Therapeutic agent for multiple myeloma

Abstract

The present invention relates to use of a pharmaceutical composition for multiple myeloma containing 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide.

Description

Treatment for multiple myeloma

[0001] This invention relates to optically active azabicyclo ring derivatives useful as pharmaceuticals, pharmaceutically acceptable salts thereof, and the use of pharmaceutical compositions containing these for multiple myeloma.

[0002] Multiple myeloma is a malignant tumor of plasma cells differentiated from B cells, characterized not only by bone marrow infiltration of monoclonal plasma cells but also by the presence of monoclonal immunoglobulins in the patient's serum or urine, and various complications such as osteolytic lesions, renal impairment, anemia, and infections. Multiple myeloma progresses from monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma and then to symptomatic myeloma. It has become clear that during this progression, changes in the surrounding bone marrow microenvironment and immune cells, in addition to genomic abnormalities caused by gene activation or inactivation due to epigenetic regulatory abnormalities such as genomic instability in myeloma cells and methylation of specific gene promoters, complicate the pathogenesis. While the introduction of proteasome inhibitors, immunomodulatory drugs (thalidomide-based), and various antibody drugs has extended patient survival in the treatment of multiple myeloma, a cure has yet to be achieved, and more alternative treatment options are needed (Non-Patent Literature 1, 2). Furthermore, there is a strong demand for safe and effective compounds and methods for treating, preventing, and managing multiple myeloma while reducing the toxicity and / or side effects associated with conventional therapies. In addition, there is a need for indicators, diagnostic methods, and detection methods for selecting patients likely to respond to agents used in the treatment of multiple myeloma.

[0003] Recently, an optically active azabicyclo ring derivative targeting the inhibition of binding between menin and MLL fusion protein was reported in Patent Document 1.

[0004] International Publication No. 2020 / 045334

[0005] Kumar, SK et al., Nat Rev Dis Primers. 3:17046 (2017)Bhatt, P. et al., Curr Oncol. 30(2):2322-2347 (2023)

[0006] The purpose of this disclosure is to provide a useful therapeutic agent and method for multiple myeloma, comprising an optically active azabicyclo ring derivative as an active ingredient, which exhibits excellent anticancer activity by inhibiting the binding of menin to MLL fusion protein.

[0007] More specifically, the inventors provide pharmaceutical-related technologies comprising 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonane-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide (hereinafter sometimes referred to as the "free form" or "free form of the compound"), or a pharmaceutically acceptable salt thereof (hereinafter sometimes referred to as the "compound" or "compound of the disclosure"). The disclosure also provides biomarkers for patient selection in the treatment of multiple myeloma, as well as methods for therapeutic intervention, diagnostic kits, and detection methods. In particular, this disclosure provides opportunities, methods, and tools that can be used to select patients with multiple myeloma who express certain biomarkers above a certain level, and / or to avoid treating patients who are unlikely to respond to treatment.

[0008] As a result of diligent research, the present inventors have found that a pharmaceutical product containing 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonane-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, exhibits therapeutic efficacy against multiple myeloma, and have completed this disclosure.

[0009] In other words, the present invention is as follows:

[0010] [Item 1] A therapeutic agent for patients with multiple myeloma containing 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, as an active ingredient. A therapeutic and / or prophylactic agent characterized in that at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 is highly expressed compared to the expression level of GAPDH in the patient.

[0011] [Item 2] The therapeutic and / or prophylactic agent according to Item 1, characterized in that at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 is expressed at a higher level compared to the expression level of GAPDH in the patient.

[0012] [Item 3] The therapeutic and / or prophylactic agent according to Item 1, characterized in that the TLR2 gene of the patient is expressed at a higher level compared to the expression level of GAPDH in the patient.

[0013] [Item 4] The therapeutic and / or prophylactic agent according to any one of items 1 to 3, characterized in that the TLR2 gene of the patient is expressed at a level at least twice as high as the expression level of GAPDH in the patient.

[0014] [Item 5] The therapeutic and / or prophylactic agent according to any one of items 1 to 3, characterized in that the TLR2 gene of the patient is expressed at a level 30 times higher than the expression level of GAPDH in the patient.

[0015] [Clause 6] The therapeutic and / or prophylactic agent according to any one of Clauses 1 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

[0016] [Clause 7] The therapeutic and / or prophylactic agent according to any one of Clauses 2 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

[0017] [Clause 8] The therapeutic and / or prophylactic agent according to any one of Clauses 3 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of the TLR2 gene in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

[0018] [Clause 9] The therapeutic and / or prophylactic agent according to any one of Clauses 2 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one protein selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the protein detected in (1).

[0019] [Item 10] A therapeutic and / or prophylactic agent for a patient with multiple myeloma, comprising 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, as an active ingredient, characterized in that the patient's IFN-β expression level is elevated after administration of the therapeutic and / or prophylactic agent compared to the patient's IFN-β expression level before administration.

[0020] [Clause 11] The therapeutic and / or prophylactic agent according to any one of Clauses 1 to 9, further characterized in that the patient's IFN-β level is elevated after administration of the therapeutic and / or prophylactic agent compared to the patient's IFN-β level before administration.

[0021] [Clause 12] The therapeutic and / or prophylactic agent according to claim 10 or 11, characterized in that after administration of the therapeutic and / or prophylactic agent, the patient's IFN-β expression is at least twice as high as the expression level of IFN-β before administration.

[0022] [Clause 13] The therapeutic and / or prophylactic agent according to claim 10 or 11, characterized in that after administration of the therapeutic and / or prophylactic agent, the patient's IFN-β expression is four times higher than the expression level of IFN-β before administration.

[0023] [Item 14] Multiple myeloma, comprising administering to a patient a therapeutically effective amount of 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or its hydrate or solvate. A method for treating and / or preventing a disease, characterized in that at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in the patient is expressed at a higher level compared to the expression level of GAPDH in the patient.

[0024] [Item 15] A method for treating a patient with multiple myeloma, comprising: (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 obtained from a patient with multiple myeloma; and (2) determining the administration of the compound based on the expression level of the gene detected in step (1), and then (3) A method comprising administering to the patient a therapeutically effective amount of 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof.

[0025] [Item 16] Use of 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, for the manufacture of therapeutic and / or prophylactic agents for patients with multiple myeloma, The use is characterized in that at least one gene selected from the patient's TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 is expressed at a higher level compared to the patient's GAPDH expression level.

[0026] [Item 17] Use of 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, for the manufacture of therapeutic and / or prophylactic agents for patients with multiple myeloma, Use is characterized by being determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in multiple myeloma cells obtained from the patient, and (2) determining the administration of the therapeutic agent and / or prophylactic agent based on the expression level of the gene detected in (1).

[0027] [Item 18] A kit for detecting the expression level of one or more genes selected from the group TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in a patient with multiple myeloma, characterized by comprising: (1) a reagent for detecting the expression level of RNA transcripts and / or protein products of one or more genes; (2) one or more gene-specific primers or probes for one or more genes; or (3) a reagent for detecting the expression level of protein products of one or more genes.

[0028] The technology of this disclosure, when used with the compounds of this disclosure as provided herein, is useful as a therapeutic and / or prophylactic agent for multiple myeloma. Furthermore, this disclosure allows for the selection of multiple myeloma patients who express certain biomarkers above a certain level in the treatment of multiple myeloma, thereby avoiding treatment of patients who are unlikely to respond to treatment.

[0029] More specifically, the present inventors provide a technology related to pharmaceuticals comprising 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonane-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof.

[0030] The cell number changes after treating each cell with DMSO or 10 μmol / L of the evaluation compound are shown. The expression of Annexin V and DAPI after treating RPMI 8226 cells and delta-47 cells with DMSO, 1 μmol / L of the evaluation compound, or 10 μmol / L of the evaluation compound for 3 days is shown in a dot plot. Based on the Gene Ontology analysis, enriched Gene Ontology terms are shown in a heatmap in order of statistical significance.

[0031] Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Therefore, singular articles (e.g., "a," "an," and "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0032] 5-Fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonane-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide has the following structure and is also referred to as "the compound represented by chemical formula (1)" in this specification. In addition, "the compound represented by chemical formula (1) or its pharmaceutically acceptable salt, or its hydrate or solvate" means the hydrate or solvate of the compound represented by chemical formula (1), and the hydrate or solvate of a pharmaceutically acceptable salt of the compound represented by chemical formula (1). The compound represented by chemical formula (1) can be prepared by referring to International Publication No. 2020 / 045334.

[0033] Examples of "pharmaceutically acceptable salts" include acid addition salts and base addition salts. For example, acid addition salts include inorganic salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, or organic salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, or organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, "pharmaceutically acceptable salts" also include amino acid salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid.

[0034] "The tartrate of the compound represented by chemical formula (1)" means 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonane-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propane-2-yl)benzamide 1L(+)-tartrate.

[0035] The compounds provided herein may encompass a variety of stereochemical forms. The compounds of this disclosure include not only optical isomers but also diastereomers, mixtures of enantiomers including, for example, racemic mixtures, as well as individual enantiomers and diastereomers resulting from structural asymmetry in particular compounds. Separation of individual isomers or selective synthesis of individual isomers can be achieved by applying various methods known to those skilled in the art.

[0036] The compounds represented by Chemical Formula (1) include various hydrates, solvates, and crystal polymorphs. Further, the compounds of the present disclosure may be substituted with isotope elements (e.g., ２ H (or D), ３ H (or T), １１ C, １３ C, １４ C, １３ N, １５ N, １５ O, ３５ S, １８ F, １２５ I, etc.), and these compounds are also included in the compounds of the present disclosure.

[0037] Furthermore, the scope of the present disclosure includes prodrugs of the compounds represented by Chemical Formula (1). In the present disclosure, a prodrug refers to a derivative that is hydrolyzed by acid or enzymatically in vivo to give a compound represented by Chemical Formula (1). For example, a prodrug can be produced by modifying the amino group of the compound represented by Chemical Formula (1) according to a conventional method. Specifically, examples include a compound in which the amino group is substituted with an alkanoyl group to form an alkanoylamino group, a compound in which the amino group is substituted with an alkoxycarbonyl group to form an alkoxycarbonylamino group, a compound in which the amino group is substituted with an alkanoyloxymethylamino group, or a compound in which the amino group is substituted with a hydroxylamine.

[0038] Also, what is provided for use in the methods herein is, for example, a pharmaceutical composition formulated for administration by a suitable route and means, containing an effective concentration of a compound such as a compound represented by Chemical Formula (1) or a pharmaceutically acceptable salt thereof.

[0039] The terms used in this specification are explained below.

[0040] In some embodiments, the subject or patient to be treated by the above method is a mammal. The present disclosure can be used in mammals, and in this specification, the term "mammal" is used in its ordinary biological sense. Thus, specifically, it includes humans, cows, horses, dogs, cats, rats, mice, but also many other species. In some further embodiments, the subject or patient is a human.

[0041] Multiple myeloma is characterized according to the International Staging System for Multiple Myeloma (ISS; https: / / www.myeloma.org / international-staging-system-iss-reivised-iss-r-iss), but it is considered to progress through two stages, namely "monoclonal gammopathy of undetermined significance (MGUS)" and "smoldering (asymptomatic) multiple myeloma", before becoming symptomatic and requiring treatment, i.e., "(symptomatic) multiple myeloma".

[0042] When obtaining a pharmaceutically acceptable salt of the compound represented by Chemical Formula (1), if the compound represented by Chemical Formula (1) is obtained in the form of a pharmaceutically acceptable salt, it may be purified as it is. If it is obtained in the free form, it may be dissolved or suspended in a suitable organic solvent, and an acid or a base may be added to form a salt by a conventional method.

[0043] "RPMI 8226 cells", "delta-47 cells", "MM.1S cells", "MM.1R cells", "KMS-12-BM cells", "KMS-11 cells", "NCI-H929 cells" and "IM-9 cells" are human multiple myeloma cell lines.

[0044] The genes described in this specification (TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAAP2-AS1) are identified by the gene names registered in the International Nucleotide Sequence Database Collaboration (https: / / www.insdc.org / ), GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), etc., and the gene sequences refer to the registered sequences. In this specification, each gene present in the genome of human cells is indicated, including point mutations, partial deletions, etc. of these registered sequences.

[0045] The compounds disclosed herein may be administered orally, parenterally, rectally, or as eye drops, and the daily dose will vary depending on the type of compound, the method of administration, and the patient's symptoms and age. For example, they may be administered in multiple doses throughout the day, or once, twice, or three times per day (24 hours).

[0046] The compound represented by chemical formula (1), or a pharmaceutically acceptable salt thereof, or its hydrate or solvate, may be administered orally, either directly or in a suitable dosage form. Examples of dosage forms include, but are not limited to, tablets, capsules, powders, granules, solutions, suspensions, injections, patches, and poultices. The preparations are manufactured by known methods using pharmaceutically acceptable additives.

[0047] Depending on the purpose, additives such as excipients, disintegrants, binders, fluidizers, lubricants, coating agents, solvents, solubilizers, thickeners, dispersants, stabilizers, sweeteners, and flavorings can be used. Specific examples of additives include lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, magnesium stearate, stearyl fumarate sodium, polyethylene glycol, propylene glycol, titanium dioxide, and talc.

[0048] A single oral dose of the compound represented by chemical formula (1), or its pharmaceutically acceptable salt, or its hydrate or solvate, converted to the free form, is 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 These are mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1010 mg, 1020 mg, 1030 mg, 1040 mg, 1050 mg, 1060 mg, 1070 mg, 1080 mg, 1090 mg, 1100 mg, 1120 mg, 1130 mg, 1140 mg, 1150 mg, 1160 mg, 1170 mg, 1180 mg, 1190 mg, and 1200 mg. It also includes a range between any two of these doses.

[0049] The amounts used for the treatment or prophylaxis of the compound represented by chemical formula (1), or its pharmaceutically acceptable salt, or its hydrate or solvate, are approximately 1 to 1000 mg / day, 1 to 500 mg / day, 1 to 200 mg / day, 1 to 100 mg / day, and 1 to 50 mg / day, when converted to the free form.

[0050] The compound represented by chemical formula (1) or a pharmaceutically acceptable salt thereof, or its hydrate or solvate, may be used in combination with other drugs or therapies, including, but not limited to, drugs, radiotherapy, chemotherapy, stem cell transplantation, cell therapy, or other non-drug-based therapies currently used for the treatment, prevention, or management of multiple myeloma.

[0051] Biological samples obtained for the purpose of investigating the responsiveness to a compound represented by chemical formula (1) or a pharmaceutically acceptable salt thereof, or its hydrate or solvate, are not particularly limited as long as they are samples from a patient, but include, for example, cancer tissue, cancer cell-containing blood samples, blood-derived products (serum and plasma), lymph, urine, tears, saliva, hair follicle cells, cerebrospinal fluid, cheek swabs, feces, synovial fluid, synovial cells, sputum, or tissue samples. Preferably, they are cancer tissue, cancer cell-containing blood samples, blood, or blood-derived products.

[0052] mRNA extraction is performed using methods known in the art. RNA isolation may be performed using a purification kit, buffer set, and protease according to the product manual. After analysis of RNA concentration, RNA repair and / or amplification steps may be included as needed, and the RNA is reverse transcribed using a gene-specific promoter and subsequent PCR. Preferably, real-time PCR is used that is suitable for both quantitative competitive PCR in which internal competitors of each target sequence are used for normalization, and quantitative comparative PCR in which normalization genes or housekeeping genes in RT-PCR are used in the sample.

[0053] Various methods for determining the expression levels of RNA transcripts (e.g., mRNA) or proteins include gene expression profiling and polymerase chain reaction (PCR), including quantitative real-time PCR (qRT-PCR), which can be performed using commercially available instruments according to the manufacturer's protocol. mRNA analysis is performed using polymerase chain reaction (PCR) techniques or by microarray analysis. When PCR is used, the preferred form of PCR is quantitative real-time PCR (qRT-PCR). Other methods include, but are not limited to, microarray analysis, gene expression analysis by sequential gene expression analysis, proteomics, and immunohistochemistry (IHC).

[0054] Expression levels may also be determined at the protein level, for example, using various types of immunoassays or proteomics techniques.

[0055] In one embodiment, a therapeutic and / or prophylactic agent for multiple myeloma is described, characterized by being determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in multiple myeloma cells obtained from a patient with multiple myeloma, and (2) determining the administration of a compound represented by chemical formula (1) based on the expression level of the gene detected in step (1). In one embodiment, the administration of a drug is determined by the degree to which at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 is expressed relative to the expression level of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0056] The determination is made by detecting the expression levels of the following genes in multiple myeloma cells: TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1, preferably TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2, more preferably TLR2. Identifying multiple myeloma cells in which elevated expression of these genes can be confirmed will increase the responsiveness to the compound represented by chemical formula (1).

[0057] Furthermore, the proteins that are the product of these genes may also be detected.

[0058] In one embodiment, the expression levels of TLR2, FAM178B, IGLON5, CXADRP2, or FAR2P2 relative to GAPDH are greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 times.

[0059] In one embodiment, the present disclosure provides a kit for detecting the expression level of one or more genes in a patient with multiple myeloma, comprising at least one container containing one or more reagents for detecting the expression level of one or more genes selected from the group TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2. The kit may have any of the following features (1) to (3): (1) The reagents include reagents for detecting the expression level of RNA transcripts and / or protein products of one or more genes. (2) The reagents include one or more gene-specific primers or probes for one or more genes. (3) The reagents include reagents for detecting the expression level of protein products of one or more genes.

[0060] In one embodiment of the method described herein, a pharmaceutical composition is administered in an amount effective for the treatment, prevention, or remission of multiple myeloma. A composition containing an effective amount of the compound or a therapeutically effective concentration of the compound is administered to a subject or patient exhibiting symptoms of multiple myeloma. These amounts are effective for the remission or elimination of one or more symptoms of multiple myeloma. As used in this disclosure, and unless otherwise specified, “to treat,” “to cure,” and “to treat” mean to reduce or lessen the severity of the disease or condition being treated, e.g., the symptoms associated with multiple myeloma. “Prevention” also includes inhibiting the symptoms of a specific disease or disorder, e.g., multiple myeloma. In some embodiments, patients with a family history of multiple myeloma are candidates for prophylactic therapy. Generally, the term “to prevent” refers to the administration of a drug before the onset of symptoms, particularly to patients at risk of multiple myeloma.

[0061] One embodiment describes a method for treating a patient with multiple myeloma, comprising the steps of (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 in multiple myeloma cells obtained from a patient with multiple myeloma, (2) determining the administration of a compound represented by chemical formula (1) based on the expression level of the gene detected in step (1), and then (3) administering an effective amount of the compound represented by chemical formula (1) to the patient.

[0062] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0063] Example 1: Efficacy evaluation test for cell proliferation RPMI 8226 cells, delta-47 cells, KMS-12-BM cells, KMS-11 cells, and IM-9 cells were obtained from the JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition (https: / / cellbank.nibiohn.go.jp / ). MM. 1S cells, MM. 1R cells, and NCI-H929 cells were obtained from the American Cell Culture and Cell Lineage Preservation Center (ATCC; https: / / www.atcc.org / ). RPMI 8226 cells, MM. 1S cells, MM. 1R cells, KMS-12-BM cells, KMS-11 cells, and IM-9 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2. ２ NCI-H929 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 0.05 mmol / L 2-mercaptoethanol, and 1% penicillin / streptomycin at 37°C and 5% CO2. ２ Delta-47 cells were cultured in RPMI1640 medium containing 20% ​​fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2. ２ Cells were cultured in the presence of the compound. Each cell was seeded at a rate of 50,000 cells per well in a 24-well plate. The evaluation compound (tartrate salt of the compound represented by chemical formula (1)) was dissolved in DMSO and added to the culture medium to a final concentration of 10 μmol / L, and the cells were cultured. The final concentration of DMSO in the culture medium was 0.1%. A similar sample of cells cultured with DMSO (final concentration in the culture medium: 0.1%) was used as a negative control. After 4, 7, 11, and 14 days following compound addition, a portion of the cultured cells was removed and the cell count was measured using a Coulter counter (Beckman Coulter).

[0064] The evaluation results for Example 1 are shown in Figure 1. A significant inhibitory effect on cell proliferation was confirmed in RPMI 8226 cells and delta-47 cells. A partial inhibitory effect on cell proliferation was confirmed in MM. 1S cells, MM. 1R cells, KMS-12-BM cells, and KMS-11 cells.

[0065] Example 2: Apoptosis Evaluation Test In Example 1, RPMI 8226 cells and delta-47 cells, which showed a significant inhibitory effect on cell proliferation, were seeded in 24-well plates at a rate of 150,000 cells per well. The evaluation compounds were added to achieve final concentrations of 1 and 10 μmol / L, respectively, and cultured for 3 days. Annexin V (BioLegend) and DAPI (BioLegend) were labeled, and the cells were analyzed using a Flow cytometer (Bay Bioscience J-SAN).

[0066] The evaluation results for Example 2 are shown in Figure 2. Dose-dependent enhancement of apoptosis was confirmed in RPMI 8226 cells and delta-47 cells following treatment with the evaluation compound.

[0067] Example 3: Analysis of gene expression changes in cells RPMI 8226 cells, delta-47 cells, MM. 1S cells, MM. 1R cells, KMS-12-BM cells, KMS-11 cells, NCI-H929 cells, and IM-9 cells were seeded at a rate of 1,000,000 cells each in a 10 cm dish, and the evaluation compound was added to a final concentration of 1 μmol / L and cultured for 3 days. Similarly, cells were cultured with DMSO (final concentration 0.1%) for 3 days and used as a negative control. After the culture period, total RNA was extracted from the cells using RNeasy® Mini Kit (QIAGEN, 74106). Using 430 ng of extracted RNA, cDNA was synthesized and the library prepared using the NEBNext Ultra II Directional RNA Library prep kit for Illumina. Subsequently, gene expression levels were comprehensively analyzed by RNA sequencing (Illumina Novaseq 6000, 2×150 bp configuration) at GENEWIZ.

[0068] In Example 1, in RPMI 8226 cells, which showed a significant inhibitory effect on cell proliferation, it was confirmed that 2227 genes showed more than a twofold increase in expression after treatment with the evaluation compound compared to DMSO treatment. Similarly, in delta-47 cells, which also showed a significant inhibitory effect on cell proliferation, it was confirmed that 1760 genes showed more than a twofold increase in expression after treatment with the evaluation compound compared to DMSO treatment. In both RPMI 8226 and delta-47 cells, 421 genes showed more than a twofold increase in expression after treatment with the evaluation compound.

[0069] Example 4: Identification of genes showing high gene expression changes in RPMI 8226 cells and delta-47 cells. In order to identify genes that were expressed even without treatment with the evaluation compound, in addition to the cell proliferation inhibitory effect shown by treatment with the evaluation compound, the expression levels of RPMI 8226 cells and delta-47 cells were compared with those of MM. 1S cells, MM. 1R cells, KMS-12-BM cells, KMS-11 cells, NCI-H929 cells and IM-9 cells that were not treated with the evaluation compound. As a result, the expression levels of GAPDH in RPMI 8226 cells and delta-47 cells were compared with those of MM. 1S cells and MM. Fourteen genes were identified as having higher expression levels compared to GAPDH in 1R cells, KMS-12-BM cells, KMS-11 cells, NCI-H929 cells, and IM-9 cells, including TLR2 (Toll-like receptor 2), FAM178B (family with sequence similarity 178 member B), IGLON5 (IgLON Family Member 5), CXADRP2 (CXADR pseudogene 2), and FAR2P2 (Fatty Acyl-CoA Reductase 2 Pseudogene 2) (Table 1). These genes can be used as markers for patient selection.

[0070] Example 5: Gene Ontology Analysis In Example 3, gene ontology analysis was performed using StrandNGS (Digital Biology) on 421 genes in both RPMI 8226 cells and delta-47 cells that showed increased gene expression after treatment with the evaluation compound compared to DMSO treatment.

[0071] The evaluation results for Example 5 are shown in Figure 3. In both RPMI 8226 cells and delta-47 cells, a significant increase in the expression of interferon alpha / beta signaling-related genes was observed after treatment with the evaluation compound. For example, in Figure 3, R-HAS-909733 (Interferon alpha / beta signaling) shows a significant increase. Furthermore, when the expression of RPMI 8226 cells and delta-47 cells in the sample from Example 3 was examined, a clear increase in IFN-β (interferon-β-b1) expression was indeed observed in both cells after treatment with the evaluation compound (Table 2).

[0072] The pharmaceuticals disclosed herein comprise optically active azabicyclo ring derivatives or pharmaceutically acceptable salts thereof, or hydrates or solvates thereof, and may be used as pharmaceuticals for the treatment or prevention of multiple myeloma.

Claims

1. A therapeutic agent for patients with multiple myeloma containing 5-fluoro-2-[(4-{7-[(1S,3S,4R)-5-methylidene-2-azabicyclo[2.2.2]octane-3-carbonyl]-2,7-diazaspiro[3.5]nonan-2-yl}pyrimidine-5-yl)oxy]-N,N-di(propan-2-yl)benzamide or a pharmaceutically acceptable salt thereof, or its hydrate or solvate, as an active ingredient. A therapeutic and / or prophylactic agent characterized in that at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 is highly expressed compared to the expression level of GAPDH in the patient.

2. The therapeutic and / or prophylactic agent according to claim 1, characterized in that at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 is expressed at a high level compared to the expression level of GAPDH in the patient.

3. The therapeutic and / or prophylactic agent according to claim 1, characterized in that the TLR2 gene of the patient is expressed at a higher level compared to the expression level of GAPDH in the patient.

4. The therapeutic and / or prophylactic agent according to any one of claims 1 to 3, characterized in that the TLR2 gene of the patient is expressed at a level at least twice as high as the expression level of GAPDH in the patient.

5. The therapeutic and / or prophylactic agent according to any one of claims 1 to 3, characterized in that the TLR2 gene of the patient is expressed at a level 30 times higher than the expression level of GAPDH in the patient.

6. The therapeutic and / or prophylactic agent according to any one of claims 1 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, FAR2P2, MAL, LOC100128885, ST8SIA6-AS1, PAGE1, LINC01287, H1F0, PTPRH, PDIA2, and BAIAP2-AS1 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

7. The therapeutic and / or prophylactic agent according to any one of claims 2 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one gene selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

8. The therapeutic and / or prophylactic agent according to any one of claims 3 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of the TLR2 gene in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the gene detected in (1).

9. The therapeutic and / or prophylactic agent according to any one of claims 2 to 5, characterized in that the patient is determined by a step including (1) detecting the expression level of at least one protein selected from TLR2, FAM178B, IGLON5, CXADRP2, and FAR2P2 in multiple myeloma cells obtained from the subject, and (2) determining the administration of the therapeutic and / or prophylactic agent based on the expression level of the protein detected in (1).

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