Therapeutic agents for malignant mesothelioma and methods for selecting patients with malignant mesothelioma

Abstract

Provided is a therapeutic agent for malignant mesothelioma and a method for selecting a patient with malignant mesothelioma that expresses a large amount of oxytocin receptor. The therapeutic agent for malignant mesothelioma contains a compound that targets the oxytocin receptor as an active ingredient, thereby solving the problem.

Description

Technical Field

[0001] The disclosures in this application relate to therapeutic agents for malignant mesothelioma and methods for selecting patients with malignant mesothelioma. Background Technology

[0002] Malignant mesothelioma is a tumor that originates from the mesothelium that covers the pleura, pericardium (cardiac membrane), and peritoneum. More than 80% of malignant mesotheliomas originate in the pleura, but both malignant pleural mesothelioma and malignant peritoneal mesothelioma have a poor prognosis.

[0003] Most malignant mesotheliomas originating in the pleura and peritoneum are caused by asbestos exposure, with a long period of 30 to 40 years preceding their onset. Therefore, the incidence of malignant mesothelioma is likely to increase in the future.

[0004] Malignant mesothelioma can develop in localized or diffuse forms. Most malignant mesotheliomas develop diffusely, infiltrating along the pleura, peritoneum, etc. Although surgical treatments, radiotherapy, and chemotherapy have been performed, no effective treatment has been established, and the prognosis is very poor. Therefore, the development of therapeutic agents and methods for malignant mesothelioma, as described in Patent Documents 1 and 2, is actively underway.

[0005] Prior art literature

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2009-013077;

[0008] Patent Document 2: Japanese Patent Application Publication No. 2014-208650. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] As described in Patent Documents 1 and 2, new therapeutic agents and methods have been reported. However, since therapeutic agents and methods for malignant mesothelioma have not yet been established, it is desirable to develop new therapeutic agents and methods. The disclosure of this application has conducted in-depth research on the development of new therapeutic agents for malignant mesothelioma, and has made the following new discoveries: (1) compounds targeting oxytocin receptors have the effect of inhibiting the proliferation of malignant mesothelioma; and (2) there are a large number of malignant mesotheliomas expressing oxytocin receptors.

[0011] Specifically, the purpose of this application is to provide a therapeutic agent for malignant mesothelioma and a method for selecting patients with malignant mesothelioma who extensively express oxytocin receptors.

[0012] Problem-solving methods

[0013] (1) A therapeutic agent for malignant mesothelioma, wherein the therapeutic agent comprises a compound targeting the oxytocin receptor as an active ingredient.

[0014] (2) The therapeutic agent according to (1) above, wherein the compound is an oxytocin receptor inhibitor.

[0015] (3) The therapeutic agent according to (2) above, wherein the oxytocin receptor inhibitor is at least one selected from the group consisting of Cligosiban, OT-R antagonist 1, L368,899 hydrochloride and atosiban.

[0016] (4) The therapeutic agent according to (2) above, wherein the oxytocin receptor inhibitor is retosiban.

[0017] (5) The therapeutic agent according to (1) above, wherein the compound is a nucleic acid.

[0018] (6) The therapeutic agent according to (5) above, wherein the nucleic acid is siRNA (Small interfering RNA) or shRNA (short hairpin RNA).

[0019] (7) The therapeutic agent according to any one of (1) to (6) above, wherein the therapeutic agent further comprises an anticancer agent.

[0020] (8) The therapeutic agent according to (7) above, wherein the anticancer agent is cisplatin.

[0021] (9) The therapeutic agent according to (7) above, wherein the anticancer agent is cisplatin and pemetrexed.

[0022] (10) A method for selecting patients with malignant mesothelioma, the method comprising:

[0023] The assay procedure measures the expression level of oxytocin receptors in malignant mesothelioma tissue.

[0024] The determination process involves determining whether the expression level of oxytocin receptors is above a certain threshold.

[0025] The selection process involves choosing patients with malignant mesothelioma whose oxytocin receptor expression levels are above the threshold.

[0026] Invention Effects

[0027] The therapeutic agent for malignant mesothelioma disclosed in this application can inhibit the proliferation of malignant mesothelioma. Attached Figure Description

[0028] Figure 1 This is a graph showing the results of inhibition of malignant mesothelioma cell proliferation induced by clegosiban using a xenograft model.

[0029] Figure 2 Figure A shows the expression levels of oxytocin receptors in malignant mesothelioma tissues from 87 patients with malignant mesothelioma; Figure B shows the Kaplan-Meier curves of overall survival relative to the high, moderate, and low expression groups of oxytocin receptors.

[0030] Figure 3 This is a figure showing the results of the WST-1 assay of malignant mesothelioma cells administered in vitro with clegosiban.

[0031] Figure 4 This is a graph showing the results of the WST-1 assay on malignant mesothelioma cells treated with an oxytocin receptor inhibitor.

[0032] Figure 5 This is a figure showing the results of the WST-1 assay in malignant mesothelioma cells with knocked-down oxytocin receptors.

[0033] Figure 6 This is a graph showing the results of a community formation assay of malignant mesothelioma cells with knocked-down oxytocin receptors.

[0034] Figure 7 This is a figure showing the results of inhibited proliferation of malignant mesothelioma cells with knocked-down oxytocin receptors using a xenograft model.

[0035] Figure 8 This is a graph showing the results of the WST-1 assay on malignant mesothelioma cells after administration of compounds and anticancer agents targeting the oxytocin receptor.

[0036] Figure 9 This is a graph showing the results of malignant mesothelioma cell proliferation inhibition using existing standard treatments, crigosiban, and a combination of existing standard treatments and crigosiban in a xenograft model. Detailed Implementation

[0037] (Implementation methods for treatment agents for malignant mesothelioma)

[0038] The following describes the therapeutic agent for malignant mesothelioma (hereinafter simply referred to as "therapeutic agent") involved in the implementation method.

[0039] The therapeutic agent involved in the implementation method is characterized in that it contains a compound that targets the oxytocin receptor as an active ingredient.

[0040] The term "compound" is not specifically limited to any compound that targets the oxytocin receptor. Furthermore, in this specification, "compound targeting the oxytocin receptor" includes compounds that bind to the oxytocin receptor and compounds that knock down the oxytocin receptor. Examples of compounds targeting the oxytocin receptor include oxytocin receptor inhibitors, nucleic acids such as siRNA and shRNA that knock down the oxytocin receptor, and antibodies against the oxytocin receptor.

[0041] As shown in the embodiments described below, the inventors have conducted repeated and in-depth studies and confirmed the effect of administering crigosiban on inhibiting the proliferation of malignant mesothelioma cells when transplanted into a nude mouse xenograft model.

[0042] Cligosiban is an oxytocin receptor inhibitor used for premature ejaculation in men, and is a compound represented by the following chemical formula (1). Furthermore, the oxytocin receptors that crigosiban acts on are known to be primarily expressed in the mammary glands and the uterus during late pregnancy.

[0043] [Chemical Formula 1]

[0044]

[0045] Oxytocin is a peptide hormone composed of nine amino acids, secreted by the posterior pituitary gland. It plays a role in milk secretion by causing contraction of mammary gland muscle fibers, smooth muscle contraction, and uterine contractions during childbirth. Furthermore, while oxytocin is known to be a hormone unique to women, it is also secreted at certain levels in men.

[0046] Since crigosiban inhibits the proliferation of malignant mesothelioma cells, the inventors focused on oxytocin receptor inhibitors and, as shown in the examples described later, conducted in vitro tests on oxytocin receptor inhibitors other than crigosiban. The oxytocin receptor inhibitors used were OTR antagonists 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, inhibition of malignant mesothelioma cell proliferation was confirmed.

[0047] [Chemical Formula 2]

[0048]

[0049] Furthermore, retosambran, represented by chemical formula (5), and OT-R antagonist 2, represented by chemical formula (6), are also known as oxytocin receptor inhibitors. Therefore, retosambran and OT-R antagonist 2 can also be expected to inhibit the proliferation of malignant mesothelioma.

[0050] [Chemical Formula 3]

[0051]

[0052] As demonstrated in the examples described later, it was confirmed that nucleic acids such as siRNA and shRNA that knock down the oxytocin receptor inhibit the proliferation of malignant mesothelioma cells. Furthermore, the siRNA, shRNA, and other nucleic acids are not particularly limited as long as they can inhibit the expression of the oxytocin receptor. It is expected that the proliferation of malignant mesothelioma can be inhibited by suppressing the expression of the oxytocin receptor.

[0053] Antibodies against the oxytocin receptor can be polyclonal or monoclonal. Furthermore, they can be complete antibody molecules or antibody fragments that specifically bind to the oxytocin receptor. Antibodies against the oxytocin receptor can be prepared using known methods. Moreover, antibodies against the oxytocin receptor are expected to inhibit the proliferation of malignant mesothelioma in the same way as oxytocin receptor inhibitors, oxytocin receptor knockdown siRNAs, and shRNAs.

[0054] Therefore, compounds targeting oxytocin receptors can be used as therapeutic agents for malignant mesothelioma. Furthermore, compounds targeting oxytocin receptors can be used alone or in combination as therapeutic agents.

[0055] The therapeutic agents involved in the implementation may contain anticancer agents in addition to compounds targeting oxytocin receptors. Compounds targeting oxytocin receptors and anticancer agents can synergistically enhance the effect of inhibiting the proliferation of malignant mesothelioma cells. Examples of added anticancer agents include cell inhibitors (cytotoxic drugs), angiogenesis inhibitors, and immune checkpoint inhibitors.

[0056] Cell inhibitors are agents that kill cancer cells, induce cell death, or reduce cell proliferation / survival rates. Examples of cell inhibitors include alkylating agents, platinum preparations, metabolic antagonists, antitumor antibiotics, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, topoisomerase inhibitors, plant alkaloids, hormones, and bacterial toxins. Examples of alkylating agents include cyclophosphamide, isophosphamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, thiotepa, procarbazin, and ranimustine. Examples of platinum preparations include cisplatin, nedaplatin, oxaliplatin, and carboplatin. Examples of metabolic antagonists include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-Gimeracil-Oteracil Potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxyurea, and mecaptopurine.Examples of antitumor antibiotics include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and zinostatin 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. Furthermore, maytansine (DM-1), represented by emtansine, and maytansine analogues, used in antibody-drug conjugates (ADCs) targeting cancer, are also preferred cell inhibitors.

[0057] Angiogenesis inhibitors are drugs that target vascular endothelial growth factor (VEGF) and inhibit cancer cell proliferation by blocking the supply of nutrients to cancer cells. Examples of angiogenesis inhibitors include bevacizumab, ramucirumab, and aflibercept.

[0058] Immune checkpoint inhibitors are drugs that bind to inhibitory receptors or their ligands that act as immune checkpoint molecules, blocking inhibitory signal transduction, thereby relieving the brakes on the immune system and enhancing the immune response against tumors. Examples of immune checkpoint inhibitors include nivolumab and pembrolizumab as anti-PD1 antibodies; atezolizumab and durvalumab as anti-PD-L1 antibodies; and ipilimumab as an anti-CTLA4 antibody.

[0059] Compounds targeting oxytocin receptors and anticancer agents can be used simultaneously or with a time difference. Alternatively, dosing schedules can be set independently for both, and the subjects can be administered them separately according to these schedules. Furthermore, the number of doses for both can be arbitrarily set, allowing for single or multiple administrations. In addition, the anticancer agent used in combination with the oxytocin receptor-targeting compound can be one, or a combination of two or more.

[0060] The method of administration of the therapeutic agent involved in the implementation method is not particularly limited as long as it is effective in malignant mesothelioma. Examples of administration methods include oral, transdermal, intravenous, intramuscular, intrapleural, intraperitoneal, and rectal routes.

[0061] Examples of dosage forms of therapeutic agents involved in the implementation methods include tablets, pills, powders, lozenges, sachet agents, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, gelatin soft and hard capsules, suppositories, sterile injectable solutions, and sterile sealed powders.

[0062] Furthermore, the therapeutic agents involved in the embodiments may also contain conventionally used additives as needed. Examples of additives include, but are not limited to, existing additives such as excipients, binders, lubricants, disintegrants, flavoring and odor-correcting agents, solvents, stabilizers, base agents, humectants, and preservatives.

[0063] The therapeutic agents involved in the implementation methods have the following effects.

[0064] (1) Therapeutic agents containing compounds that target the oxytocin receptor as active ingredients can inhibit the proliferation of malignant mesothelioma cells.

[0065] (2) By further containing anticancer agents in the therapeutic agent, the proliferation of malignant mesothelioma cells is synergistically inhibited.

[0066] (Implementation method of patient selection for malignant mesothelioma)

[0067] The following describes the method for selecting patients with malignant mesothelioma involved in the implementation method.

[0068] The method for selecting patients with malignant mesothelioma involved in the implementation includes: (1) a measurement step, measuring the expression level of oxytocin receptor in malignant mesothelioma tissue; (2) a judgment step, determining whether the expression level of oxytocin receptor is above a threshold; and (3) a selection step, selecting patients with malignant mesothelioma whose expression level of oxytocin receptor is above the threshold. Furthermore, this selection method is performed in the order of (1), (2), and (3).

[0069] In the assay process, the method for determining the expression level of oxytocin receptors in malignant mesothelioma tissue is not particularly limited as long as the expression level of oxytocin receptors can be measured. Examples of methods for determining the expression level of oxytocin receptors include PCR (Polymerase Chain Reaction), RT-PCR (Reverse Transcription-PCR), Northern blot, microarrays, DNA chips, and RNA chips.

[0070] In the judgment process, it is determined whether the expression level of oxytocin receptor measured by the measurement process is above a threshold. The threshold in the judgment process can be set appropriately.

[0071] In the selection process, patients with malignant mesothelioma tissue whose oxytocin receptor expression level was above the threshold in the judgment process were selected.

[0072] The method for selecting patients with malignant mesothelioma involved in the implementation method has the following effects.

[0073] (1) Patients with malignant mesothelioma who express oxytocin receptors extensively in their malignant mesothelioma tissue can be selected. Therefore, companion diagnostics can be performed for therapeutic agents containing compounds that target oxytocin receptors as active ingredients.

[0074] The following examples illustrate the implementation methods disclosed in this application, but these examples are for illustrative purposes only and do not limit or restrict the scope of the invention disclosed in this application.

[0075] Example

[0076] [Crigosiban inhibits the proliferation of malignant mesothelioma cells using a xenograft model]

[0077] (Example 1)

[0078] In vivo studies investigated whether crigosiban inhibited the proliferation of malignant mesothelioma cells.

[0079] The steps are as follows.

[0080] (1) 5.0 × 10 6 One malignant mesothelioma cell line NCI-H2052 (purchased from ATCC) was administered subcutaneously to the left buttock of nude mice (BALB / c nude females, 6-8 weeks old, purchased from Charles River, Japan).

[0081] (2) Three days after subcutaneous administration, 60 mg / kg of crigosiban (purchased from MedChemExpress) was administered orally 10 times every other day.

[0082] (3) One week after the 10th oral administration, nude mice were dissected and the weight of subcutaneous tumors was measured.

[0083] (Comparative Example 1)

[0084] No crigosiban was administered, except that the procedure was carried out in the same manner as in Example 1.

[0085] The results are shown in Figure 1 Furthermore, Example 1 used four nude mice. Therefore, each nude mouse is referred to as Example 1-1 to Example 1-4. The same applies to Comparative Example 1. Figure 1 The results show that the subcutaneous tumors of Example 1, which were treated with crigosiban, were smaller in weight compared to Comparative Example 1, which was not treated with crigosiban. Furthermore, Example 1 was shown to statistically significantly inhibit subcutaneous tumor proliferation compared to Comparative Example 1. Therefore, it is demonstrated that crigosiban, which targets the oxytocin receptor, can be used as a therapeutic agent for malignant mesothelioma.

[0086] [Analysis of oxytocin receptor expression in patients with malignant mesothelioma]

[0087] (Example 2)

[0088] The results from Example 1 show that crigosiban, as an oxytocin receptor inhibitor, suppressed the proliferation of malignant mesothelioma cells. Therefore, the expression of oxytocin receptors in malignant mesothelioma was analyzed.

[0089] The database used was analyzed from 87 patients with malignant mesothelioma. The database used was The Cancer Genome Atlas (TCGA; https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga), a comprehensive database that analyzes genomic / methylation abnormalities and gene / protein expression abnormalities.

[0090] Figure 2 Figure A shows a graph illustrating the expression levels of oxytocin receptors in malignant mesothelioma tissues from 87 patients with malignant mesothelioma. It shows that approximately 40% of the 87 patients had more than 1000-fold higher expression levels compared to cases with low expression. Furthermore, Figure 2 Figure B shows the Kaplan-Mel curves for overall survival in the high-expression, medium-expression, and low-expression groups of the oxytocin receptor. Figure 2B indicates that cases with higher oxytocin receptor expression tend to have a worse prognosis. Therefore, this demonstrates the presence of high oxytocin receptor expression in malignant mesotheliomas with poor prognosis.

[0091] [In vitro inhibition of malignant mesothelioma cell proliferation by crigosiban]

[0092] (Example 3)

[0093] Next, we investigated whether crigosiban, an oxytocin receptor inhibitor, inhibited the proliferation of malignant mesothelioma cells in vitro.

[0094] The steps are as follows.

[0095] (1) The malignant mesothelioma cell line NCI-H2052 was placed in a culture dish and cultured for one day.

[0096] (2) Administer crigosiban to a culture dish at a concentration of 0, 5, 10, 15, 20 or 25 μM.

[0097] (3) After 5 days of culture, an evaluation based on the WST-1 assay was performed. The WST-1 assay is an assay method for evaluating cell proliferation by administering the cell proliferation assay drug WST-1 (product number: 11644807001, purchased from Roche) and using a colorimetric method.

[0098] (Example 4)

[0099] The malignant mesothelioma cell line was replaced with NCI-2373 (purchased from ATCC), and crigosiban was administered to the culture dish at a concentration of 0, 20, 30, 40, 50 or 60 μM, otherwise the procedure was the same as in Example 3.

[0100] Figure 3 The results of the WST-1 tests for Examples 3 and 4 are shown. Figure 3 It is shown that either Example 3 or Example 4 inhibited the proliferation of malignant mesothelioma cells with increasing crigosiban concentration. Therefore, the inhibition of malignant mesothelioma cell proliferation in vivo and in vitro is demonstrated.

[0101] [Inhibition of malignant mesothelioma cell proliferation by oxytocin receptor inhibitors other than crigosiban]

[0102] For crigosiban, whose efficacy was confirmed in mice, since crigosiban was also confirmed to be useful against two types of malignant mesothelioma cells in vitro, an in vitro experimental system was used to investigate whether oxytocin receptor inhibitors other than crigosiban inhibited the proliferation of malignant mesothelioma cells.

[0103] (Example 5)

[0104] The oxytocin receptor inhibitor was replaced with OTR antagonist 1 (purchased from MedChemExpress) and administered to the culture dish at concentrations of 0, 20, 40, 60 or 80 μM, otherwise the procedure was the same as in Example 3.

[0105] (Example 6)

[0106] The oxytocin receptor inhibitor was replaced with OTR antagonist 1 and administered to the culture dish at concentrations of 0, 20, 40, 60 or 80 μM, otherwise the procedure was the same as in Example 4.

[0107] (Example 7)

[0108] The oxytocin receptor inhibitor was replaced with L368,899 hydrochloride (purchased from MedChemExpress), otherwise the procedure was carried out in the same manner as in Example 5.

[0109] (Example 8)

[0110] The oxytocin receptor inhibitor was replaced with L368,899 hydrochloride, and otherwise the procedure was carried out in the same manner as in Example 6.

[0111] (Example 9)

[0112] The oxytocin receptor inhibitor was administered with atosiban (purchased from MedChemExpress), except that the procedure was the same as in Example 5.

[0113] (Example 10)

[0114] The oxytocin receptor inhibitor was replaced with atosiban, and otherwise the procedure was carried out in the same manner as in Example 6.

[0115] Figure 4 The results of the WST-1 test in Examples 5 to 10 are shown. Figure 4 Examples 5 through 10 show that any of them inhibited the proliferation of malignant mesothelioma cells with increasing oxytocin receptor inhibitor concentration. Therefore, it is demonstrated that oxytocin receptor inhibitors can be used as therapeutic agents for malignant mesothelioma.

[0116] [Inhibition of malignant mesothelioma cell proliferation caused by oxytocin receptor knockdown (1)]

[0117] The study investigated whether siRNA, a compound targeting the oxytocin receptor, inhibits the proliferation of malignant mesothelioma cells.

[0118] (Example 11)

[0119] The proliferation of malignant mesothelioma cells with oxytocin receptor knockdown via siRNA was evaluated using the WST-1 assay.

[0120] The steps are as follows.

[0121] (1) The malignant mesothelioma cell line NCI-H2373 was placed in a culture dish and cultured for one day.

[0122] (2) Oxytocin receptors were knocked down using siRNA1 (product number s9947, purchased from Thermo Fisher Scientific).

[0123] (3) After 4 days of incubation, an evaluation based on the WST-1 experiment was conducted.

[0124] (Example 12)

[0125] Replace siRNA1 with siRNA2 (product number s9948, purchased from Thermo Fisher Scientific), otherwise perform the same steps as in Example 11.

[0126] (Example 13)

[0127] The malignant mesothelioma cell line was replaced with NCI-H2052, otherwise the same steps as in Example 11 were performed.

[0128] (Example 14)

[0129] The malignant mesothelioma cell line was replaced with NCI-H2052, otherwise the same steps as in Example 12 were performed.

[0130] (Comparative Example 2)

[0131] (1) The malignant mesothelioma cell line was replaced with the normal mesothelial cell line MeT-5A (purchased from ATCC) without death. (2) Oxytocin receptor was not knocked down. Otherwise, the same steps as in Example 11 were performed.

[0132] (Comparative Example 3)

[0133] The oxytocin receptor was knocked down using siRNA1, otherwise the procedure was performed in the same manner as in Comparative Example 2.

[0134] (Comparative Example 4)

[0135] The oxytocin receptor was knocked down using siRNA2, otherwise the procedure was performed in the same manner as in Comparative Example 2.

[0136] (Comparative Example 5)

[0137] Oxytocin receptors were not knocked down; otherwise, the procedure was carried out in the same manner as in Example 11.

[0138] (Comparative Example 6)

[0139] Oxytocin receptors were not knocked down; otherwise, the procedure was carried out in the same manner as in Example 13.

[0140] Figure 5 The results of the WST-1 test for Examples 11 to 14 and Comparative Examples 2 to 6 are shown. Figure 5 Examples 11-14, showing knockdown of oxytocin receptors in malignant mesothelioma cells, compared to Comparative Examples 5 and 6, showing no knockdown of oxytocin receptors in malignant mesothelioma cells, showed inhibition of malignant mesothelioma cell proliferation. Furthermore, in... Figure 5 In normal mesothelial cell lines, Comparative Examples 3 and 4, with oxytocin receptor knockdown, showed no significant difference in proliferation inhibition compared to Comparative Example 2, which did not have oxytocin receptor knockdown. Therefore, it is considered that oxytocin receptor-targeting siRNAs are useful for inhibiting the proliferation of malignant mesothelioma cells expressing the oxytocin receptor. Thus, oxytocin receptor-targeting siRNAs could be used as therapeutic agents for malignant mesothelioma.

[0141] [Inhibition of malignant mesothelioma cell proliferation due to oxytocin receptor knockdown (2)]

[0142] (Example 15)

[0143] The proliferation of malignant mesothelioma cells with oxytocin receptor knockdown using siRNA was evaluated using a community formation assay.

[0144] The steps are as follows.

[0145] (1) The malignant mesothelioma cell line NCI-H2373 was placed in a culture dish and cultured for one day.

[0146] (2) Use siRNA1 to knock down the oxytocin receptor.

[0147] (3) After 48 hours, the NCI-H2373 with knocked-down oxytocin receptors was placed in a new culture dish.

[0148] (4) After two weeks of cultivation, the community formation experiment was evaluated by counting the number of communities formed.

[0149] (Example 16)

[0150] Replace siRNA1 with siRNA2, otherwise follow the same steps as in Example 15.

[0151] (Example 17)

[0152] The malignant mesothelioma cell line was replaced with NCI-H2052, otherwise the same steps as in Example 15 were performed.

[0153] (Example 18)

[0154] The malignant mesothelioma cell line was replaced with NCI-H2052, otherwise the same steps as in Example 16 were performed.

[0155] (Comparative Example 7)

[0156] Oxytocin receptors were not knocked down; otherwise, the procedure was carried out in the same manner as in Example 15.

[0157] (Comparative Example 8)

[0158] Oxytocin receptors were not knocked down; otherwise, the procedure was carried out in the same manner as in Example 17.

[0159] Figure 6 A and Figure 6 B shows the results of community formation tests for Examples 15 to 18, Comparative Example 7, and Comparative Example 8. Figure 6 A and Figure 6 Example B shows that Examples 15-18, in which the oxytocin receptor of malignant mesothelioma cells was knocked down, inhibited community 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 are shown to inhibit the proliferation of malignant mesothelioma cells in the same way as Examples 11-14.

[0160] [Inhibition of malignant mesothelioma cell proliferation induced by oxytocin receptor knockdown using a xenograft model]

[0161] (Example 19)

[0162] In vivo studies investigated whether constant knockdown of oxytocin receptor shRNA inhibited the proliferation of malignant mesothelioma cells.

[0163] The steps are as follows.

[0164] (1) Lentiviruses were created by transfecting the 293FT cell line (purchased from Thermo Fisher Scientific) with three plasmids: pMD2.G (plasmid #12259, purchased from Addgene), psPAX2 (plasmid #12260, purchased from Addgene), and pLKO.1puro (pLKO.1puro with shRNA construct, plasmid #10878, purchased from Addgene) which contains a sequence that knocks down the oxytocin receptor.

[0165] (2) The malignant mesothelioma cell line NCI-H2373 was placed in a culture dish and cultured for one day.

[0166] (3) Using the lentivirus prepared in (1) above, shRNA was introduced into NCI-H2373 to knock down the oxytocin receptor.

[0167] (4) NCI-H2373 cells with knocked-down oxytocin receptor were cultured.

[0168] (5) 1.5 × 10 6 Subcutaneous injection of NCI-H2373-treated nude mice (BALB / c nude (nu / nu) females, 6 weeks old, purchased from Chaslife Japan) with knocked-down oxytocin receptors was applied to the left buttock.

[0169] (6) One month after subcutaneous administration, nude mice were dissected and the weight of the subcutaneous tumor was measured.

[0170] (Example 20)

[0171] The malignant mesothelioma cell line was replaced with NCI-H2052, otherwise the same steps as in Example 19 were performed.

[0172] (Comparative Example 9)

[0173] The lentivirus was prepared by replacing the pLKO.1puro with shRNA construct containing a sequence that knocks down the oxytocin receptor with Scramble shRNA (plasmid #1864, purchased from Addgene), without knocking down the oxytocin receptor, and otherwise carried out in the same manner as in Example 19.

[0174] (Comparative Example 10)

[0175] The procedure was carried out using a lentivirus prepared by replacing the pLKO.1puro with shRNA construct containing a sequence that knocks down the oxytocin receptor with a scramble shRNA, without knocking down the oxytocin receptor, except that the procedure was the same as in Example 20.

[0176] The results are shown in Figure 7 . Figure 7 Examples 19 and 20, where the oxytocin receptor was knocked down, showed that the subcutaneous tumors were smaller compared to Comparative Examples 9 and 10, where the oxytocin receptor was not knocked down. Therefore, shRNAs targeting the oxytocin receptor can be used as therapeutic agents for malignant mesothelioma.

[0177] [Inhibition of malignant mesothelioma cell proliferation induced by the combined use of oxytocin receptor-targeting compounds and anticancer agents]

[0178] (Example 21)

[0179] The WST-1 assay evaluated the inhibition of malignant mesothelioma cell proliferation caused by the combined use of crigosiban and cisplatin.

[0180] The steps are as follows.

[0181] (1) The malignant mesothelioma cell line NCI-H2373 was placed in a culture dish and cultured for one day.

[0182] (2) 45 μM clegosiban and 6 μM cisplatin (product number 033-20091, purchased from Fujifilm and Koh Genuine Chemicals Co., Ltd.) were administered to the culture dish.

[0183] (3) After 5 days of cultivation, an evaluation based on the WST-1 experiment was conducted.

[0184] (Example 22)

[0185] Only 45 μM crigosiban was administered, otherwise the procedure was carried out in the same manner as in Example 21.

[0186] (Example 23)

[0187] The malignant mesothelioma cell line was replaced with NCI-H2052, and 15 μM clegosiban and 0.8 μM cisplatin were administered to the culture dish, otherwise the same procedure as in Example 21 was followed.

[0188] (Example 24)

[0189] The malignant mesothelioma cell line was replaced with NCI-H2052, and 15 μM crigosiban was administered. Otherwise, the procedure was carried out in the same manner as in Example 22.

[0190] (Comparative Example 11)

[0191] Untreated crigosiban 45 μM and cisplatin 6 μM were administered, except as described in Example 21.

[0192] (Comparative Example 12)

[0193] Only 6 μM cisplatin was administered, otherwise the procedure was carried out in the same manner as in Example 21.

[0194] (Comparative Example 13)

[0195] Untreated 15 μM clegosiban and 0.8 μM cisplatin were administered, except as described in Example 23.

[0196] (Comparative Example 14)

[0197] Only 0.8 μM of cisplatin was administered, otherwise the procedure was carried out in the same manner as in Example 23.

[0198] Figure 8 The results of the WST-1 test for Examples 21 to 24 and Comparative Examples 11 to 14 are shown. Figure 8 Examples 21-24, Comparative Examples 12 and 14, which administered crigasiban and cisplatin or either one to malignant mesothelioma cells, showed inhibition of malignant mesothelioma cell proliferation compared to Comparative Examples 11 and 13, which did not administer crigasiban and cisplatin. Furthermore, Examples 21 and 23, which administered both crigasiban and cisplatin, showed further inhibition of malignant mesothelioma cell proliferation compared to Examples 22, 24, Comparative Examples 12 and 14, which administered either crigasiban or cisplatin.

[0199] As demonstrated in Examples 1 to 24 above, compounds targeting the oxytocin receptor can be used as therapeutic agents for malignant mesothelioma. Furthermore, compounds targeting the oxytocin receptor are effective against malignant mesothelioma expressing the oxytocin receptor. Therefore, by measuring the expression level of the oxytocin receptor in the malignant mesothelioma tissue of patients with malignant mesothelioma, the possibility of companion diagnostics using this therapeutic agent was shown.

[0200] [Inhibition of malignant mesothelioma cell proliferation caused by existing standard treatments for malignant mesothelioma, crigosiban, and the combination of existing standard treatments and crigosiban]

[0201] (Example 25)

[0202] Further investigation was conducted on the inhibition of malignant mesothelioma cell proliferation when cisplatin and pemetrexed, which are the current standard treatments for malignant mesothelioma, were used in combination with crigosiban.

[0203] The steps are as follows.

[0204] (1) 5.0 × 10 6 One malignant mesothelioma cell line, NCI-H2052 (purchased from ATCC), was administered subcutaneously to the left buttock of nude mice (BALB / c nude females, 6-8 weeks old, purchased from Chasliwa, Japan).

[0205] (2) Seven days after subcutaneous administration, 60 mg / kg of crigosiban (purchased from MedChemExpress) was administered orally 10 times every other day.

[0206] (3) Seven days after subcutaneous administration, 2 mg / kg of cisplatin (product number 033-20091, purchased from Fujifilm and Hikari Pure Chemical Industries Co., Ltd.) and 25 mg / kg of pemetrexed disodium heptahydrate (product number 161-26263, purchased from Fujifilm and Hikari Pure Chemical Industries Co., Ltd.) were injected once into the tail vein.

[0207] (4) Two days after the 10th oral administration of crigosiban, nude mice were dissected and the weight of subcutaneous tumors was measured.

[0208] (Example 26)

[0209] No cisplatin or pemetrexed was administered, except that the procedure was carried out in the same manner as in Example 25.

[0210] (Comparative Example 15)

[0211] No crigosiban, cisplatin, or pemetrexed were administered; the procedure was otherwise performed in the same manner as in Example 25.

[0212] (Comparative Example 16)

[0213] No crigosiban was administered, except that the procedure was carried out in the same manner as in Example 25.

[0214] The results are shown in Figure 9 Furthermore, Example 25 used three nude mice. Therefore, each tumor removed from the nude mice was labeled as Example 25-1 to Example 25-3. In addition, two nude mice were used in Example 26, three in Comparative Example 15, and two in Comparative Example 16, and were labeled with the same numbers as in Example 25.

[0215] like Figure 9As shown, the weight of the subcutaneous tumor in Example 26, which was treated with crigosiban, was smaller than that in Comparative Example 16, which was treated with existing standard treatments for malignant mesothelioma (i.e., cisplatin and pemetrexed). Surprisingly, crigosiban was more effective than existing standard treatments even when used alone.

[0216] Furthermore, it was also shown that in Example 25, when crigosiban, along with cisplatin and pemetrexed, the weight of the subcutaneous tumor was reduced compared to Example 26 and Comparative Example 16. Therefore, it was demonstrated that crigosiban, when used in combination with existing treatments for malignant mesothelioma, can achieve significant therapeutic effects that were unpredictable with the prior art.

[0217] Industrial applicability

[0218] This is useful in the treatment of malignant mesothelioma, for which effective therapeutic agents and treatments have not yet been established.

Claims

1. The use of an oxytocin receptor-targeting compound in the manufacture of a therapeutic agent for malignant mesothelioma, wherein, The compound is at least one selected from the group consisting of crigosiban, OT-R antagonist 1, L368,899 hydrochloride, atosiban, and nucleic acids, wherein the nucleic acid is at least one selected from siRNA1 and siRNA2, wherein the product number of siRNA1 is s9947 and it is purchased from Thermo Fisher Scientific, and the product number of siRNA2 is s9948 and it is purchased from Thermo Fisher Scientific.

2. The use according to claim 1, wherein, The therapeutic agent also includes an anticancer agent.

3. The use according to claim 2, wherein, The anticancer agent is cisplatin.

4. The use according to claim 2, wherein, The anticancer agents are cisplatin and pemetrexed.

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