Treatment drugs for malignant mesothelioma

Abstract

To provide a treatment for malignant mesothelioma that can specifically kill cancer cells and has few side effects. [Solution] A therapeutic agent for malignant mesothelioma comprising cerurenin and / or its derivatives, or KN-93 and / or its derivatives, as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for malignant mesothelioma. Specifically, it relates to the application to malignant mesothelioma, which is a novel use of selenurin and / or its derivatives, or KN-93 and / or its derivatives.

Background Art

[0002] Malignant mesothelioma is a refractory malignant tumor derived from mesothelial cells. Mesothelium refers to the membranous tissue that covers the surface of the body cavities such as the thoracic cavity, pericardium, and abdominal cavity. Malignant mesothelioma is a malignant tumor that develops from mesothelial cells arranged in the membrane covering the thoracic cavity and the like. Malignant mesothelioma is classified into malignant pleural mesothelioma, malignant pericardial mesothelioma, malignant peritoneal mesothelioma, etc. according to the location where it occurs.

[0003] Although the mechanism of occurrence of malignant mesothelioma is still unclear in many aspects, it is known that asbestos is involved in its onset. Asbestos is easy to process and resistant to heat and chemicals, so it has been used in various fields, especially in construction materials, from the 1970s to the 1980s. At present, the use of asbestos is completely prohibited, but since malignant mesothelioma develops after a long latent period of 30 to 40 years from asbestos exposure, the number of patients with malignant mesothelioma in Japan is predicted to increase in the future. In fact, the number of deaths due to malignant mesothelioma was less than 1,000 in 2000, but it has been increasing steadily to less than 1,500 in 2019 (from the annual trend of the number of deaths due to mesothelioma in the Ministry of Health, Labour and Welfare). On the other hand, compared with about �5,000 cases of lung cancer and about 51,000 cases of colorectal cancer (both from the 2019 national registration of cancer data of the National Cancer Center, National Institutes of Biomedical Innovation, Health and Nutrition), which are the death cases of cancer in Japanese people, the number of cases of malignant mesothelioma is small. Therefore, it is positioned as a rare cancer, and due to its rarity, the systems for research, diagnosis and treatment, and the development of therapeutic agents are not well established. For example, even if a substance has been experimentally shown to have an effect on breast cancer or prostate cancer (see, for example, Patent Document 1 and Non-Patent Documents 1 to 3), nothing is known about its relationship with malignant mesothelioma.

[0004] Malignant mesothelioma rarely shows any noticeable symptoms, making it difficult to detect in its early stages. Diagnosis is often made when the disease has progressed significantly. The survival period after diagnosis is very short, typically 6-12 months. Treatment options for malignant mesothelioma vary depending on the stage of the disease at diagnosis, but generally include surgery, radiation therapy, and chemotherapy. Malignant pleural mesothelioma, in particular, is often discovered as a thickening or lump in the pleura, making chemotherapy a common treatment option. Combination therapy with cisplatin and pemetrexed is the standard chemotherapy regimen.

[0005] In malignant mesothelioma, NF2, p16, and BAP1 are known to be frequently mutated genes, and these genes are considered to play an extremely important role in the development of malignant mesothelioma. All of the aforementioned genes are tumor suppressor genes. It is thought that cancer develops primarily due to abnormalities in these tumor suppressor genes, with additional and complex genomic abnormalities occurring. For example, malignant mesothelioma patients are known to have a deletion in at least one gene selected from the group consisting of NF2, p16, and BAP1. More specifically, it has been found that approximately 40% of malignant mesothelioma patients have a BAP1 gene deletion, and approximately 40-60% have a double deletion of the NF2 / p16 gene. Alternatively, there are reports that as many as 60% or more of malignant mesothelioma patients have a BAP1 gene deletion or mutation. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2006-507306 [Non-patent literature]

[0007] [Non-Patent Document 1] Oskar Rokhlin, et al., “KN-93 inhibits androgen receptor activity and induces cell death irrespective of p53 and Akt status in prostate cancer” Cancer Biology & Therapy 9:3, 224-235; February 1, 2010 [Non-Patent Document 2] Xuesong Liu, et al., “Inhibition of the phosphatidylinositol 3-kinase / Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis” Mol Cancer Ther 2006;5(3). March 2006 [Non-Patent Document 3] Na Young Jeong, et al., “Fatty acid synthase inhibitor cerulenin inhibits topoisomerase I catalytic activity and augments SN-38-induced apoptosis” Apoptosis (2013) 18:226-237 [Overview of the project] [Problems that the invention aims to solve]

[0008] Malignant mesothelioma is known to frequently involve NF2, p16, and BAP1 gene deficiencies, with NF2 / p16 double deletion or BAP1 gene deficiency being particularly common. However, current treatments are uniform regardless of the type of gene mutation and are not always effective. For example, in surgical treatment for malignant mesothelioma, even if the lesion can be completely removed, a cure by surgical intervention alone is very difficult to achieve. Furthermore, because malignant pleural mesothelioma often involves lesions over a wide area, radiation therapy, which is used as a local treatment, cannot be expected to be sufficiently effective.

[0009] While chemotherapy is applied to many patients, the response rate for combination therapy with cisplatin and pemetrexed has been reported to be only about 40%, indicating limited effectiveness and difficulty in predicting its efficacy. Furthermore, despite the extremely short life expectancy after a diagnosis of malignant mesothelioma, it has become clear that even this standard chemotherapy treatment does not significantly extend survival time. Therefore, the development of more effective treatments has been needed.

[0010] Non-patent document 1 describes that the combination of the CamKII inhibitor KN-93 and doxorubicin is effective against prostate cancer. However, it is said that long-term administration of doxorubicin increases the likelihood of developing secondary cancers (cancers originating from cells different from those of the primary site). This is presumed to be because the DNA damaging effect of doxorubicin makes normal cells more susceptible to becoming cancerous. Therefore, even if the combination of KN-93 and doxorubicin is effective against prostate cancer, the administration of doxorubicin, even at low doses, presents the challenge of increasing the risk of developing new cancers. [Means for solving the problem]

[0011] To solve the above problems, the inventors focused on selenin and / or its derivatives or KN-93 and / or its derivatives and examined their effectiveness in detail. As a result, it was found that selenin and / or its derivatives or KN-93 and / or its derivatives have a therapeutic effect on malignant mesothelioma, and the present invention was completed. That is, the present invention has been made to solve at least a part of the above problems, and an embodiment of the present invention may include at least a part of the following configurations.

[0012] [1] By stratifying patients based on the presence or absence of specific gene deficiencies, As for the aforementioned specific gene deletion, (i) Patients with malignant mesothelioma who are deficient in the BAP1 gene and the NF2 gene, or (ii) Malignant mesothelioma patients with deficiencies in the BAP1 gene and the p16 gene for the treatment of KN-93 containing as an active ingredient a therapeutic agent for malignant mesothelioma. 2 Furthermore, patients with malignant mesothelioma (i) who lack the p16 gene, or Furthermore, it is used to treat patients with malignant mesothelioma who lack the NF2 gene (ii), [1] A drug for treating malignant mesothelioma. [3] The malignant mesothelioma is malignant pleural mesothelioma, 1] or [2] The therapeutic agent for malignant mesothelioma according to the above. [4] For intravenous administration, the therapeutic agent for malignant mesothelioma according to any one of [1] to 3 . [5] Administered in combination with an immune checkpoint inhibitor, the therapeutic agent for malignant mesothelioma according to any one of [1] to 4 .

Effects of the Invention

[0013] According to the therapeutic agent of the present invention, it has a cell growth inhibitory effect and a cell killing (apoptosis induction) effect, and can specifically kill cancer cells. It also has few side effects, and effective treatment of malignant mesothelioma is expected.

Brief Description of the Drawings

[0014] [Figure 1A] ​​Method for creating NF2 and / or p16 gene knockout strains and for confirming knockouts. [Figure 1B] Method for creating BAP1 gene-deficient strains and for verifying the deficiency. [Figure 2] The concentration-dependent inhibitory effect of cerurenin on cell proliferation in specific gene-deficient cell lines compared to conventional drugs (cisplatin or pemetrexed). [Figure 3] Cell proliferation inhibitory effects of cerurenin and conventional drugs (cisplatin or pemetrexed) on NF2 / p16 double knockout cells [Figure 4] Cell proliferation inhibitory effect of KN-93 and conventional drugs (cisplatin or pemetrexed) on BAP1-deficient cells [Figure 5] Concentration-dependent inhibitory effect of KN-93 on BAP1-deficient cell proliferation. [Figure 6] Inhibitory effect of FAS inhibitors on cell proliferation in specific gene-deficient strains [Figure 7] Inhibitory effect of CamKII inhibitors on cell proliferation in specific gene-deficient strains [Figure 8] Apoptosis induction effect of cerulenin in NF2 / p16 double knockout cells [Figure 9] Apoptosis induction effect of KN-93 in BAP1-deficient strains [Figure 10] Confirmation of the degree of side effects of cerurenin or KN-93 in mice bearing mesothelioma tumors. [Figure 11] Antitumor effects of cerurenin or KN-93 in mice bearing mesothelioma [Figure 12] Effect of cerurenin addition on reducing FAS protein expression in NF2 / p16 double knockout strains [Modes for carrying out the invention]

[0015] The present invention relates to a therapeutic agent for malignant mesothelioma. More specifically, the therapeutic agent of the present invention contains cerurenin and / or its derivatives, or KN-93 and / or its derivatives, as an active ingredient. In this specification, "contains as an active ingredient" means containing an effective amount of cerurenin and / or its derivatives, or KN-93 and / or its derivatives, for therapeutic purposes.

[0016] <Malignant mesothelioma> Malignant mesothelioma is a malignant tumor derived from mesothelial cells. Due to its small patient population, it is classified as a rare cancer, which has made it difficult to establish a research system. Furthermore, because malignant mesothelioma is difficult to treat and has an extremely poor prognosis, there has been a strong need for effective treatments. Recent molecular biological studies have revealed that in malignant mesothelioma, three tumor suppressor genes—neurofibroma type II (NF2), cyclin-dependent kinase inhibitor 2A (p16), and BAP1—frequently undergo genetic changes. In particular, BAP1 gene deficiency is found in approximately 40%, or even over 60%, of malignant mesothelioma patients, and NF2 / p16 gene double deletion is found in approximately 40-60%.

[0017] Thus, while NF2, p16, and BAP1 are known causative genes for malignant mesothelioma, all of these are tumor suppressor genes. Therefore, developing molecularly targeted therapies for malignant mesothelioma that directly target these genes is considered difficult. Generally, cancer treatment drugs are broadly classified into four types: anticancer drugs, molecularly targeted therapies, hormone therapy, and immunostimulants. For example, breast cancer and prostate cancer, which are among the most common cancers in Japan, are thought to be related to hormones, and hormone therapy is used in their treatment. Specifically, aromatase inhibitors are widely used as treatments for breast cancer, and androgen receptor inhibitors are widely used as treatments for prostate cancer, and some therapeutic results have been obtained. In lung cancer, it is known that mutations occur in the oncogenes ALK and EGFR, so molecularly targeted therapies targeting these genes have been developed. On the other hand, cisplatin and pemetrexed, which are used to treat malignant mesothelioma, are anticancer drugs and are said to have significant side effects because they affect not only cancer cells but also normal cells. Therefore, there is a need to develop drugs with fewer side effects and high therapeutic efficacy.

[0018] The inventors of this invention have found that cerurenin and / or its derivatives, or KN-93 and / or its derivatives, have a high therapeutic effect on malignant mesothelioma. Because of the confirmed high therapeutic effect, the therapeutic agent of this invention is useful for malignant mesothelioma in which at least one gene selected from the gene group consisting of NF2, p16, and BAP1 is deficient, and is also useful for the treatment of malignant pleural mesothelioma. In particular, cerurenin and / or its derivatives are useful for the treatment of NF2 / p16 gene double deletion type malignant mesothelioma, and KN-93 and / or its derivatives are useful for the treatment of BAP1 gene deletion type malignant mesothelioma. Note that NF2 / p16 gene double deletion type is also called NF2 and p16 gene deletion type, and is a type in which the NF2 and p16 genes are deficient.

[0019] <Methods for diagnosing malignant mesothelioma> The general diagnostic method for malignant tumors is pathological examination of tissue obtained by biopsy. Specifically, tumor tissue obtained by biopsy is subjected to HE staining or immunohistochemical staining (IHC) using formalin-fixed paraffin-embedded sections to make a definitive diagnosis. If the expression of mesothelial markers such as Calretinin, WT1, and D2-40 is confirmed, and the cellular dysplasia of normal mesothelium is extremely high, and it is confirmed that the tumor originates from the mesothelium, then all of these are diagnosed as malignant mesothelioma.

[0020] In the present invention, it is preferable to further stratify patients diagnosed with malignant mesothelioma by conventional tests based on the presence or absence of NF2, p16, and BAP1 through genomic diagnosis. Genomic diagnosis can be any method capable of detecting the three gene mutations, and examples include immunohistochemical staining to confirm the expression level of proteins encoded by the three genes, gene panel testing methods that can comprehensively examine many gene mutations from a patient at once using next-generation sequencing (NGS), and fluorescence in situ hybridization (FISH) to analyze the copy number of the three gene loci. If NF2 and / or p16 (MTAP) are negative, cerurenin and / or its derivatives are provided as therapeutic agents, and if BAP1 is negative, KN-93 and / or its derivatives are provided as therapeutic agents.

[0021] Immunohistochemical staining uses antibodies that specifically recognize proteins encoded by the three genes mentioned above to confirm the localization of antigenic substances within tissues. It can be performed in accordance with general immunohistochemical staining methods.

[0022] Gene panel testing is a method that uses a next-generation sequencer (a device that reads large amounts of genomic information at high speed) on cancer tissue collected through biopsy or surgery. Many genes can be examined simultaneously in a single test. Specifically, DNA is extracted from formalin-fixed, paraffin-embedded sections of tumor tissue, a library is created, and analysis is performed using a next-generation sequencer to check for mutations or genomic deletions in the tumor cell DNA sequence. While there are no particular limitations on the system capable of gene panel testing, it is preferable to use the NCC OncoPanel System (Sysmex), which takes into account the characteristics of cancer genome mutations in the Japanese population.

[0023] FISH is a method that allows for the direct visualization of chromosomes and genes in tissues and cells. It can be performed in accordance with general FISH procedures. Furthermore, by analyzing genomes extracted from paraffin-embedded sections using polymerase chain reaction (PCR) or next-generation sequencing (NGS) methods, gene mutations can also be identified.

[0024] <Cerulenin> The cerulenin and / or derivatives of the present invention are useful for the treatment of malignant mesothelioma. They are useful for the treatment of NF2 or p16 genotype malignant mesothelioma, and in particular for the treatment of NF2 / p16 double-deficient malignant mesothelioma.

[0025] The inventors of this invention confirmed that fatty acid synthase (FAS) is highly expressed in patients with NF2 and / or p16 gene-deficient malignant mesothelioma, and hypothesized that FAS-mediated signaling is related to cancer cell survival. Believing that FAS inhibitors have therapeutic effects, the inventors conducted a screening test using a compound library (a standard inhibitor kit provided by the Ministry of Education, Culture, Sports, Science and Technology's Grant-in-Aid for Scientific Research, Innovative Areas, Cancer Support, Chemotherapy Infrastructure Support Activity Group) and confirmed that NF2 and / or p16 gene-deficient malignant mesothelioma cells were more sensitive to cerurenin, thus discovering its potential as a therapeutic agent.

[0026] More specifically, when cerurenin is administered to NF2 and / or p16 gene-deficient malignant mesothelioma cells, the cell viability decreases and an apoptosis-inducing effect is achieved. Generally, cancer cells are considered to have a high viability rate and can proliferate abnormally or avoid cell death by having abnormalities in the apoptosis-inducing pathway. However, when cerurenin is administered to NF2 and / or p16 gene-deficient malignant mesothelioma cells, cell proliferation can be suppressed and an apoptosis-inducing effect is achieved. When the therapeutic agent containing cerurenin and / or its derivative as an active ingredient of the present invention is administered at a concentration of 10 μM to malignant mesothelioma cell lines or human mesothelial cells with knocked-out NF2 and / or p16 genes and evaluated by an MTT assay, the cell viability after 72 hours of culture at 37°C is preferably 60% or less, and more preferably 40% or less, compared to the untreated group.

[0027] Cerurenin is (2R,3S)-3-[(4E,7E)-1-oxo-4,7-nonadien-1-yl]-2-oxilancarboxamide and is a type of fatty acid synthase inhibitor. The mechanism by which cerurenin is effective in treating malignant mesothelioma, particularly NF2 / p16 gene double-deficient malignant mesothelioma, is not clear, but it is presumed that cerurenin acts on the FAS protein, which is highly expressed in malignant mesothelioma, to reduce fatty acid production, thereby altering the balance of energy metabolism and exhibiting apoptosis induction and cell proliferation inhibition.

[0028] In the present invention, a derivative of cerulenin may be included as an active ingredient. The epoxide structure of cerulenin is essential for FAS inhibitory activity, and it has been reported that the double bond in the side chain also contributes to the inhibitory activity. Therefore, substituents may be present as long as these basic frameworks are maintained. The number of substituents may be one or more. If there are two or more substituents, the substituents may be the same or different. Substituents include, for example, halogen atoms, hydroxyl groups, cyano groups, nitro groups, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C1-C7 alkoxy groups, amino groups, acyl groups, acyloxy groups, carboxyl groups, C7-C14 aralkyl groups, aromatic hydrocarbon groups, saturated heterocyclic groups, unsaturated heterocyclic groups, and the like. The therapeutic agent of the present invention is cerurenin and / or a derivative thereof, but cerurenin is preferred due to its availability. Cerenin and / or its derivatives may be synthesized according to art known in the art, or may be commercially available.

[0029] <kn-93> KN-93 and / or its derivatives in this invention are useful for the treatment of malignant mesothelioma. In particular, they are useful for the treatment of BAP1 gene-deficient malignant mesothelioma.

[0030] The inventors of this invention confirmed that CamKIID is highly expressed in patients with BAP1 gene-deficient malignant mesothelioma and hypothesized that signaling mediated by CamKIID is related to cancer cell survival. Believing that compounds that inhibit CamKIID have therapeutic effects, the inventors conducted a screening test using the aforementioned compound library and confirmed that KN-93 sensitivity is increased in BAP1 gene-deficient malignant mesothelioma, thus identifying its potential as a therapeutic agent.

[0031] More specifically, when KN-93 is administered to BAP1 gene-deficient malignant mesothelioma cells, it reduces cell viability and induces apoptosis. When a therapeutic agent containing KN-93 and / or its derivatives as an active ingredient of the present invention is administered at a concentration of 10 μM to a malignant mesothelioma cell line or human mesothelial cells with the BAP1 gene knocked out and evaluated by an MTT assay, the cell viability after 72 hours of culture at 37°C is preferably 80% or less, more preferably 60% or less, and even more preferably 40% or less compared to the untreated group.

[0032] CamKIID is a protein kinase (Ca) of the calcium calmodulin-dependent protein kinase (CamK). 2+ CamKII is a member of the calmodulin-dependent protein kinase (CKI) family and is an important intracellular phosphorylation enzyme that can cause heart failure and arrhythmias. Among them, CamKII is known to be involved in the pathogenesis of heart failure, and CamKII inhibitors such as KN-93 are considered effective in treating heart failure. It is not known to be used as a treatment for malignant mesothelioma. KN-93 is (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methyl]amino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide) and is a type of CamKII inhibitor. The mechanism by which KN-93 is effective in treating malignant mesothelioma, particularly BAP1 gene-deficient malignant mesothelioma, is not clear, but it is thought to be because KN-93 has an inhibitory effect on CamKII.

[0033] In the present invention, derivatives of KN-93 may be included as active ingredients. Substituents may be present as long as the basic skeleton of KN-93 is maintained. The number of substituents may be one or more. If there are two or more substituents, the substituents may be the same or different. Substituents include, for example, halogen atoms, hydroxyl groups, cyano groups, nitro groups, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C1-C6 alkoxy groups, amino groups, acyl groups, acyloxy groups, carboxyl groups, C7-C14 aralkyl groups, aromatic hydrocarbon groups, saturated heterocyclic groups, unsaturated heterocyclic groups, and the like. The therapeutic agent of the present invention is KN-93 and / or a derivative thereof, but KN-93 is preferred due to its availability. KN-93 and / or its derivatives may be synthesized according to art known in the art, or may be commercially available.

[0034] <Therapeutic preparations> In one embodiment of the present invention, a therapeutic agent containing cerurenin and / or its derivatives, or KN-93 and / or its derivatives, as an active ingredient may be formulated by adding other pharmaceutically acceptable components, such as carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, physiological saline, etc., to the extent that its efficacy is not compromised. When formulating, it can be prepared by commonly known methods.

[0035] The therapeutic agents or therapeutic formulations of the present invention are intended for administration to humans and non-human mammals in whom treatment of malignant mesothelioma 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 for administration.

[0036] The dosage form of the therapeutic agent or therapeutic preparation is not particularly limited, and examples include oral preparations (tablets, coated tablets, powders, granules, capsules, liquids, etc.), respiratory preparations, intraperitoneal preparations, intravenous preparations, injections, suppositories, patches, ointments, etc., but oral preparations, respiratory preparations, or intravenous preparations are preferred. In humans, intravenous preparations are preferred.

[0037] <Dosage and method of administration> The dosage of the therapeutic agent of the present invention can be appropriately adjusted considering the purpose of use, the target of administration, the target's sex, age, weight, the condition of the affected area, etc. From the viewpoint of achieving efficacy with minimal side effects, the dosage of cerurenin and / or its derivatives or KN-93 and / or its derivatives is preferably within the following ranges. Specifically, the dosage of cerurenin and / or its derivatives is preferably 10 to 40 mg / kg, more preferably 10 to 30 mg / kg, and more preferably 10 to 20 mg / kg, for 3 days, either as a single dose or in divided doses. The dosage of KN-93 and / or its derivatives is preferably 10 to 50 mg / kg, more preferably 10 to 40 mg / kg, and more preferably 10 to 30 mg / kg, for 3 days, either as a single dose or in divided doses. In either case, the administration interval when administered in divided doses is not particularly limited, but it is preferable to administer them at intervals of, for example, 2 to 5 days.

[0038] Examples of administration methods include oral administration, airway administration, and intravenous administration, but intravenous administration is preferred when administering to humans. Since intraperitoneal administration in animals is known to be the same route of administration as intravenous administration in humans, intraperitoneal administration is preferred in non-human mammals.

[0039] When administering the therapeutic agent of the present invention, it is preferable to administer it in combination with an immune checkpoint inhibitor. Since immune checkpoint inhibitors and the therapeutic agent of the present invention have different mechanisms of action, a synergistic effect on the treatment of malignant mesothelioma can be expected. Examples of immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. More specifically, examples of PD-1 inhibitors include nivolumab and pembrolizumab. More specifically, examples of PD-L1 inhibitors include avelumab and atezolizumab. More specifically, examples of CTLA-4 inhibitors include ipilimumab. It is preferable to use the present invention in combination with a PD-L1 inhibitor and / or a CTLA-4 inhibitor. More preferably, it is preferable to use the present invention in combination with nivolumab and / or ipilimumab. [Examples]

[0040] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to these examples. <cell> In the following examples, normal mesothelial cell lines, human mesothelial cell lines with the target gene knocked out, and malignant mesothelioma cell lines were used. Since malignant mesothelioma cell lines may also have deficiencies in genes other than the target gene, cell lines with only the target gene knocked out (human mesothelial cell lines) were also used. The relationship between the deficiency gene and the cell line name is shown in Table 1. Two inactivated human mesothelial cell lines, MeT-5A (pleural mesothelial) and HOMC-B1 (omental mesothelial), and eight malignant mesothelioma cell lines were used. All cell lines were treated and subcultured in RPMI-1640 (Wako) medium containing 10% fetal bovine serum (Sigma) and 1% penicillin / streptomycin (Wako) at 37°C under a 5% CO2 humidified atmosphere. Normal human mesothelial cell lines and malignant mesothelioma cell lines were provided by the Aichi Cancer Center.

[0041] Using the CRISPR / Cas9 system, NF2 knockout cells (NF2-KO#1 and #2), p16 knockout cells (p16-KO#1 and #2), NF2 / p16 double knockout cells (DKO#1 and #2), and BAP1 knockout cells (BAP1-KO#1 and #2) were created from the aforementioned human mesothelial cell line. Figure 1A shows the method for creating NF2 and / or p16 knockout cells and the results of confirming the deficiency, and Figure 1B shows the method for creating BAP1 knockout cells and the results of confirming the deficiency.

[0042] Single guide RNAs (sgRNAs) were selected using CRISPR design. The sgRNA sequence used for NF2 was 5'-AAACATCTCGTACAGTGACA-3', and the sequence used for p16 was 5'-A CCGTAACTATTCGGTGCGT-3', corresponding to exons 8 and 1, respectively. The sgRNA sequence used for BAP1 was 5'-TCAAATGGATCGAAGAGCGC-3', corresponding to exon 4. Plasmids expressing hCas9 and sgRNA were prepared by ligating oligonucleotides to the BbsI site of PX458 (NF2 / PX458 and p16 / PX458). Knockout cells were treated with 1 μg of NF2 / PX458 or p16 / PX458 plasmid using a 4D-Nucleofector system (Lonza Japan) at a rate of 1 × 10⁶ 6 The process was established by electroporation of individual cells. Three days after transfection, cells expressing green fluorescent protein were selected using BD FACSARIA III (BD bioscience) and used in the experiment. Western blotting was used to confirm gene deletion.

[0043] As shown in Figure 1A, in NF2 and / or p16 knockout cells, the single guide RNA sequence was designed for exon 8 of the NF2 gene (left) and exon 1 of the CDKN2A(p16) gene (right). Western blotting confirmed that the proteins encoded by NF2 and p16 were not expressed in the resulting double knockout cells (DKO). As shown in Figure 1B, in BAP1 knockout cells, the single guide RNA sequence was designed for exon 4 of the BAP1 gene. Western blotting confirmed that the proteins encoded by BAP1 were not expressed in the resulting knockout cells (BAP1-KO). GAPDH was used as an internal standard. Details of the cell lines used in subsequent experiments are shown in Table 1.

[0044] [Table 1]

[0045] <Cell viability measurement> Normal cells, knockout cells, and malignant mesothelioma cells 3 × 10⁻¹⁰ 3 Cells were seeded into 96-well cell culture plates. They were cultured at 37°C for 24 hours, and then cerurenin (Fujifilm WAKO, Cat. 031-18181) and KN-93 (Fujifilm WAKO, Cat. 115-00641) were administered at varying concentrations of 20, 15, 10, 7.5, 5, 2.5, 1.25, and 0.625 (μM), and cultured at 37°C for 72 hours. The cell viability of each cell line was evaluated using an MTT assay. For comparative examples, cisplatin (SIGMA, 479306) and pemetrexed (Selleck, S5971), currently used as treatments for malignant mesothelioma (also known as conventional drugs), were added at similarly varying concentrations. Each knockdown group and inhibitor-treated group was normalized based on the absorbance of the treatment group (with the group that did not receive the drug set as 100% for each cell line) and compared.

[0046] Figures 2 to 5 show the results of measuring cell viability and confirming whether the therapeutic agent is effective against malignant mesothelioma. Statistical significance was measured using Student's t-test. An asterisk (*) is used when there is a significant difference in viability between normal mesothelial cell lines and comparison cell lines. An asterisk indicates that the probability (P) is less than 0.05. As is clear from Figure 2, cerurenin suppressed cell viability in a dose-dependent manner. Compared to the administration of conventional drugs such as cisplatin and pemetrexed, cerurenin administration resulted in a significant decrease in viable cells. In particular, in NF2 / p16 double knockout strains, efficacy was observed with cerurenin administration at concentrations of 2.5 μM or higher. The fact that therapeutic effects were observed at low concentrations is desirable from the standpoint of side effects.

[0047] Figures 3 and 4 show cell viability after the addition of 10 μM of the drug. At a concentration of 10 μM, administration of cerurenin resulted in a significant decrease in viable cells, particularly in NF2 / p16 double-deficient cells, compared to administration of conventional drugs (see Figure 3). More specifically, administration of 10 μM cerurenin reduced the cell viability of NF2 / p16 double-deficient cells to below 60%. Administration of KN-93 resulted in a significant decrease in viable cells in BAP1-deficient cells compared to administration of conventional drugs (see Figure 4). Interestingly, the cell proliferation inhibitory effect of KN-93 was observed at least in BAP1-deficient cells, including BAP1 / NF2 double-deficient and BAP1 / p16 double-deficient cells. Administration of 10 μM KN-93 reduced the cell viability of BAP1-deficient cells to below 80%.

[0048] Figure 5 shows the relationship between KN-93 administration concentration and cell viability. In BAP1-deficient strains, administration of KN-93 at concentrations of 2.5 μM or higher resulted in a reduction in viable cells. The fact that therapeutic effects can be observed at low concentrations is desirable from the standpoint of minimizing side effects.

[0049] Figure 6 shows a comparison of cell viability with that of cells treated with 10 μM cerulenin and, as a comparative example, cells treated with 10 μM C75 (ALEXIS), a fatty acid synthase inhibitor similar to cerulenin. Similarly, Figure 7 shows the cell viability with 10 μM KN-93, KN-62 (WAKO), a CamKIID inhibitor similar to KN-93, and 10 μM Lavendustin C (Calbio Chem). The experimental method is the same as described above. Figure 6 shows that cerurenin addition resulted in a significantly greater reduction in viable cells compared to C75 addition. Although both cerurenin and C75 are lipid synthesis enzyme inhibitors, it is presumed that C75 does not have a viable cell reduction effect due to structural differences. Figure 7 shows that a particularly significant reduction in viable cells in BAP1-deficient strains was observed only in KN-93. This is likely due to structural differences.

[0050] <Measurement of Apoptosis Induction Rate> Mesothelial cells or mesothelioma cell lines were cultured at 37°C for 48 hours after adding 7.5 μM of cerulenin or KN-93. 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. Cells 2 x 10 5 The cells were adjusted to a cell / mL concentration, seeded in a 12-well plate, and incubated at 37°C for 48 hours. PI staining was performed using PI (SIGMA) suspended in PBS at a concentration of 100 μg / mL. Annexin-V-FITC (MBL) was used. BD FACSCanto TM II. The percentage of apoptotic cell death was measured using a flow cytometer (BD bioscience). The results are shown in Figures 8 and 9.

[0051] Figures 8A and 9A show the results of flow cytometry. The vertical axis shows the fluorescence intensity of PI, and the horizontal axis shows 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 in both annexin V-FITC and PI are cells in the late stages of apoptosis. As is clear from Figure 8A, adding cerulenin to the NF2 / p16 double knockout cell line increases cells, particularly those in the late apoptotic stage. Similarly, as shown in Figure 9A, adding KN-93 to the BAP1 knockout cell line increases cells in both the early and late apoptotic stages. Figure 8B is a graph created based on the results of Figure 8A, showing the percentage of cells that underwent apoptosis. Figure 8B also clearly shows that cerulenin enhances the apoptosis-inducing effect in the NF2 / p16 double knockout cell line. Similarly, Figure 9B shows that KN-93 enhances the apoptosis-inducing effect in the BAP1 knockout cell line.

[0052] <Survey to confirm the degree of side effects> The degree of side effects of the therapeutic agent of the present invention was confirmed using a Xenograft model in which MSTO-211H (p16-deficient malignant mesothelioma cell line) and Y-MESO-9 (p16 / BAP1-deficient malignant mesothelioma cell line) were transplanted into immunodeficient mice (n=6). A large amount of weight loss in the mice was considered to indicate a large side effect. After transplanting cancer cells into the mice, once the tumor volume reached 100 mm3 (Day-0), mice transplanted with MSTO-211H were administered cerurenin at a dose of 20 mg / kg every 3 days for a total of 5 doses. Mice transplanted with Y-MESO-9 were administered KN-93 at a dose of 30 mg / kg twice a week for 21 days (a total of 6 doses). The weight of the mice was measured every 3 days, and the average value of the 6 individuals was calculated. The results are shown in Figure 10. In all cases, there was no increase or decrease in the mice's body weight, and it remained at a nearly constant value, so we believe that no side effects (weight loss) were observed.

[0053] <Tumor suppression effect confirmation test> For the mice whose weight was measured as described above, the transplanted tumor site was incised, the tumor was removed, and its volume was measured. The longest and shortest diameters of the tumor were measured with calipers, and the volume was calculated by multiplying the longest diameter by the shortest diameter and dividing the shortest diameter by two, and then divided by the volume of the mouse at the time of treatment. The results are shown in Figure 11. A reduction in tumor volume was observed with administration of cerurenin (Figure 11A) and KN-93 (Figure 11B). As is clear from Figure 11A, cerurenin also showed a tumor-suppressing effect in the p16-only deficient strain (MSTO-211H).

[0054] <Confirmation of effectiveness against malignant tumor-causing proteins> Western blotting was used to examine the increase or decrease in FAS protein (FASN), CD24, and cleaved PARP (c-PARP) levels in normal human mesothelial cell lines (parent) and NF2 / p16 double knockout cells (DKO) upon addition of cerurenin. Parent and DKO cells were cultured for 24 hours after adding 7.5 μM of cerurenin, and then protein extracts were prepared. Antibodies specifically recognizing FAS protein (FASN), CD24, and cleaved PARP (c-PARP) were used. The results are shown in Figure 12. In the group not treated with cerurenin, increases in FASN and CD24 were observed in the DKO cell line. These expression levels decreased upon cerurenin administration. CD24 is a glycosylphosphatidylinositol-binding sialoprotein, and recent studies have shown it to be highly expressed in NF2 / p16 double knockout cells; therefore, it was used as a target for malignant mesothelioma development. The decrease in CD24 levels following cerurenin administration suggests that cerurenin has an inhibitory effect on cell proliferation in malignant mesothelioma, particularly NF2 / p16 double-deficient malignant mesothelioma. Furthermore, cerurenin administration increased cleaved PARP, an apoptosis marker, suggesting that cerurenin induces apoptosis. [Industrial applicability]

[0055] The therapeutic agent of the present invention can effectively treat malignant mesothelioma. It has cell proliferation inhibitory and cytotoxic (apoptosis induction) effects, and can specifically kill cancer cells, resulting in fewer side effects.

Claims

1. Stratifying patients based on the presence or absence of a specific gene defect, As for the aforementioned specific gene deletion, (i) Patients with malignant mesothelioma who are deficient in the BAP1 gene and the NF2 gene, or (ii) Used in the treatment of malignant mesothelioma patients who are deficient in the BAP1 gene and the p16 gene, Contains KN-93 as the active ingredient. A drug used to treat malignant mesothelioma.

2. A malignant mesothelioma patient (i) who is further deficient in the p16 gene, or Furthermore, it is used to treat patients with malignant mesothelioma who have a deficiency in the NF2 gene (ii), The therapeutic agent for malignant mesothelioma according to claim 1.

3. The therapeutic agent for malignant mesothelioma according to claim 1 or 2, wherein the malignant mesothelioma is malignant pleural mesothelioma.

4. A therapeutic agent for malignant mesothelioma according to any one of claims 1 to 3, for intravenous administration.

5. A therapeutic agent for malignant mesothelioma according to any one of claims 1 to 4, administered in combination with an immune checkpoint inhibitor.

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