Treatments for malignant mesothelioma and selection methods for malignant mesothelioma patients

Abstract

The present invention addresses the issue of providing a malignant mesothelioma therapeutic agent and a method for selecting malignant mesothelioma patients. The issue can be solved by a malignant mesothelioma therapeutic agent that includes, as an effective component thereof, a compound that targets the oxytocin receptor.

Description

Technical Field

[0001] The disclosure in the present application relates to a therapeutic agent for malignant mesothelioma and a method for selecting malignant mesothelioma patients.

Background Art

[0002] Malignant mesothelioma is a tumor that develops from the mesothelium covering the surfaces of the pleura, pericardium, and peritoneum. More than 80% of malignant mesotheliomas occur in the pleura, but both malignant pleural mesothelioma and malignant peritoneal mesothelioma have a poor prognosis.

[0003] Many malignant mesotheliomas that occur in the pleura and peritoneum are known to be caused by asbestos exposure and have a long period of 30 to 40 years until onset. Therefore, the incidence of malignant mesothelioma will continue to increase in the future.

[0004] The growth pattern of malignant mesothelioma is either localized or diffuse. Most malignant mesotheliomas have a diffuse growth pattern and infiltrate in a disseminated manner along the pleura, peritoneum, etc. Surgical therapy, radiation therapy, chemotherapy, etc. are being carried out, but at present, no effective treatment method has been established and the prognosis is very poor. Therefore, the development of therapeutic agents and treatment methods for malignant mesothelioma as described in Patent Document 1 and Patent Document 2 is being actively carried out.

Prior Art Documents

Patent Documents

[0005] [[ID=XXX]] [[ID=XXX]]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] As described in Patent Documents 1 and 2, new therapeutic agents and treatments have been reported. However, since no established therapeutic agents or treatments for malignant mesothelioma have yet been established, the development of new therapeutic agents and treatments is desired. Disclosure in this application is the result of diligent research into the development of new therapeutic agents for malignant mesothelioma, in which we have newly discovered that (1) compounds targeting oxytocin receptors have the effect of suppressing the proliferation of malignant mesothelioma, and (2) there are malignant mesotheliomas that express a large amount of oxytocin receptors.

[0007] In other words, the purpose of the disclosure of this application is to provide a therapeutic agent for malignant mesothelioma and a method for selecting malignant mesothelioma patients who express a large amount of oxytocin receptors. [Means for solving the problem]

[0008] (1) A therapeutic agent for malignant mesothelioma, The therapeutic agent contains a compound that targets oxytocin receptors as its active ingredient. A therapeutic agent. (2) The compound is an oxytocin receptor inhibitor. The therapeutic agent described in (1) above. (3) The oxytocin receptor inhibitor is at least one selected from the group consisting of crigosiban, OT-R antagonist 1, L368,899 hydrochloride, and atosiban. The therapeutic agent described in (2) above. (4) The oxytocin receptor inhibitor is letociban. The therapeutic agent described in (2) above. (5) The compound is a nucleic acid. The therapeutic agent described in (1) above. (6) The nucleic acid is either siRNA or shRNA. The therapeutic agent described in (5) above. (7) In addition, including anticancer drugs, A therapeutic agent listed in any one of the above (1) to (6). (8) The anticancer drug is cisplatin. The therapeutic agent described in (7) above. (9) The anticancer agents are cisplatin and pemetrexed. The therapeutic agent according to (7) above. (10) A method for selecting a malignant mesothelioma patient, The selection method is as follows: A measurement step of measuring the expression level of oxytocin receptor in malignant mesothelioma tissue; A determination step of determining whether the expression level of oxytocin receptor is above a threshold value; A selection step of selecting a malignant mesothelioma patient with an expression level of oxytocin receptor above the threshold value; including A method for selecting a malignant mesothelioma patient.

Effect of the Invention

[0009] The therapeutic agent for malignant mesothelioma disclosed in this application can suppress the growth of malignant mesothelioma.

Brief Description of the Drawings

[0010] [Figure 1] A diagram showing the results of inhibiting the growth of malignant mesothelioma cells by cligosiban using a xenograft model. [Figure 2] A: A diagram showing the expression level of oxytocin receptor in malignant mesothelioma tissues of 87 malignant mesothelioma patients. B: A diagram showing the Kaplan-Meier curves for the overall survival periods of the high-expression group, moderate-expression group, and low-expression group of oxytocin receptor. [Figure 3] A diagram showing the results of the WST-1 assay of malignant mesothelioma cells administered with cligosiban in vitro. [Figure 4] A diagram showing the results of the WST-1 assay of malignant mesothelioma cells administered with an oxytocin receptor inhibitor. [Figure 5] A diagram showing the results of the WST-1 assay of malignant mesothelioma cells with knocked-down oxytocin receptor. [Figure 6] A diagram showing the results of the colony formation assay of malignant mesothelioma cells with knocked-down oxytocin receptor. [Figure 7]Figure showing the results of suppressing the growth of malignant mesothelioma cells with knockdown of the oxytocin receptor using a xenograft model. [Figure 8] Figure showing the results of the WST-1 assay of malignant mesothelioma cells administered with a compound targeting the oxytocin receptor and an anticancer agent. [Figure 9] Figure showing the results of suppressing the growth of malignant mesothelioma cells using an existing standard therapeutic agent for malignant mesothelioma, crizotinib, and in combination with an existing standard therapeutic agent and crizotinib in a xenograft model.

Mode for Carrying Out the Invention

[0011] (Embodiment of the Therapeutic Agent for Malignant Mesothelioma) Hereinafter, a therapeutic agent for malignant mesothelioma according to an embodiment (hereinafter, may be simply referred to as "therapeutic agent") will be described.

[0012] The therapeutic agent according to the embodiment is characterized by containing a compound targeting the oxytocin receptor as an active ingredient.

[0013] The compound is not particularly limited as long as it targets the oxytocin receptor. In this specification, the "compound targeting the oxytocin receptor" includes a compound that binds to the oxytocin receptor as a target and a compound that knocks down the oxytocin receptor as a target. Examples of the compound targeting the oxytocin receptor include an oxytocin receptor inhibitor, nucleic acids such as siRNA and shRNA that knock down the oxytocin receptor, and antibodies against the oxytocin receptor.

[0014] As shown in the examples described later, the inventors have conducted intensive research and confirmed the effect of suppressing the growth of malignant mesothelioma cells when crizotinib was administered to a xenograft model in which malignant mesothelioma cells were transplanted into nude mice.

[0015] Cligosiban is an oxytocin receptor inhibitor used for premature ejaculation in men, and is a compound represented by the following chemical formula (1). It is also known that the oxytocin receptors on which cligosiban acts are mainly expressed in the mammary glands and the uterus in late pregnancy.

[0016] [ka]

[0017] Oxytocin is a peptide hormone secreted from the posterior pituitary gland, consisting of nine amino acids. It plays a role in contracting muscle fibers in the mammary glands to secrete milk, and in contracting smooth muscles, which causes uterine contractions during childbirth. Although oxytocin was discovered as a hormone unique to women, it is known to be secreted in men at certain levels as well.

[0018] Since cligosiban inhibited the proliferation of malignant mesothelioma cells, the inventors focused on oxytocin receptor inhibitors and conducted in vitro tests on oxytocin receptor inhibitors other than cligosiban, as shown in the examples described below. The oxytocin receptor inhibitors used were OT-R antagonist 1 (chemical formula (2)), L368,899 hydrochloride (chemical formula (3)), and atosiban (chemical formula (4)), represented by the following chemical formulas (2) to (4). As a result, it was confirmed that they inhibited the proliferation of malignant mesothelioma cells.

[0019] [ka]

[0020] Furthermore, letosiban, represented by the following chemical formula (5), and OT-R antagonist 2, represented by the chemical formula (6), are also known as oxytocin receptor inhibitors. Therefore, letosiban and OT-R antagonist 2 can also be expected to suppress the proliferation of malignant mesothelioma.

[0021] [ka]

[0022] Nucleic acids such as siRNA and shRNA that knock down the oxytocin receptor have been confirmed to suppress the proliferation of malignant mesothelioma cells, as shown in the examples described later. Furthermore, there are no particular restrictions on the nucleic acids used, as long as they can suppress the expression of the oxytocin receptor. Suppressing the expression of the oxytocin receptor is expected to suppress the proliferation of malignant mesothelioma.

[0023] Antibodies against oxytocin receptors may be polyclonal or monoclonal antibodies. They may also be complete antibody molecules or antibody fragments that can specifically bind to oxytocin receptors. Antibodies against oxytocin receptors can be produced by known methods. Antibodies against oxytocin receptors are expected to suppress the proliferation of malignant mesothelioma, similar to oxytocin receptor inhibitors and siRNAs and shRNAs that knock down oxytocin receptors.

[0024] Therefore, compounds that target oxytocin receptors can be used as therapeutic agents for malignant mesothelioma. Furthermore, compounds that target oxytocin receptors may be used individually as therapeutic agents, or multiple types may be used in combination as therapeutic agents.

[0025] The therapeutic agent according to this embodiment may include an anticancer agent in addition to a compound that targets oxytocin receptors. The combination of the oxytocin receptor-targeting compound and the anticancer agent can synergistically enhance the effect of suppressing the proliferation of malignant mesothelioma cells. Examples of anticancer agents that may be added include cytotoxic agents, angiogenesis inhibitors, and immune checkpoint inhibitors.

[0026] Cytotoxic drugs are medications that kill cancer cells, induce cell death, or reduce the proliferation / survival rate of cells. Examples of cytotoxic drugs include alkylating agents, platinum-based drugs, antimetabolites, antitumor antibiotics, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, topoisomerase inhibitors, plant alkaloids, hormones, and bacterial toxins. Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, thiotepa, procarbazine, and ranimustine. Examples of platinum-based drugs include cisplatin, nedaplatin, oxaliplatin, and carboplatin. Examples of antimetabolites include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphonate, nerarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, and mercaptopurine. Examples of antitumor antibiotics include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and dinostatin stimalamer. Examples of microtubule polymerization inhibitors include vinblastine, vincristine, vinorelbine, and vindesine. Examples of microtubule depolymerization inhibitors include paclitaxel and docetaxel. Examples of topoisomerase inhibitors include irinotecan, nogitecan, etoposide, and sobuzoxane. In addition, meitasinoids and meitasinoid analogs, such as emtansine (DM-1), which is used in ADCs (anticoagulants) for cancer, are also preferred cytotoxic drugs.

[0027] Angiogenesis inhibitors are drugs that target vascular endothelial growth factor, inhibiting the supply of nutrients to cancer cells and suppressing their proliferation. Examples of angiogenesis inhibitors include bevacizumab, ramucirumab, and aflibercept.

[0028] Immune checkpoint inhibitors are drugs that enhance the immune response to tumors by binding to inhibitory receptors or their ligands, which are immune checkpoint molecules, and blocking inhibitory signaling, thereby releasing the brakes on the immune system. Examples of immune checkpoint inhibitors include the anti-PD1 antibodies nivolumab and pembrolizumab, the anti-PD-L1 antibodies atezolizumab and durvalumab, and the anti-CTLA4 antibody ipilimumab.

[0029] Compounds targeting oxytocin receptors and anticancer drugs may be used simultaneously or with a time difference. Administration schedules can also be set independently for both, and each can be administered to the target according to these schedules. Furthermore, the number of administrations for both can be arbitrarily set, and single or multiple administrations are possible. Additionally, the anticancer drug used in combination with the compound targeting oxytocin receptors may be a single type or a combination of two or more types.

[0030] The method of administering the therapeutic agent according to the embodiment is not particularly limited as long as it is effective against malignant mesothelioma. Examples of administration methods include oral, transdermal, intravenous, intramuscular, intrathoracic, intraperitoneal, and transrectal routes.

[0031] Examples of dosage forms of therapeutic agents according to the embodiment include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), ointments, gelatin soft and hard capsules, suppositories, sterile injection solutions, sterile encapsulated powders, and the like.

[0032] Furthermore, the therapeutic agent according to the embodiment may include conventionally used additives as needed. Examples of additives include, but are not limited to, existing additives such as excipients, binders, lubricants, disintegrants, flavoring and deodorizing agents, solvents, stabilizers, bases, wetting agents, and preservatives.

[0033] The therapeutic agent according to this embodiment provides the following effects. (1) Therapeutic agents containing compounds that target oxytocin receptors as active ingredients suppress the proliferation of malignant mesothelioma cells. (2) The addition of an anticancer drug to the treatment synergistically suppresses the proliferation of malignant mesothelioma cells.

[0034] (Implementation of a method for selecting patients with malignant mesothelioma) The following describes the method for selecting malignant mesothelioma patients according to the embodiment.

[0035] The method for selecting malignant mesothelioma patients according to this embodiment includes (1) a measurement step of measuring the expression level of oxytocin receptors in malignant mesothelioma tissue, (2) a determination step of determining whether the expression level of oxytocin receptors is above a threshold, and (3) a selection step of selecting malignant mesothelioma patients whose expression level of oxytocin receptors is above a threshold. The selection method is performed in the order of (1), (2), and (3).

[0036] The method for measuring oxytocin receptor expression levels in malignant mesothelioma tissue during the measurement process is not particularly limited, as long as the oxytocin receptor expression level can be measured. Examples of methods for measuring oxytocin receptor expression levels include PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR), Northern blotting, microarrays, DNA chips, RNA chips, etc.

[0037] The determination step determines whether the expression level of oxytocin receptors measured in the measurement step is above a threshold. The threshold in the determination step may be set as appropriate.

[0038] The selection process involves selecting patients with malignant mesothelioma tissue whose oxytocin receptor expression level is above a threshold, based on the determination process.

[0039] The method for selecting malignant mesothelioma patients according to this embodiment has the following effects. (1) It is possible to select malignant mesothelioma patients in whom oxytocin receptors are highly expressed in malignant mesothelioma tissue. Therefore, companion diagnostics can be performed for therapeutic agents containing compounds that target oxytocin receptors as active ingredients.

[0040] The embodiments disclosed in this application are described below in detail by illustrating the embodiments, but these embodiments are solely for illustrative purposes and are not intended to limit or restrict the scope of the invention disclosed in this application. [Examples]

[0041] [Inhibition of malignant mesothelioma cell proliferation by crigosiban using a xenograft model] (Example 1) We investigated whether crigosiban inhibits the proliferation of malignant mesothelioma cells in vivo. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2052 (purchased from ATCC), 5.0 × 10 6 The drug was administered subcutaneously to the left rump of nude mice (BALB / c nude (nu / nu) female, 6-8 weeks old: purchased from Charles River Japan). (2) Three days after subcutaneous administration, crigosiban (purchased from MedChemExpress) was administered orally at a dose of 60 mg / kg every other day for a total of 10 doses. (3) One week after the 10th oral administration, nude mice were dissected and the weight of the subcutaneous tumors was measured.

[0042] (Comparative Example 1) The procedure was the same as in Example 1, except that crigosiban was not administered.

[0043] Figure 1 shows the results. Note that Example 1 was conducted using four nude mice. Therefore, each nude mouse was designated as Example 1-1 to Example 1-4. The same applies to Comparative Example 1. Figure 1 shows that the weight of the subcutaneous tumors in Example 1, where cligosiban was administered, was smaller than that of Comparative Example 1, where cligosiban was not administered. Furthermore, Example 1 showed a statistically significant suppression of subcutaneous tumor growth compared to Comparative Example 1. Therefore, it was shown that cligosiban, which targets the oxytocin receptor, can be used as a therapeutic agent for malignant mesothelioma.

[0044] [Analysis of oxytocin receptor expression in patients with malignant mesothelioma] (Example 2) The results of Example 1 showed that the proliferation of malignant mesothelioma cells was suppressed by the oxytocin receptor inhibitor, cligosiban. Therefore, we analyzed the expression of oxytocin receptors in malignant mesothelioma.

[0045] We analyzed 87 cases of malignant mesothelioma using a database. The database used was The Cancer Genome Atlas (TCGA; https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga), which comprehensively analyzes genomic and methylation abnormalities, as well as gene and protein expression abnormalities.

[0046] Figure 2A shows a graph of oxytocin receptor expression levels in malignant mesothelioma tissue from 87 patients with malignant mesothelioma. Approximately 40% of the 87 patients showed expression levels more than 1000 times higher than those with low expression. Figure 2B shows Kaplan-Meier curves for overall survival in the high, medium, and low oxytocin receptor expression groups. Figure 2B indicates that patients with high oxytocin receptor expression tended to have a worse prognosis. Therefore, it was suggested that malignant mesothelioma with a poor prognosis exhibits high oxytocin receptor expression.

[0047] [In vitro inhibition of malignant mesothelioma cell proliferation by crigosiban] (Example 3) Next, we investigated whether the oxytocin receptor inhibitor cligosiban suppresses the proliferation of malignant mesothelioma cells in vitro. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2052 was seeded in a dish and cultured for 1 day. (2) Cligosiban was administered to dishes at concentrations of 0, 5, 10, 15, 20, or 25 μM. (3) After culturing for 5 days, the cells were evaluated using the WST-1 assay. The WST-1 assay is an assay method that evaluates cell proliferation by colorimetric method after administering the cell proliferation test drug WST-1 (product number: 11644807001, purchased from Roche).

[0048] (Example 4) The procedure was the same as in Example 3, except that the malignant mesothelioma cell line was replaced with NCI-2373 (purchased from ATCC), and cligosiban was administered to dishes at concentrations of 0, 20, 30, 40, 50, or 60 μM.

[0049] Figure 3 shows the results of the WST-1 assay for Examples 3 and 4. Figure 3 shows that in both Examples 3 and 4, the proliferation of malignant mesothelioma cells was suppressed as the concentration of cligosiban increased. Therefore, it was demonstrated that cligosiban suppresses the proliferation of malignant mesothelioma cells both in vivo and in vitro.

[0050] [Inhibition of malignant mesothelioma cell proliferation by oxytocin receptor inhibitors other than crigosiban] Since crigosiban, whose effectiveness was confirmed in mice, was also found to be useful in two types of malignant mesothelioma cells in vitro, we investigated whether other oxytocin receptor inhibitors besides crigosiban could suppress the proliferation of malignant mesothelioma cells using an in vitro experimental system. (Example 5) The procedure was the same as in Example 3, except that the oxytocin receptor inhibitor was replaced with OT-R antagonist 1 (purchased from MedChemExpress) and administered to the dish at concentrations of 0, 20, 40, 60, or 80 μM.

[0051] (Example 6) The procedure was the same as in Example 4, except that the oxytocin receptor inhibitor was replaced with OT-R antagonist 1 and administered to the dish at concentrations of 0, 20, 40, 60, or 80 μM.

[0052] (Example 7) The procedure was the same as in Example 5, except that the oxytocin receptor inhibitor was replaced with L368,899 hydrochloride (purchased from MedChemExpress).

[0053] (Example 8) The procedure was the same as in Example 6, except that the oxytocin receptor inhibitor was replaced with L368,899 hydrochloride.

[0054] (Example 9) The procedure was the same as in Example 5, except that the oxytocin receptor inhibitor was replaced with atosiban (purchased from MedChemExpress).

[0055] (Example 10) The procedure was the same as in Example 6, except that the oxytocin receptor inhibitor was replaced with atosiban.

[0056] Figure 4 shows the results of the WST-1 assay for Examples 5 to 10. Figure 4 demonstrates that in all of Examples 5 to 10, the proliferation of malignant mesothelioma cells was suppressed as the concentration of the oxytocin receptor inhibitor increased. Therefore, it was shown that oxytocin receptor inhibitors can be used as therapeutic agents for malignant mesothelioma.

[0057] [Inhibition of malignant mesothelioma cell proliferation by knockdown of oxytocin receptors (1)] We investigated whether siRNA, a compound that targets oxytocin receptors, suppresses the proliferation of malignant mesothelioma cells. (Example 11) The proliferation of malignant mesothelioma cells in which the oxytocin receptor was knocked down by siRNA was evaluated using the WST-1 assay. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2373 was seeded in a dish and cultured for 1 day. (2) The oxytocin receptor was knocked down using siRNA1 (product number s9947, purchased from Thermo Fisher Scientific). (3) After culturing for 4 days, evaluation was performed using the WST-1 assay.

[0058] (Example 12) The procedure was the same as in Example 11, except that siRNA1 was replaced with siRNA2 (product number s9948, purchased from Thermo Fisher Scientific).

[0059] (Example 13) The procedure was the same as in Example 11, except that the malignant mesothelioma cell line was replaced with NCI-H2052.

[0060] (Example 14) The procedure was the same as in Example 12, except that the malignant mesothelioma cell line was replaced with NCI-H2052.

[0061] (Comparative Example 2) The procedure was the same as in Example 11, except that (1) the malignant mesothelioma cell line was replaced with the immortalized normal mesothelial cell line MeT-5A (purchased from ATCC), and (2) the oxytocin receptor was not knocked down.

[0062] (Comparative Example 3) The procedure was the same as in Comparative Example 2, except that the oxytocin receptor was knocked down using siRNA1.

[0063] (Comparative Example 4) The procedure was the same as in Comparative Example 2, except that the oxytocin receptor was knocked down using siRNA2.

[0064] (Comparative Example 5) The procedure was the same as in Example 11, except that the oxytocin receptor was not knocked down.

[0065] (Comparative Example 6) The procedure was the same as in Example 13, except that the oxytocin receptor was not knocked down.

[0066] Figure 5 shows the results of the WST-1 assay for Examples 11-14 and Comparative Examples 2-6. Figure 5 shows that Examples 11-14, in which the oxytocin receptor of malignant mesothelioma cells was knocked down, suppressed the proliferation of malignant mesothelioma cells more effectively than Comparative Examples 5 and 6, in which the oxytocin receptor of malignant mesothelioma cells was not knocked down. Furthermore, Figure 5 shows that in normal mesothelial cell lines, Comparative Examples 3 and 4, in which the oxytocin receptor was knocked down, did not show a significant difference in proliferation suppression compared to Comparative Example 2, in which the oxytocin receptor was not knocked down. Therefore, it was found that siRNA targeting the oxytocin receptor is useful for suppressing the proliferation of malignant mesothelioma cells that express the oxytocin receptor. Thus, it was demonstrated that siRNA targeting the oxytocin receptor can be used as a therapeutic agent for malignant mesothelioma.

[0067] [Inhibition of malignant mesothelioma cell proliferation by knockdown of oxytocin receptors (2)] (Example 15) The proliferation of malignant mesothelioma cells in which the oxytocin receptor was knocked down by siRNA was evaluated using a colony formation assay. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2373 was seeded in a dish and cultured for 1 day. (2) The oxytocin receptor was knocked down using siRNA1. (3) After 48 hours, NCI-H2373 with the oxytocin receptor knocked down was spread onto a new dish. (4) After culturing for two weeks, the colonies were evaluated using a colony formation assay, which counts the number of colonies formed.

[0068] (Example 16) The procedure was carried out in the same manner as in Example 15, except that siRNA1 was replaced with siRNA2.

[0069] (Example 17) The procedure was the same as in Example 15, except that the malignant mesothelioma cell line was replaced with NCI-H2052.

[0070] (Example 18) The procedure was the same as in Example 16, except that the malignant mesothelioma cell line was replaced with NCI-H2052.

[0071] (Comparative Example 7) The procedure was the same as in Example 15, except that the oxytocin receptor was not knocked down.

[0072] (Comparative Example 8) The procedure was the same as in Example 17, except that the oxytocin receptor was not knocked down.

[0073] Figures 6A and 6B show the results of the colony formation assays for Examples 15-18, Comparative Example 7, and Comparative Example 8. From Figures 6A and 6B, it was shown that Examples 15-18, in which the oxytocin receptor of malignant mesothelioma cells was knocked down, inhibited colony formation compared to Comparative Examples 7 and 8, in which the oxytocin receptor of malignant mesothelioma cells was not knocked down. Therefore, Examples 15-18 were shown to suppress the proliferation of malignant mesothelioma cells, similar to Examples 11-14.

[0074] [Inhibition of malignant mesothelioma cell proliferation by knockdown of oxytocin receptor using a xenograft model] (Example 19) We investigated whether shRNAs that constitutively knock down the oxytocin receptor in vivo suppress the proliferation of malignant mesothelioma cells. The procedure is as follows. (1) Lentiviruses were generated by transfecting 293FT cell lines (purchased from Thermo Fisher) with three plasmids: pMD2.G (Plasmid#12259, purchased from addgene), psPAX2 (Plasmid#12260, purchased from addgene), and pLKO.1 puro with shRNA construct (Plasmid#10878, purchased from Addgene), which incorporates a sequence to knock down the oxytocin receptor. (2) Malignant mesothelioma cell line NCI-H2373 was seeded in a dish and cultured for 1 day. (3) Using the lentivirus prepared in (1) above, shRNA was introduced into NCI-H2373 to constitutively knock down the oxytocin receptor. (4) NCI-H2373 cells with the oxytocin receptor knocked down were cultured. (5) NCI-H2373 with cultured oxytocin receptor knocked down, 1.5 × 10 6 The drug was administered subcutaneously to the left rump of nude mice (BALB / c nude (nu / nu) female, 6 weeks old: purchased from Charles River Japan). (6) Nude mice were dissected one month after subcutaneous administration and the weight of the subcutaneous tumors was measured.

[0075] (Example 20) The procedure was the same as in Example 19, except that the malignant mesothelioma cell line was replaced with NCI-H2052.

[0076] (Comparative Example 9) The procedure was the same as in Example 19, except that the lentivirus prepared by replacing the pLKO.1 puro with shRNA construct, which incorporates a sequence to knock down the oxytocin receptor, with Scramble shRNA (Plasmid#1864, purchased from addgene) was used to not knock down the oxytocin receptor.

[0077] (Comparative Example 10) The procedure was the same as in Example 20, except that the oxytocin receptor was not knocked down using a lentivirus prepared by replacing the pLKO.1 puro with shRNA construct, which incorporates a sequence to knock down the oxytocin receptor, with Scramble shRNA.

[0078] The results are shown in Figure 7. Figure 7 shows that in both Example 19 and Example 20, in which the oxytocin receptor was knocked down, the weight of the subcutaneous tumor was smaller than in Comparative Examples 9 and 10, in which the oxytocin receptor was not knocked down. Therefore, it was shown that shRNA targeting the oxytocin receptor can be used as a therapeutic agent for malignant mesothelioma.

[0079] [Inhibition of malignant mesothelioma cell proliferation by combining compounds targeting oxytocin receptors with anticancer drugs] (Example 21) The proliferation of malignant mesothelioma cells induced by the combined use of crigosiban and cisplatin was evaluated using the WST-1 assay. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2373 was seeded in a dish and cultured for 1 day. (2) 45 μM of crigosiban and 6 μM of cisplatin (product number 033-20091, purchased from Fujifilm Wako Pure Chemical Corporation) were administered into a dish. (3) After culturing for 5 days, evaluation was performed using the WST-1 assay.

[0080] (Example 22) The procedure was the same as in Example 21, except that only 45 μM of cligosiban was administered.

[0081] (Example 23) The procedure was the same as in Example 21, except that the malignant mesothelioma cell line was replaced with NCI-H2052 and 15 μM cligosiban and 0.8 μM cisplatin were administered to the dish.

[0082] (Example 24) The procedure was the same as in Example 22, except that the malignant mesothelioma cell line was replaced with NCI-H2052 and 15 μM of cricosiban was administered.

[0083] (Comparative Example 11) The procedure was the same as in Example 21, except that 45 μM cligosiban and 6 μM cisplatin were not administered.

[0084] (Comparative Example 12) The procedure was the same as in Example 21, except that only 6 μM cisplatin was administered.

[0085] (Comparative Example 13) The procedure was the same as in Example 23, except that 15 μM cligosiban and 0.8 μM cisplatin were not administered.

[0086] (Comparative Example 14) The procedure was the same as in Example 23, except that only 0.8 μM cisplatin was administered.

[0087] Figure 8 shows the results of the WST-1 assay for Examples 21-24 and Comparative Examples 11-14. From Figure 8, it was shown that Examples 21-24, Comparative Examples 12 and 14, in which malignant mesothelioma cells were administered either or both cligosiban and cisplatin, suppressed the proliferation of malignant mesothelioma cells more effectively than Comparative Examples 11 and 13, in which neither cligosiban nor cisplatin was administered. Furthermore, Examples 21 and 23, in which both cligosiban and cisplatin were administered, suppressed the proliferation of malignant mesothelioma cells more effectively than Examples 22 and 24, Comparative Examples 12 and 14, in which either cligosiban or cisplatin was administered.

[0088] Examples 1 to 24 demonstrate that compounds targeting oxytocin receptors can be used as therapeutic agents for malignant mesothelioma. Furthermore, compounds targeting oxytocin receptors were effective against malignant mesothelioma expressing oxytocin receptors. Therefore, measuring the expression level of oxytocin receptors in malignant mesothelioma tissue from malignant mesothelioma patients suggests the possibility of using this therapeutic agent as a companion diagnostic.

[0089] [Inhibition of malignant mesothelioma cell proliferation by existing standard treatments for malignant mesothelioma, crigosiban, and combination therapy with existing standard treatments and crigosiban] (Example 25) We investigated the inhibitory effect of adding crigosiban to cisplatin and pemetrexed, existing standard treatments for malignant mesothelioma, on the suppression of malignant mesothelioma cell proliferation. The procedure is as follows. (1) Malignant mesothelioma cell line NCI-H2052 (purchased from ATCC), 5.0 × 10 6 The drug was administered subcutaneously to the left rump of nude mice (BALB / c nude (nu / nu) female, 6-8 weeks old: purchased from Charles River Japan). (2) Starting 7 days after subcutaneous administration, crigosiban (purchased from MedChemExpress) was administered orally at a dose of 60 mg / kg every other day for a total of 10 doses. (3) Seven days after subcutaneous administration, cisplatin (product number 033-20091, purchased from Fujifilm Wako Pure Chemical Industries, Ltd.) was administered at a dose of 2 mg / kg and pemetrexed disodium heptahydrate (product number 161-26263, purchased from Fujifilm Wako Pure Chemical Industries, Ltd.) was administered once via tail vein injection. (4) Two days after the 10th oral administration of crigosiban, nude mice were dissected and the weight of subcutaneous tumors was measured.

[0090] (Example 26) The procedure was the same as in Example 25, except that cisplatin and pemetrexed were not administered.

[0091] (Comparative Example 15) The procedure was the same as in Example 25, except that cligosiban, cisplatin, and pemetrexed were not administered.

[0092] (Comparative Example 16) The procedure was the same as in Example 25, except that crigosiban was not administered.

[0093] The results are shown in Figure 9. Note that Example 25 used three nude mice. Therefore, each tumor excised from the nude mice is numbered from Example 25-1 to Example 25-3. Similarly, Example 26 used two nude mice, Comparative Example 15 used three, and Comparative Example 16 used two nude mice, all numbered in the same manner as Example 25.

[0094] As shown in Figure 9, the weight of subcutaneous tumors in Example 26, which was administered with cligosiban, was smaller than the weight of subcutaneous tumors in Comparative Example 16, which was administered with cisplatin and pemetrexed, existing standard treatments for malignant mesothelioma. Surprisingly, cligosiban was shown to be more useful than existing standard treatments even when used alone.

[0095] Furthermore, in Example 25, where cligosiban was used in combination with cisplatin and pemetrexed, the weight of the subcutaneous tumor was shown to be significantly smaller than in Example 26 and Comparative Example 16. Therefore, it was demonstrated that cligosiban, when used in combination with existing treatments for malignant mesothelioma, can produce remarkable therapeutic effects that were not predictable from conventional techniques. [Industrial applicability]

[0096] It is useful in the field of treating malignant mesothelioma, for which no effective treatments or therapies have been established to date.

Claims

1. It is a treatment for malignant mesothelioma, The therapeutic agent contains a compound that targets oxytocin receptors as its active ingredient. The compound is an oxytocin receptor inhibitor, The oxytocin receptor inhibitor is at least one selected from the group consisting of crigosiban, OT-R antagonist 1, L368,899 hydrochloride, atosiban, and letosiban. A therapeutic agent.

2. A therapeutic agent for malignant mesothelioma, The therapeutic agent contains nucleic acids that knock down oxytocin receptors as its active ingredient. Nucleic acids are either siRNA or shRNA. A therapeutic agent.

3. Furthermore, including anticancer drugs, The therapeutic agent according to claim 1 or 2.

4. The anticancer drug is cisplatin. The therapeutic agent according to claim 3.

5. The anticancer drugs are cisplatin and pemetrexed. The therapeutic agent according to claim 3.

6. A method for selecting patients with malignant mesothelioma, The selection method is: A measurement step for measuring the expression level of oxytocin receptors in malignant mesothelioma tissue, A determination step to determine whether the expression level of the oxytocin receptor is above a threshold, A selection process to select malignant mesothelioma patients whose oxytocin receptor expression level is above a threshold, including, Selection methods for malignant mesothelioma patients.

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