, and have also been found in lung cancer, colorectal cancer, multiple myeloma, and uterine cancer. NRAS gene mutations have been found in skin cancer (malignant melanoma), multiple myeloma, and thyroid cancer. HRAS gene mutations have been reported in bladder cancer and thyroid cancer.

[0004] Although RAS gene mutations are found in many cancers, they do not exhibit characteristic changes in the amino acid sequence, and there are no drug binding sites on the surface of the RAS protein. As a result, despite extensive research by many scientists, no effective RAS inhibitors have reached clinical trials. Therefore, the RAS gene has been recognized as a therapeutic target that "cannot be used as a drug."

[0005] Lumakeras® (generic name: sotrasib), a treatment for relapsed or refractory non-small cell lung cancer patients positive for the KRAS G12C mutation, was first approved in the United States in 2021 and in Japan in 2022 (see, for example, Non-Patent Document 1). KRAS G12C is a type of KRAS gene mutation in which the glycine (G) at codon 12 is replaced with cysteine ​​(C).

[0006] The overall response rate for sotrasib in patients with non-small cell lung cancer was 37.1%, and side effects such as diarrhea (31.7%), nausea (19.0%), elevated ALT (15.1%), and elevated AST (15.1%) were reported in 69.8% of patients (see, for example, Non-Patent Document 2). Furthermore, the KRAS G12C mutation has also been identified in colorectal cancer patients. The overall response rate for sotrasib in colorectal cancer patients was reported to be 9.7% (see, for example, Non-Patent Document 3).

[0007] On the other hand, proteasome inhibitors are sometimes used as anticancer drugs. Proteasome inhibitors work by inhibiting the function of proteasomes in cancer cells, causing abnormal proteins to accumulate in cancer cells and ultimately leading to the death of cancer cells. Compounds with a boronic acid structure are considered useful as proteasome inhibitors, and bortezomib, a type of proteasome inhibitor, was approved in Japan in 2006 as a treatment for multiple myeloma, and its indications have been expanded to date. Ixazomib is also a proteasome inhibitor with a boronic acid structure, and in Japan, ixazomib citrate was approved in 2017 as a treatment for multiple myeloma.

[0008] However, NRAS mutant cancer patients have a significantly lower response to bortezomib (see, for example, Patent Document 1), and experiments on the response to ixazomib have reported that wild-type KRAS cells are sensitive to ixazomib, while KRAS mutant cells are resistant to it, although the administered cell lines differ (see, for example, Patent Documents 2-3). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Special Publication No. 2014-533114 [Patent Document 2] Special Publication No. 2014-533112 [Patent Document 3] Special Publication No. 2016-527202 [Non-patent literature]

[0010] [Non-Patent Document 1] David S. Hong, et al., “KRASG12C Inhibition with Sotorasib in Advanced Solid Tumors”, The New England Journal of Medicine, 2020; 383:1207-1217 [Non-Patent Document 2] Skoulidis F, et al., “Sotorasib for Lung Cancers with KRAS p.G12C Mutation”, N Engl J Med., 2021 Jun 24;384(25):2371-2381 [Non-Patent Document 3] Marwan G Fakih, et al., “Sotorasib for previously treated colorectal cancers with KRAS G12C mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial”, Lancet Oncol. 2022; 23(1):115-124 [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] Many cancer patients have RAS gene mutations, but effective treatments are still limited to certain mutations, and their response rates are low. Specifically, the only drug approved in Japan is sotrasib, but sotrasib is only effective for cancers with the KRAS G12C mutation, and no effective drugs have been developed for cancers with other mutations. Furthermore, according to Patent Documents 1-3, bortezomib, a proteasome inhibitor, shows significantly low responsiveness to NRAS mutant strains, and ixazomib, also a proteasome inhibitor, shows resistance to KRAS mutant strains. Even proteasome inhibitors, which are expected to have anticancer effects, have little effect on RAS mutant cancers. However, since the cells used as comparison subjects differ in the examples in Patent Documents 1-3, it cannot be simply concluded that KRAS mutations alone are the cause of resistance to bortezomib and ixazomib, and further investigation is needed. Thus, the treatment of RAS gene mutant cancers is still in its early stages, and the development of effective treatments is desired. [Means for solving the problem]

[0012] Since KRAS, HRAS, and NRAS all share a common mechanism of action and common mutation sites, it is presumed that drugs effective against KRAS gene mutant cancers can also be applied to HRAS gene mutant cancers or NRAS gene mutant cancers.

[0013] Therefore, the inventors focused on KRAS gene mutant cancer, which has the highest incidence. They conducted a library screening of 1507 compounds to search for a drug that selectively inhibits cell proliferation or reduces cell viability in KRAS gene mutant cell lines, and found that ixazomib citrate analog, represented by the following formula (1), exerts that effect, thus completing the present invention.

[0014] In other words, the present invention has been made to solve the above-mentioned problems, and embodiments of the present invention may include the following configurations. (1) A therapeutic agent for RAS gene mutant cancer, comprising an ixazomib citrate analog represented by the following formula (1). [Chemical formula] (2) The therapeutic agent for RAS gene mutant cancer according to (1), wherein the RAS gene mutant cancer is a cancer containing a KRAS gene mutation. (3) The therapeutic agent for RAS gene mutant cancer according to (1) or (2), wherein the RAS gene mutant cancer is one or more cancers selected from the group consisting of pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer, skin cancer, uterine cancer, thyroid cancer, and gastric cancer. [Advantages of the invention]

[0015] According to the cancer therapeutic agent of the present invention, it is possible to inhibit the differentiation, proliferation, and migration of cancer cells of RAS gene mutant cancer, and selectively suppress the proliferation of cancer cells. Among them, it is effective for the treatment of cancers containing KRAS gene mutations. In addition, the cancer therapeutic agent of the present invention has fewer side effects than conventional chemotherapeutic agents. Furthermore, since it can be administered orally, the administration is simple and can improve the quality of life of patients. Since it is a low-molecular compound, there is also an advantage that the manufacturing cost can be kept low unlike biopharmaceuticals. [Brief description of the drawings]

[0016] [Figure 1A] Outline of the Prime Editing method for recombining glycine (G) at codon 12 of KRAS into aspartic acid (D). [Figure 1B] Figure for confirming the success of knock-in in the created KRAS gene mutant cell line. [Figure 2A] Figure showing the cell proliferation ability of the KRAS gene mutant cell line G12D. [Figure 2B] Figure showing the migration ability of the KRAS gene mutant cell line G12D. [Figure 2C] Figure showing the invasion ability of the KRAS gene mutant cell line G12D. [Figure 3]Figure showing the search results of compounds that cause a decrease in cell viability of KRAS gene mutant cell lines using the MTT assay. [Figure 4] Figure showing the results of cell viability according to the difference in drug types. [Figure 5] Figure showing the results of cell viability of various KRAS gene mutant cell lines by administration of ixazomib citrate analog. [Figure 6A] Figure showing the results of apoptosis induction rate (flow cytometry) by administration of ixazomib citrate analog. [Figure 6B] Graph of the flow cytometry results of Figure 6A. [Figure 7] Figure showing the detection results of ubiquitinated proteins and Cleaved caspase 3 by administration of ixazomib citrate analog. [Figure 8A] Photograph showing the state of a mouse transplanted with a pancreatic cancer cell line after administration of ixazomib citrate analog. The arrow indicates the tumor site. [Figure 8B] Figure showing the tumor growth of a mouse transplanted with a pancreatic cancer cell line in the administration of ixazomib citrate analog. [Figure 8C] Figure showing the body weight change of a mouse transplanted with a pancreatic cancer cell line in the administration of ixazomib citrate analog. [Figure 8D] Figure showing the effect on mouse liver function by administration of ixazomib citrate analog.

Mode for Carrying Out the Invention

[0017] Hereinafter, the embodiments of the present invention will be described in detail, but the scope of the present invention is not limited to these embodiments.

[0018] <RAS gene mutant cancer> The RAS protein is a low-molecular-weight guanosine triphosphate (GTP)-binding protein of approximately 21 kDa, consisting of 188-189 amino acids. There are three types of RAS genes that encode this protein: KRAS, HRAS, and NRAS, which constitute the gene family most frequently mutated in cancer (see Table 1). [Table 1]

[0019] Table 1 was created based on Adrienne D. Cox, et al., “Drugging the undruggable Ras: mission possible?” National Library of Medicine, 2014 Nov;13(11): 828-51.

[0020] To date, the UK database COSMIC has identified over 500 oncogenes, and it has become clear that the three RAS genes—KRAS, HRAS, and NRAS—constitute the family of oncogenes that most frequently mutate in human cancers.

[0021] Among these, KRAS gene mutations occur at a significantly higher frequency than NRAS and HRAS gene mutations. According to the COSMIC database, the incidence of KRAS gene mutations is 83.3%, NRAS gene mutations are 13.0%, and HRAS gene mutations are 3.7% (Prior IA et al., “The Frequency of Ras Mutations in Cancer”, Cancer Research, 2020, 80(14): 2969-2974). Looking at cancer types, KRAS gene mutations are found in approximately 25-30% of human lung adenocarcinoma, approximately 70-100% of human pancreatic cancer, and approximately 50% of human colorectal cancer.

[0022] Here, the KRAS gene refers to the gene encoding KRAS-GTPase, the NRAS gene refers to the gene encoding NRAS-GTPase, and the HRAS gene refers to the gene encoding HRAS-GTPase. The KRAS gene is located on chromosome 12, NRAS on chromosome 1, and HRAS on chromosome 11, and each consists of four exons and three introns.

[0023] A common mechanism in KRAS, NRAS, and HRAS is that the RAS gene acts as an on / off switch in cell signaling. Upstream stimuli such as EGFR cause guanosine diphosphate (GDP) to detach from the RAS molecule, and GTP binds from the cytoplasm, activating RAS. Activated RAS binds to up to 20 effector proteins, including RAF, PI3K, and RALGDS, activating the downstream signaling cascade. Normally, activated RAS becomes inactive due to its own GTP hydrolysis activity (GTPase), but if a mutation occurs in the RAS gene, resulting in amino acid substitution, the function of RAS as a GTPase decreases, leading to a constitutively activated state and continuous signaling downstream. This can cause cells to proliferate continuously and potentially develop into cancer.

[0024] The RAS gene is characterized by mutations that primarily encode single amino acid substitutions at glycine-12 (G12), glycine-13 (G13), or glutamine-61 (Q61) residues. These mutation sites are common to KRAS, NRAS, and HRAS. For example, according to Prior IA et al., “The Frequency of Ras Mutations in Cancer”, Cancer Research, 2020, 80(14): 2969-2974, in KRAS, 81% of mutations are at the G12 residue, 14% at the G13 residue, and 2% at the Q61 residue. In NRAS, 23% of mutations are at the G12 residue, 11% at the G13 residue, and 62% at the Q61 residue. In HRAS, 26% of mutations are at the G12 residue, 23% at the G13 residue, and 38% at the Q61 residue. Thus, KRAS, HRAS, and NRAS share common mutation sites. These mutations cause RAS to become GTP-bound, leading to activation independent of extracellular stimuli, and excessive stimulation of the signaling pathway, resulting in cancer cell proliferation. Note that the frequency of mutations at the G12, G13, and Q61 residues varies depending on the type of cancer.

[0025] <Ixazomib citrate analog> The inventors conducted a library screening of 1507 compounds to search for a drug that selectively inhibits cell proliferation or reduces cell viability in KRAS gene mutant cell lines. They discovered that ixazomib citrate analog, a type of proteasome inhibitor, exerts this effect, thus completing the present invention.

[0026] Here, the structural formulas of ixazomib, ixazomib citrate, and the ixazomib citrate analog of the present invention are shown. [ka]

[0027] Ixazomib citrate is already approved as a treatment for multiple myeloma. Patent documents 2 and 3 describe the results of administering ixazomib to cell lines with KRAS mutations, but reports indicate resistance to KRAS mutations. Although ixazomib citrate is also given as an example in patent documents 2 and 3, ixazomib is used in the examples because ixazomib citrate is immediately hydrolyzed to ixazomib in aqueous solution.

[0028] A key feature of this invention is the use of an "ixazomib citrate ester analog" instead of ixazomib itself. An "analog," also known as an "analog," is a compound that is structurally similar to another compound (a so-called "lead compound") but is recognized as a separate compound. Examples include compounds in which one atom is replaced by an atom of another element, compounds in which one functional group is replaced by another functional group, and compounds in which the absolute stereochemistry of one or more chiral centers of the lead compound is different. This invention contains an ixazomib citrate ester analog represented by formula (1). [ka]

[0029] <Method for producing ixazomib citrate analog> The ixazomib citrate ester of the present invention can be obtained commercially and used. It is available from Selleck Biotechnology Co., Ltd.

[0030] <Isazomib citrate analogs and other RAS gene mutation-related cancer treatments> The cancer therapeutic agent of the present invention can be used to treat RAS gene mutant cancers. Preferably, it is useful for treating RAS gene mutant cancers in which the frequency of RAS gene mutations is 10% or more. Preferably, it is used to treat cancers in which the frequency of RAS gene mutations is 20% or more, and more preferably, it is used to treat cancers in which the frequency is 50% or more. The frequency of RAS gene mutations referred to here is the sum of the frequencies of the three RAS genes: KRAS, HRAS, and NRAS. Examples of cancer types in which the incidence of RAS gene mutations is 50% or more include pancreatic ductal adenocarcinoma and colorectal adenocarcinoma.

[0031] Furthermore, RAS gene mutation cancers can be detected through genetic testing. The incidence rate can also be determined through the same genetic testing. For example, cancer gene panel testing using next-generation sequencing or the RAS gene mutation detection kit OncoBEAM™ RAS CRC kit (Sysmex Corporation) can be used.

[0032] In another embodiment, the cancer therapeutic agent of the present invention can be used to treat cancers including KRAS gene mutations. In yet another embodiment, the cancer therapeutic agent of the present invention is used to treat one or more cancers selected from the group consisting of pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer, skin cancer, uterine cancer, thyroid cancer, and gastric cancer. All of these cancers are RAS gene mutant cancers. Examples of pancreatic cancer include pancreatic ductal adenocarcinoma, colorectal cancer includes colorectal adenocarcinoma, and lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, and small cell lung cancer. Examples of skin cancer include cutaneous malignant melanoma, and examples of uterine cancer include endometrioid carcinoma of the uterine body, uterine carcinosarcoma, and cervical adenocarcinoma. The cancer therapeutic agent of the present invention can be used to treat the above cancers, and is particularly useful in the treatment of pancreatic cancer, which is said to have the worst prognosis.

[0033] The target population for the cancer treatment drug of the present invention is humans and non-human mammals for whom cancer treatment is desired or required. Non-human mammals include, for example, monkeys, pigs, cattle, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters, and include pet animals, livestock, and laboratory animals. Humans are a preferred target population.

[0034] (Prescription) The RAS gene mutant cancer therapeutic agent of the present invention contains an ixazomib citrate analog. Furthermore, it may contain pharmaceutically acceptable excipients as long as they do not impair the effects of the present invention. pharmaceutically acceptable excipients are formulated by mixing with solid, semi-solid, or liquid diluents, dispersants, fillers, and carriers. Further examples of excipients as long as they do not impair the effects of the present invention include stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, flow enhancers, flavoring agents, colorants, fragrance preservatives, media, and physiological saline.

[0035] (Dosage form) The dosage form of the RAS gene mutant cancer treatment drug of the present invention is not particularly limited, and examples include oral formulations (e.g., solid formulations such as capsules, tablets, granules, and powders; liquid formulations such as syrups, emulsions, and suspensions), respiratory formulations, intraperitoneal formulations, intravenous formulations, injections, suppositories, patches, and ointments. Oral formulations, respiratory formulations, or intravenous formulations are preferred. Examples of intravenous formulations include intravenous injection formulations and intravenous drip infusion formulations. In humans, oral formulations are preferred from the viewpoint of ease of handling. (Dosage / Dosage) The dosage of the RAS gene mutant cancer treatment drug of the present invention can be appropriately adjusted considering the intended use, the target population, the sex, age, weight, and stage of cancer progression of the target population. The effective therapeutic dose for humans can also be determined from animal models. For example, a human dose can be formulated to match a concentration known to be effective in animals. Specifically, when administered to humans, 1 to 20 mg of the ixazomib citrate analog of the present invention is preferred per dose, more preferably 2 to 10 mg, and even more preferably 3 to 4 mg. Within this range, the effects of the present invention are easily achieved, and toxicity is low, resulting in fewer side effects.

[0036] As a dosing regimen, it is preferable to administer the amount within the above range three times once a day and once every week. For example, a method of repeating a 28-day cycle in which 4 mg is administered once a day once a week for 3 weeks (days 1, 8, and 15) and then drug withdrawal is performed for 13 days can be mentioned. Note that since this dosage and the number of administrations vary under various conditions, there may be cases where a dosage and number of administrations less than the above range are sufficient, and there may also be cases where a dosage and number of administrations exceeding the above range are required. The concentration of the administered ixazomib citrate analog can be adjusted according to the patient's disease state so as to fall within an effective range.

[0037] (Combined agent) The cancer therapeutic agent of the present invention can be used alone, but can also be used in combination with anti-cancer therapies and anti-cancer agents with different mechanisms. For example, it may be used in combination with hormone therapy or radiation therapy. Alternatively, it can be administered in combination with drugs such as chemotherapeutic agents, hormone therapy agents, and immunotherapy agents for cancer treatment. It may also be used in combination with anti-cancer agents (5-FU, irinotecan, oxaliplatin, levofolinate, gemcitabine nab-paclitaxel, S-1) used in lung cancer and pancreatic cancer. Note that combined administration means administering at the same time as the administration of the cancer therapeutic agent of the present invention, or with a time difference before or after the administration of the cancer therapeutic agent of the present invention. Alternatively, the cancer therapeutic agent of the present invention and the above other cancer therapeutic agents can be mixed to form a single preparation. The dosage of the combined agent can be appropriately selected based on the clinically used dosage. In addition, the mixing ratio of the cancer therapeutic agent of the present invention and the combined agent can be appropriately selected according to the administration target, administration route, target disease, symptoms, combination, etc.

Example

[0038] The present invention will be specifically described by the examples shown below, but the present invention is not limited thereto.

[0039] <Establishment of KRAS gene mutant cell line> Using the KRAS gene from the normal pancreatic duct-derived cell line HPNE as the wild type, a new gene editing technique called Prime Editing was used to replace glycine (G) at codon 12 of KRAS with aspartic acid (D). Figure 1A shows an overview of this technique. In this technique, Prime Editing guide RNA (pegRNA) first attaches to the site where genome editing will occur, opening up the DNA. At this site, Cas9 nicksase makes a cut (nick) in the opposite strand, and then the C-terminal end of the pegRNA attaches to the cut DNA. Using this as a primer, reverse transcriptase synthesizes a new DNA strand that copies the sequence of the pegRNA. Specifically, the pU6-tevopreq1-GG-acceptor plasmid was first cleaved with the restriction enzyme BsaI to create KRAS-PegRNA incorporating the following sequences: (sgRNA sequence: 5'-TTTAACTTGCTATTTCTAGCTCTAAAACGCTCCAACTACCACAAGTTT-3', Scaffold sequence: 5'-TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG-3', donor sequence (PBS RT G12D: 5'-TGCCTACGCCATCAGCTCCAACTACCACAA-3'). Next, pCMV-PEmax-P2A-GFP and the created KRAS-PegRNA plasmid were co-expressed in HPNE cells to perform genetic modification. Plasmid introduction was performed using a 4D-Nucleofector (Lonza Japan), with 1 μg of each plasmid being introduced in 1 × 10⁶ cells. 6 The plasmid was introduced into individual cells. Three days after plasmid introduction, cells expressing green fluorescent protein were selected using BD FACS Aria III (BD bioscience) and seeded into 96-well plates at a density of 1 cell / well. Subsequently, KRAS was identified in the cells of each well by Sanger sequencing. The results are shown in Figure 1B. From Figure 1B, it can be seen that in the created KRAS gene mutant cell line, GGT of the wild-type cell line is replaced with GAT. This mutation resulted in a recombination from glycine (G) to aspartic acid (D).

[0040] Furthermore, the cell proliferation, migration, and invasive abilities of the established KRAS gene mutant cell lines were investigated. The measurement methods and results are shown in Figures 2A to 2C. In Figure 2, WT refers to the wild type, and Parental refers to the parent cell line possessing the wild-type KRAS-WT.

[0041] (Cell proliferation ability) Control cells and KRAS gene mutant cell lines 2 × 10 3 The cells were seeded in a 96-well cell culture plate. They were cultured at 37°C for 3 hours (day-0), 24 hours (day-1), 72 hours (day-3), 120 hours (day-5), and 168 hours (day-7), and cell viability over time was evaluated using the MTT assay. The results are shown in Figure 2A. From the results in Figure 2A, it can be seen that the cell proliferation ability of the created KRAS gene mutant cell line is higher than that of the wild type.

[0042] (Migration ability (soft agar colony forming ability)) First, a bottom-agar layer (0.6% agarose-containing medium, 2 mL / well) was prepared in a 6-well microplate. Next, a soft-agar layer (0.4% agarose-containing control cell line or KRAS gene mutant cell line, 1 × 10⁶) was placed on top of the bottom-agar layer. 4 Cell suspension (1 mL / well) was added and allowed to solidify in a cool place. Medium (1 mL / well) was added to the soft-agar layer and soft-agar culture was performed in a CO2 incubator (37°C, 5% CO2). After about 3 weeks, the medium was removed, MTT reagent was added, and the cells were left to stand in the CO2 incubator for 6 hours (cell staining). The number of colonies was analyzed using colony counter software under a microscope (N = 3). The results are shown in Figure 2B. From the results in Figure 2B, it can be seen that the migratory ability of the created KRAS gene mutant cell line is higher than that of the wild type.

[0043] (infiltration ability) 1 × 10⁶ suspended in 100 μL of serum-free medium 4One control cell or knock-in cell was added to the upper chamber of a Trans well (8 μm pore, Corning) and cultured. Serum-containing culture medium was added to the lower chamber layer. After 24 hours, the cells were fixed with formalin and stained with 0.1% crystal violet. The number of colonies was observed under a microscope (N = 3). The results are shown in Figure 2C. From the results in Figure 2C, it can be seen that the invasiveness of the created KRAS gene mutant cell line is higher than that of the wild type.

[0044] <Exploration of Ixazomib Citrate Analogues> In this invention, a screening was performed using a library of 1507 compounds (Selleck Cat. Cherry Pick Library (96-well)-L2000-Z546325) to search for drugs that selectively induce cell proliferation inhibition or a decrease in cell viability in KRAS gene mutant cell lines.

[0045] Specifically, 3000 HPNE-KRAS WT Or HPNE-KRAS G12D KRAS gene mutant cell lines were seeded in 96-well cell culture plates. They were cultured at 37°C for 24 hours, then a 1507-component compound library was added to a final concentration of 5.0 μM, and the cells were cultured at 37°C for 72 hours. After culturing, cell viability was measured using an MTT assay. Cell viability without drug addition was set as 100%, and the cell viability with each drug added was analyzed. The results are shown in Figure 3. It was confirmed that ixazomib citrate analog, a proteasome inhibitor, has a selective inhibitory effect on cell proliferation in KRAS gene mutant cells.

[0046] <Effects of ixazomib citrate analog on KRAS gene mutant cell lines> The effects of ixazomib citrate analogs on KRAS gene mutant cell lines in which the glycine (G) at codon 12 of KRAS was replaced with aspartic acid (D) were investigated.

[0047] (Confirmation of Effect 1: Comparison of Cell Viability by Different Drug Types) For proteasome inhibitors (bortezomib, delanzomib, ixazomib) having a structure similar to the ixazomib citrate analog of the present invention, the cell viability was evaluated. The method for measuring cell viability is as follows.

[0048] 3×10 normal cells and KRAS mutants 3 cells were seeded in a 96-well cell culture plate. As normal cells, a normal pancreatic duct-derived cell line (HPNE-KRAS WT ) without KRAS gene mutation was used, and as the KRAS mutant, a normal pancreatic duct-derived cell line HPNE-KRAS G12D was used. These cell lines were cultured at 37°C for 24 hours, and then bortezomib (Selleck, Cat. S1013), delanzomib (Selleck, Cat S1157), ixazomib (Selleck, Cat. S2180), and the ixazomib citrate analog of the present invention (Selleck, Cat. S2181) were administered at 5 μM and cultured at 37°C for 72 hours. The cell viability of each cell line was evaluated using the MTT assay. In each cell line, the group without drug administration was set as 100% for comparative study. The results are shown in Figure 4. As is clear from Figure 4, in vitro, the ixazomib citrate analog showed a superior cell growth inhibitory effect in the KRAS gene mutant cell line.

[0049] The significant cell growth inhibitory effect of the ixazomib citrate analog on the KRAS mutant cell line was observed more than that of ixazomib. It is presumed that the difference in structure between ixazomib and the ixazomib citrate analog increased the sensitivity to the KRAS mutant cell line. At present, the pharmacokinetics of the ixazomib citrate analog are unknown, but it is presumed that the chemical structure of the ixazomib citrate analog affects the stability as a drug, the metabolic pathway and metabolic time in the body, etc., and they appear as the cell growth inhibitory effect.

[0050] (Effect Verification 2: Comparison of Cell Viability in Various Cell Lines) The cell viability of various cell lines was evaluated after administration of an ixazomib citrate analog. The method for measuring cell viability is as follows:

[0051] Normal cells and KRAS mutant cells 3 × 10 3 The cells were seeded into 96-well cell culture plates. The KRAS mutant cell lines used were pancreatic cancer cell lines with KRAS gene mutation (G12D) (Panc04.03, PL8, PANC-1, Panc10.05, KP-4), pancreatic cancer cell line with KRAS gene mutation (G12C) (MIAPaCa2), pancreatic cancer cell line with KRAS gene mutation (G12R) (Hup-T3), pancreatic cancer cell line with KRAS gene mutation (G12V) (CFPAC-1), and normal pancreatic duct-derived cell line with KRAS gene mutation (HPNE-KRASSG12D). As wild-type cells, normal pancreatic duct-derived cell lines without KRAS gene mutation (HPNE-KRASWT, HPDE-4) and bronchial epithelial cell line with normal KRAS gene (HBEC3-KT) were used. These cell lines were cultured at 37°C for 24 hours, and then administered ixazomib citrate analog (Selleck, Cat. S2181) at concentrations of 20, 15, 10, 7.5, 5, 2.5, 1.25, 0.625, and 0.3125 (μM), followed by culture at 37°C for 72 hours. Cell viability for each cell line was evaluated using the MTT assay. For each cell line, the group without drug administration was set as 100%, and comparisons were made. In particular, the normal pancreatic duct-derived cell line HPNE-KRASG12D with a KRAS gene mutation and the normal pancreatic duct-derived cell line without a KRAS gene mutation (HPNE-KRAS-WT, HPDE-4) were created by introducing the KRAS mutation into the same cell line, allowing for a more rigorous evaluation of drug sensitivity.

[0052] As shown in Figure 5, it was revealed that ixazomib citrate analogs exhibit a concentration-dependent inhibitory effect on cell proliferation in various KRAS gene mutant cell lines. In particular, a significant inhibitory effect on cell viability was observed with ixazomib citrate analogs at concentrations of 0.315 μM or higher. The selective therapeutic effect on KRAS gene mutant cell lines at low concentrations is desirable from the standpoint of minimizing side effects.

[0053] (Effect confirmation 3: Induction rate of apoptosis by ixazomib citrate analog) To confirm that the ixazomib citrate analog of the present invention induces apoptosis in KRAS gene mutant cell lines, the apoptosis induction rate was also measured. 5 μM of the ixazomib citrate analog was added to normal pancreatic duct-derived cell lines without KRAS gene mutations (HPNE-KRAS-WT), normal pancreatic duct-derived cell lines with KRAS gene mutations (HPNE-KRASG12D), and pancreatic cancer cell lines with KRAS gene mutations (PANC-1, Panc04.03, PL8), and the cells were cultured at 37°C for 18 hours. The cell death (apoptosis) induction effect was analyzed by flow cytometry using a double staining method of PI (Propidium iodide) staining and Annexin-V-FITC staining.

[0054] Specifically, cells 2 x 10 5 The cells were adjusted to a cell / mL concentration, seeded in a 6-well plate, and cultured at 37°C for 12 hours. PI staining was performed using PI (SIGMA) suspended in PBS at a concentration of 100 μg / mL. Annexin-V-FITC (MBL) was also used. The percentage of apoptotic cell death was measured using a BD FACSCanto™ II flow cytometer (BD bioscience). The results are shown in Figure 6.

[0055] Figure 6 shows the flow cytometry results. The vertical axis represents the fluorescence intensity of PI, and the horizontal axis represents the fluorescence intensity of annexin V-FITC. Cells with high annexin V-FITC fluorescence and low PI fluorescence are cells in the early stages of apoptosis, while cells with high fluorescence values ​​for both annexin V-FITC and PI are cells in the late stages of apoptosis.

[0056] As is clear from Figure 6A, when ixazomib citrate analogs were added to HPNE-KRASG12D, PANC-1, Panc04.03, and PL8 cell lines, the number of apoptotic cells increased, demonstrating that ixazomib citrate analogs induce apoptosis in KRAS gene mutant cell lines. Figure 6B is a graph of the results from Figure 6A. As is clear from the graph in Figure 6B, it can be seen that the number of apoptotic cells increased when ixazomib citrate analogs were administered.

[0057] Furthermore, various cell lines were cultured for 24 hours after adding 5 μM of ixazomib citrate analog, and protein extracts were prepared. Ubiquitinated proteins and cleaved caspase 3 were detected by Western blotting. The results are shown in Figure 7. From Figure 7, ubiquitinated proteins were detected in cells administered with ixazomib citrate analog. Since ubiquitinated proteins are more easily recognized by the proteasome, it is thought that the amount of protein degraded by the proteasome increases when ixazomib citrate analog is administered. Also from Figure 7, it can be seen that cleaved caspase 3, which executes apoptosis, increased in cells administered with ixazomib citrate analog. From this, it is suggested that ixazomib citrate analog induces apoptosis in KRAS gene mutant cell lines.

[0058] (Effect Confirmation 4: Confirmation of tumor formation inhibitory effect and side effects in vivo) The extent of side effects of the therapeutic agent of the present invention was confirmed using a Xenograft model in which Panc04.03 (KRAS MUT pancreatic cancer cell line) was transplanted into immunodeficient mice (N = 5). Specifically, 0.5 × 10⁶ cells were transplanted into the back of 5-week-old nude female mice. 7 After subcutaneous transplantation of 1 Panc04.03 cells, the tumor volume increased to 100 mm². 3 After reaching [a certain threshold] (Day 0), ixazomib citrate analog was administered intraperitoneally at a dose of 7.5 mg / kg every two days for a total of seven doses (N = 5). The control group received phosphate-buffered saline with 10% DMSO.

[0059] Figure 8A shows photographs of mice after a total of seven administrations, and Figure 8B shows the results of measuring relative tumor volume. Relative tumor volume was calculated by measuring the longest and shortest diameters of the tumor with calipers, multiplying the longest diameter by the shortest diameter by the shortest diameter, dividing the volume by two, and then dividing by the mouse's volume at the time of treatment. The mouse's body weight was also measured, and the results are shown in Figure 8C. Significant weight loss in mice was considered indicative of a high degree of side effects.

[0060] Figure 8A shows that, even visually, the tumors in mice treated with the ixazomib citrate analog were smaller than those in the control group. This is also evident from Figure 8B. The administration of the ixazomib citrate analog significantly reduced tumor volume. Furthermore, Figure 8C shows that there was no increase or decrease in the mice's body weight, and it remained almost constant, suggesting that there were no side effects (weight loss) associated with the administration of the ixazomib citrate analog.

[0061] Furthermore, the presence or absence of side effects was also confirmed by the levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). This is based on the fact that many drugs are metabolized in the liver, and the liver itself can be damaged during this process. Severe drug-induced liver injury can lead to liver failure and can also cause brain damage. When liver damage occurs, AST and ALT leak into the bloodstream, so the risk of liver damage was confirmed by detecting these levels. Specifically, mice were administered ixazomib citrate analog at a dose of 7.5 mg / kg body weight intraperitoneally once every two days for a total of seven doses, then subjected to general anesthesia, and plasma was obtained.

[0062] Biochemical tests were requested from Fujifilm-Wako Pure Chemical Corporation to measure AST and ALT, which are liver damage markers. The results are shown in Figure 8D. Since the levels of AST and ALT did not significantly increase even after administration of the ixazomib citrate analog, it was concluded that liver damage was not a side effect.

[0063] This embodiment demonstrates that the therapeutic agent containing the ixazomib citrate analog of the present invention can effectively suppress the proliferation of RAS gene mutant cancer cells, particularly KRAS gene mutant cancer cells. Furthermore, in vivo results showed that the agent was safe, as it did not cause weight loss in subjects and did not exhibit serious side effects. Based on these results, the cancer therapeutic agent of the present invention can be expected to be an effective treatment for RAS gene mutant cancers.

Claims

1. (1) A therapeutic agent for RAS gene mutant cancer comprising an ixazomib citrate analog represented by the following formula (1). 【Chemistry 1】

2. The RAS gene mutant cancer treatment drug according to claim 1, wherein the RAS gene mutant cancer is a cancer containing a KRAS gene mutation.

3. The RAS gene mutant cancer treatment drug according to claim 1 or 2, wherein the RAS gene mutant cancer is one or more cancers selected from the group consisting of pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer, skin cancer, uterine cancer, thyroid cancer, and gastric cancer.

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