JP2016088863A - Therapeutic agent to ret inhibitor-resistant cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel cancer therapeutic agent or preventive agent for tumors which have a RET gene abnormality, particularly a secondary mutation in a fusion gene of a RET gene and other genes, and are refractory to RET inhibitors except alectinib and the like, or resistant to such RET inhibitors.SOLUTION: The invention provides a therapeutic agent or a preventive agent containing as active ingredients a compound (alectinib) and the like represented by formula (II) for tumors which have a RET gene abnormality and are refractory to RET inhibitors except the compound or resistant to such RET inhibitors, or for the metastasis of the tumors.SELECTED DRAWING: Figure 2

Description

  The present invention belongs to the technical field of molecular targeted drugs for cancer patients having a RET (Rearranged Duration Transcription) gene abnormality. The present invention relates to a novel therapeutic agent or preventive agent for a tumor having a RET gene abnormality, wherein the RET inhibitor is ineffective or resistant to the RET inhibitor. Specifically, the present invention relates to a tumor having an RET gene abnormality, which contains, as an active ingredient, alectinib (Alectinib, trade name: Alecensa) represented by the following formula (II), and is a RET inhibitor other than alectinib or the like Relates to a novel therapeutic or preventive agent for tumors that are ineffective or resistant to RET inhibitors.

  The chemical name of Alectinib is 9-ethyl-6,6-dimethyl-8- (4-morpholin-4-yl-piperidin-1-yl) -11-oxo-6,11-dihydro-5H-benzo [b Carbazole-3-carbonitrile or 9-ethyl-6,6-dimethyl-8- [4- (morpholin-4-yl) piperidin-1-yl] -11-oxo-6,11-dihydro-5H- Benzo [b] carbazole-3-carbonitrile.

  Alectinib is a therapeutic agent approved as an effective treatment for non-small cell lung cancer having a receptor tyrosine kinase ALK (anaplastic lymphoma kinase) fusion gene belonging to the insulin receptor family. Inventions relating to such alectinib are disclosed, for example, in Patent Publications 1 to 3 and the like, including manufacturing methods.

Alectinib has been reported to inhibit a number of kinases including RET (see Non-Patent Document 1). RET gene abnormalities (point mutations, chromosomal translocations, chromosomal inversions, gene amplifications) are known to be involved in carcinogenesis, with about 1-2% of lung adenocarcinomas and about 10% of thyroid cancers. It is said to be a cancer having a RET fusion gene. RET is one of the proto-oncogenes.
Therefore, since compounds having an inhibitory action on RET tyrosine kinase are extremely useful for the prevention and treatment of cancer, alectinib is considered to be a preventive or therapeutic agent for cancer in RET gene abnormality. In fact, alectinib has been reported to be useful as a RET inhibitor or RET tyrosine kinase inhibitor, a cancer having a mutation in RET, or a preventive or therapeutic agent for diseases including cancer metastasis (see Patent Document 4).

Examples of other RET tyrosine kinase inhibitors include multi-kinase inhibitors T5 which express multi-kinase RE, such as sorafenib, Sunitinib, cabozantinib (Cabozantinib, XL184), vandetanib (Vandetanib), and ponatinib IF. It has been reported to show cell growth inhibitory action against strains (see Non-Patent Document 2). In addition, it has been reported that cabozantinib, which is a multikinase inhibitor, showed partial response to two RET fusion gene-positive non-small cell lung cancer patients (see Non-Patent Document 3).
However, even a RET inhibitor may become ineffective or relapse after a response to a tumor having a RET gene abnormality.

JP 2011-16830 A International Publication No. 2010/143664 JP 2012-126711 A International Publication No. 2014/050781

Cancer cell, 19 (5), p. 679-690, 2011, Supplemental Information J Clin Oncol 30, 2012, suppl; Abstract no: 7510 Cancer Discov, 3 (6), Jun 2013, p. 630-5

  The present invention mainly relates to a RET gene abnormality, particularly a tumor having a secondary mutation in a fusion gene between a RET gene and another gene, and a RET inhibitor other than alectinib is ineffective or resistant to such a RET inhibitor. It is an object to provide a novel cancer therapeutic agent or preventive agent for tumors.

  As a result of intensive studies, the present inventors have also found that alectinib is effective against lung cancer cells and thyroid cancer cells having an RET gene abnormality in which other RET inhibitors are ineffective or resistant to other RET inhibitors. It has been found that the antitumor effect is effectively exhibited, and the present invention has been completed.

（式中、Ｒ１は、炭素数１〜６のアルキルを表す。） Examples of the present invention include the following.
[1] A therapeutic or prophylactic agent containing a compound represented by formula (I) (hereinafter referred to as “the above-mentioned compound”) or a salt thereof, or a solvate thereof as an active ingredient, wherein the RET gene abnormality A therapeutic agent or a preventive agent for a tumor in which a RET inhibitor excluding the compound is ineffective or resistant to such a RET inhibitor, or a metastasis of the tumor.

(Wherein R1 represents alkyl having 1 to 6 carbon atoms.)

[2] The therapeutic or preventive agent according to [1] above, wherein the tumor is a tumor having a fusion gene of a RET gene and another gene and / or a fusion protein of a RET protein and another protein.

[3] The tumor is acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, brain tumor, neuroblastoma, glioma, thyroid cancer, bone marrow abnormality Plastic syndrome, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, mesothelioma, pancreatic cancer, liver cancer, gallbladder cancer, skin cancer, malignant melanoma, renal cancer, renal pelvis The therapeutic or preventive agent according to the above [1] or [2], which is selected from the group consisting of ureteral cancer, bladder cancer, uterine cancer, testicular cancer, prostate cancer, and a tumor metastasized from the tumor.
[4] The therapeutic or prophylactic agent according to any one of [1] to [3] above, wherein the tumor is thyroid cancer, lung cancer, or mesothelioma.

[5] The RET inhibitor excluding the compound is vandetanib (Landatinib), lenvatinib, sunitinib, sorafenib, or any one of the above [1] to [4] A therapeutic or prophylactic agent.

  According to the present invention, a RET gene abnormality, particularly a tumor having a secondary mutation in a fusion gene between a RET gene and another gene, wherein a RET inhibitor other than the above-mentioned compound such as alectinib is invalid or such a RET inhibitor Can inhibit the growth of tumor cells resistant to. Specific examples of the tumor include leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, etc.), malignant lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma, etc.), brain tumor, neuroblast Tumor, glioma, thyroid cancer, myelodysplastic syndrome, head and neck cancer, esophageal cancer, stomach cancer, colorectal cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, mesothelioma, pancreatic cancer, liver cancer, gallbladder cancer, Examples include various cancers such as skin cancer, malignant melanoma, renal cancer, renal pelvic and ureteral cancer, bladder cancer, uterine cancer, testicular cancer, and prostate cancer. Furthermore, the invasion / metastasis of solid cancer as described above can be suppressed.

Represents cell viability. The vertical axis represents the survival rate (%) of the cells, and the horizontal axis represents the concentration (nM) of vandetanib. The solid curve of the black triangle shows the result in Ba / F3 cells into which the KIF5B-RET fusion gene was introduced, and the broken curve in white circle shows the result in the Ba / F3 parent strain. Represents cell viability. (A) The figure shows the result of vandetanib, and (B) the figure shows the result of alectinib. In both figures, the vertical axis represents the survival rate (%) of the cells, and the horizontal axis represents the drug concentration (nM). Represents cell viability. (A) shows the results of vandetanib in LC2 / ad parent strain and LC2 / ad VRA-2 cells, and (B) shows the results of vandetanib and alectinib in LC2 / ad VRA-2 cells. In both figures, the vertical axis represents the survival rate (%) of the cells, and the horizontal axis represents the drug concentration (nM). Represents cell viability. The vertical axis represents the survival rate (%) of the cell, and the horizontal axis represents the drug concentration (μM). The white circle solid line shows the results for vandetanib, the black circle solid line shows the results for alectinib, and the white triangle solid line shows the results for lenvatinib.

The present invention is described in detail below.
I. Explanation of Terms In the present invention, the “C1-C6 alkyl” according to R1 refers to linear and branched alkyl having 1 to 6 carbon atoms, specifically, for example, methyl, Ethyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, 2,3-dimethylpropyl, hexyl, 2,3-dimethylhexyl, 1,1-dimethylpentyl, heptyl, octyl Can be mentioned. Preferably it is a C1-C4 alkyl, More preferably, it is ethyl. The one in which R1 corresponds to ethyl is alectinib.

Examples of the salt of the compound include sulfonic acids such as hydrochloride, hydrobromide, hydroiodide, phosphate, phosphonate, sulfate, methanesulfonate, and p-toluenesulfonate. Salts, acetates, citrates, malates, tartrate, succinates, salicylates and other carboxylates, or alkali metal salts such as sodium and potassium salts; alkaline earths such as magnesium and calcium salts Metal salts; ammonium salts such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, tetraalkylammonium salts, and the like. Preferred are hydrochloride and methanesulfonate, and more preferred is hydrochloride.
These salts can be produced by bringing the compound into contact with an acid or base that can be used for the production of a pharmaceutical product.

  The compound or a salt thereof may be an anhydride, or may form a solvate such as a hydrate. “Solvation” as used herein refers to a phenomenon in which a solute molecule or ion strongly attracts a solvent molecule adjacent to it in a solution to form a single molecular group. For example, hydration is performed when the solvent is water. The solvate may be either a hydrate or a non-hydrate. As the non-hydrate, alcohol (for example, methanol, ethanol, n-propanol), dimethylformamide and the like can be used.

The compounds and salts thereof can also exist in several tautomeric forms, such as enol and imine forms, keto and enamine forms, and mixtures thereof. Tautomers exist as a mixture of tautomeric sets in solution. In the solid form, one tautomer is usually predominant. Although one tautomer may be described, the present invention includes all tautomers of the compounds.
The present invention includes all stereoisomers (eg, enantiomers, diastereomers (including cis and trans geometric isomers)), racemates of the isomers, and other mixtures. For example, the compounds may have one or more asymmetric points, and the present invention includes racemic mixtures, diastereomeric mixtures, and enantiomers of such compounds.

When the compound is obtained as a free form, it can be converted into a salt which may be formed by the compound or a hydrate or solvate thereof according to a conventional method.
When the compound is obtained as a salt, hydrate, or solvate of the compound, it can be converted into a free form of the compound according to a conventional method.

The substance used in the present invention also includes prodrugs of the above compounds. Here, “prodrug” means a derivative of the compound that is converted to the compound or a pharmaceutically acceptable salt thereof by enzymatic or non-enzymatic degradation under physiological conditions after administration. Prodrugs may be inactive when administered to a patient, but are present converted to the active compound in vivo.
For example, a prodrug is converted to the desired drug form when a specific pH is reached or by the action of an enzyme.

  In the present invention, the “RET gene” means a gene encoding a RET (Rearranged Duration Transcription) tyrosine kinase. The RET gene according to the present invention means any RET gene.

  In the present invention, “RET protein” means a protein consisting of amino acids constituting RET tyrosine kinase. The RET protein of the present invention means a RET protein of any origin. RET tyrosine kinase is known to contain three types of proteins, RET9, RET43, and RET51, due to differences in carboxyl-terminal splicing (TRENDS in Genetics, 2006, Vol. 22, p. 627-636). Polypeptides consisting of amino acids constituting these three types of proteins are also included in the “RET protein”.

  In the present invention, “a fusion gene of a RET gene and another gene” refers to all or a part of the RET gene and another gene (for example, KIF5B (kinesin family protein 5B) gene, CCDC6 (coiled-coildomain containing 6)). It refers to a gene in which all or a part of the gene, NCOA4 (nuclear receptor coactivator 4) gene, TRIM33 (triplicate motif-containing 33) gene) is fused. Specifically, for example, a gene in which all or part of the RET gene and all or part of the KIF5B gene are fused (KIF5B-RET fusion gene), all or part of the RET gene and all or part of the CCDC6 gene A gene in which all or part of the RET gene and all or part of the NCOA4 gene are fused (NCOA4-RET fusion gene), all or part of the RET gene and TRIM33 A gene in which all or part of the gene is fused (TRIM33-RET fusion gene) can be mentioned.

  In the present invention, “a fusion protein of a RET protein and another protein” means all or part of the RET protein and all or part of another protein (for example, KIF5B protein, CCDC6 protein, NCOA4 protein, TRIM33 protein). Refers to a protein fused with Specifically, for example, a protein in which all or part of the RET protein and all or part of the KIF5B protein are fused (KIF5B-RET fusion protein), all or part of the RET protein and all or part of the CCDC6 protein And a fusion protein of all or part of the RET protein and all or part of the NCOA4 protein (NCOA4-RET fusion protein), all or part of the RET protein and TRIM33 A protein in which all or part of the protein is fused (TRIM33-RET fusion protein) can be mentioned.

  In the present invention, “having a point mutation in the fusion gene” refers to a mutation in which one nucleotide base in the fusion gene is replaced with another nucleotide base, and “having a point mutation in the fusion protein” A mutation in which one amino acid in the fusion protein is replaced with another amino acid. This includes secondary mutations with resistance acquired by administration of anticancer agents. Specifically, examples of RET protein mutation include C634W, C634Y, E768D, V804M, V804L, and M918T mutations in the amino acid sequence of RET. Here, “C634W”, “C634Y”, “E768D”, “V804M”, “V804L”, and “M918T” have single-letter symbols of amino acids before and after the mutation before and after the numbers representing specific positions. This is an expression representing amino acid mutation. For example, “C634W” means that the 634th amino acid from the N-terminal of the specific amino acid sequence is substituted from Cys to Trp. That is, the number represents the position of the amino acid counted from the N-terminus of the specific amino acid sequence, the one-letter code of the amino acid described before that is the amino acid before the substitution, and the one-letter code of the amino acid described after that is the one after the substitution Represents an amino acid.

  Examples of the “RET inhibitor” other than the above compound include any drug that inhibits the activity of RET kinase or a drug that binds to RET kinase and has an action of inhibiting the activity. For example, Sorafenib, Sunitinib, Cabozantinib (XL184), Vandetanib, Ponatinib, and Lenvatinib can be mentioned.

  Whether or not a RET inhibitor is “invalid” and whether or not it is “resistant” is based on, for example, the so-called RECIST (Response Evaluation Criteria in Solid Tumors) guidelines for determining the therapeutic effect of solid cancer. Can be evaluated. Further, those skilled in the art can appropriately evaluate based on experimental data obtained by a conventional method.

“Therapeutic agent” means an agent that directly or indirectly improves, alleviates, cures, or prevents a target disease, symptom, or associated symptoms. Specifically, it means a drug used for inhibiting the growth of tumor tissue, reducing the effect, suppressing metastasis, reducing tumor markers, improving systemic symptoms of patients, extending the survival time, and the like.
“Prophylactic agent” means that a patient who suffers from, develops symptoms of, or may develop symptoms associated with the target disease, avoids it, delays it, or reduces its risk. Means a drug used to pre-treat to do.
“Tumor metastasis” means that the primary tumor metastasizes to other tissues. The therapeutic agent for “metastasis of the tumor” means an agent that inhibits or suppresses metastasis of the tumor, or an agent that suppresses or reduces the growth of a tumor that has recurred due to metastasis. The prophylactic agent for "metastasis of the tumor" means a drug used for pretreatment so that the tumor does not metastasize or metastasize and recur.

II. Method for producing the compound
Although the said compound which is an active ingredient of the therapeutic agent or preventive agent of this invention can be manufactured in accordance with the method as described in international publication WO2010 / 143664 pamphlet, the manufacturing method of the said compound is not limited to these. .

III. Therapeutic agent / preventive agent of the present invention The “therapeutic agent” or “preventive agent” according to the present invention refers to a drug used for treatment, prevention, or treatment and prevention, specifically, treatment of the target disease, It is a drug used for prevention, suppression of progression of disease state (prevention of deterioration and maintenance of the current situation), and the like.
The therapeutic / prophylactic agent of the present invention can be used as a pharmaceutical composition containing a pharmaceutically acceptable carrier in addition to the selected compound useful for the present invention.

  As used herein, the term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid shaped diluents or encapsulating materials suitable for administration to mammals. To do. As used herein, the term “acceptable” refers to the composition in such a way that under normal conditions of use, reactions that do not substantially reduce the pharmaceutical effectiveness of the composition do not occur with each other. This means that the component in the product and the target compound can be mixed. The pharmaceutically acceptable carrier should of course have a sufficiently high purity and a sufficiently low toxicity so that it is suitable for administration to the animal to be treated, preferably an animal, more preferably a mammal.

  Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; Tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, cocoa oil; propylene glycol, glycerin, sorbitol, mannitol And polyhydric alcohols such as polyethylene glycol; alginic acid; emulsifiers such as TWEEN; wetting agents such as lecithin; coloring agents; flavors; tableting agents; stabilizers; Pyrogen-free water; isotonic saline solution; and phosphate buffer.

  The therapeutic agent or prophylactic agent of the present invention can be administered orally, rectally, parenterally (intravenous, intramuscular, subcutaneous), intracisternally, intravaginally, intraperitoneally, intravesically. Topical (infusion, powder, ointment, gel or cream) administration and inhalation (oral or nasal spray). The dosage forms include solid preparations such as tablets, capsules, granules, powders, pills, aqueous and non-aqueous oral solutions and suspensions, and containers adapted to be subdivided into individual doses. And a liquid preparation such as a parenteral solution filled in a lyophilizate, which can be dissolved and used at the time of use. The dosage form can also be adapted to various modes of administration including controlled release formulations such as subcutaneous implantation.

Said formulation is manufactured by a well-known method using additives, such as an excipient | filler, a lubricant (coating agent), a binder, a disintegrating agent, a stabilizer, a corrigent, a diluent.
Examples of excipients include starches such as starch, potato starch, and corn starch, lactose, crystalline cellulose, and calcium hydrogen phosphate.
Examples of the coating agent include ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, and paraffin.
Examples of the binder include polyvinyl pyrrolidone, macrogol and the same compound as the excipient.
Examples of the disintegrant include the same compounds as the above excipients and chemically modified starch / celluloses such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.
Examples of the stabilizer include paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; Mention may be made of dehydroacetic acid; and sorbic acid.
Examples of the flavoring agent include sweeteners, acidulants, and fragrances that are commonly used.
Moreover, ethanol, phenol, chlorocresol, purified water, distilled water, or the like can be used as a solvent for producing the liquid agent.
Examples of the surfactant or emulsifier include polysorbate 80, polyoxyl 40 stearate, sodium lauryl sulfate, lauromacrogol and the like.

The use amount of the therapeutic agent or prophylactic agent of the present invention varies depending on symptoms, age, body weight, relative health, presence of other medications, administration method and the like. For example, for patients (warm-blooded animals, particularly humans), the generally effective amount is preferably 0.001 to 1 kg of body weight per day in the case of an oral preparation as an active ingredient (such as the compound or a salt thereof). 3000 mg, more preferably within the range of 0.01 to 300 mg per kg body weight, and the daily usage is preferably within the range of 1 to 1200 mg for adult patients with normal body weight. In the case of a parenteral preparation, it is preferably within the range of 0.001 to 1000 mg per kg body weight per day, and more preferably within the range of 0.01 to 300 mg per kg body weight. It is desirable to administer this once or several times a day according to the symptoms.
The therapeutic agent or prophylactic agent of the present invention can also be formulated by the method described in International Publication WO2012 / 023597.

  In addition, the therapeutic agent or prophylactic agent of the present invention is one or more drugs selected from other chemotherapeutic agents, hormonal therapeutic agents, immunotherapeutic agents or molecular targeted drugs (hereinafter sometimes abbreviated as concomitant drugs). And may be used in combination. These active ingredients (drugs) may be low molecular compounds, high molecular proteins, polypeptides, antibodies, or vaccines. Two or more active ingredients (drugs) may be mixed and used at an appropriate ratio.

  For example, examples of the “chemotherapeutic agent” include anticancer agents such as alkylating agents, platinum preparations, antimetabolites, topoisomerase inhibitors, anticancer antibiotics, plant-derived anticancer agents and the like. Examples of the “alkylating agent” include nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitoblonitol, Faran, dacarbazine, ranimustine, sodium estramustine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipbloman, etoglucid, altretamine, ambmustine, dibrospdium hydrochloride, fotemustine, predonimustine, pumitepa, ribomuthrezo Listed are trophosphamide, dinostatin stimamarer, adzeresin, systemustin, vizeresin, etc. It is done. Examples of the “platinum preparation” include carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin and the like. Examples of the “antimetabolite” include, for example, mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enocitabine, cytarabine, cytarabine okphosphat, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, Doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid, butosine, folinate calcium, levofolinate calcium, cladribine, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, pyritrexim, idoxyuridine, mitoguanzone, thiazoline azofumuthone Etc. Topoisomerase I inhibitors (eg, irinotecan, topotecan, etc.), topoisomerase II inhibitors (eg, sobuzoxane, etc.), and “anticancer antibiotics” include, for example, anthracycline anticancer agents (doxorubicin hydrochloride, daunorubicin hydrochloride, hydrochloric acid hydrochloride) Actinubicin, pirarubicin hydrochloride, epirubicin hydrochloride, etc.), actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromycin sulfate, neocartinostatin, mythramycin, sarcomycin, carcinophilin, mitotane, Zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride and the like can be mentioned. Examples of “plant-derived anticancer agents” include vinca alkaloid anticancer drugs (vinblastine sulfate, vincristine sulfate, vindesine sulfate, etc.), taxane anticancer drugs (paclitaxel, docetaxel, etc.), etoposide, etoposide phosphate, teniposide, vinorelbine, etc. Is mentioned.

  Examples of the “hormone therapeutic agent” include adrenocortical hormone drugs (eg, dexamethasone, prednisolone, betamethasone, triamcinolone, etc.), and prednisolone is particularly preferable.

  Examples of the “immunotherapeutic agent (BRM)” include, for example, picibanil, krestin, schizophyllan, lentinan, ubenimex, interferon, interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, lymphotoxin, BCG vaccine, corynebacterium parvum , Levamisole, polysaccharide K, procodazole and the like.

  The “molecular target drug” includes “a drug that inhibits the action of a cell growth factor and its receptor” and the like, and any “cell growth factor” may be used as long as it is a substance that promotes cell growth. In general, a peptide having a molecular weight of 20,000 or less and a factor that exerts its action at a low concentration by binding to a receptor can be mentioned. Specifically, (1) EGF (epidermal growth factor) or substantially (Eg, EGF, haregulin (HER2 ligand), etc.), (2) insulin or a substance having substantially the same activity [eg, insulin, IGF (insulin-like growth factor) -1 , IGF-2, etc.], (3) FGF (fibroblast growth factor) or a substance having substantially the same activity as the FGF (eg, acidic FGF, basic FGF, KGF (keratinocyte growth factor), FGF-10 etc.], (4) VEGF (vascular endothelial growth factor), (5) Other cell growth factors [eg, CSF (colony stimulating factor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growth factor), PDGF (platelet-derived growth factor), TGFβ (transforming growth factorβ), HGF (hepatocyte growth factor), and the like.

The “cell growth factor receptor” may be any receptor capable of binding to the above-mentioned cell growth factor, and specifically includes an EGF receptor, a haregulin receptor (HER2). Insulin receptor, IGF receptor, FGF receptor-1 or FGF receptor-2, HGF receptor (c-met), VEGF receptor, SCF receptor (c-kit) and the like. Examples of the “drug that inhibits the action of cell growth factor” include Herceptin (HER2 antibody), GLEEVEC (c-kit, abl inhibitor), Tarceva (EGF receptor inhibitor) and the like.
Also included are drugs that inhibit the action of multiple cell growth factors with a single drug, and drugs that block intracellular information emitted by cell growth factors.

In addition to the above drugs, L-asparaginase, acegraton, procarbazine hydrochloride, protoporphyrin / cobalt complex, mercury hematoporphyrin / sodium, differentiation inducer (eg, retinoid, vitamin D), angiogenesis inhibitor, α-blocker (Eg, tamsulosin hydrochloride, etc.) can also be used.
Among the above-mentioned combination drugs, platinum complexes (eg, carboplatin, cisplatin, oxaliplatin, etc.), antimetabolites (eg, gemcitabine, pemetrexed, etc.), topoisomerase I inhibitors (eg, irinotecan, topotecan, etc.), plant-derived Anticancer drugs (taxane drugs (eg, paclitaxel, docetaxel), vinorelbine), anticancer antibiotics (eg, mitomycin C), etc., hormone therapy drugs (eg, prednisolone), immunotherapy drugs (eg, picibanil, krestin, etc.) ), Molecular target drugs (eg, anti-VEGF antibodies such as bevacizumab, EGFR inhibitors such as erlotinib, and VEGFR inhibitors such as sunitinib) are preferred. Furthermore, cisplatin, gemcitabine, paclitaxel, bevacizumab and the like are preferable. It can also be used in combination with a combination therapy of these drugs. Examples include cisplatin and vinblastine and mitomycin C, cisplatin and vinorelbine, cisplatin and paclitaxel, cisplatin and gemcitabine, carboplatin and paclitaxel, pemetrexed and cisplatin, bevacizumab and cisplatin and pemetrexed.

In the present invention, the administration timing of the active ingredient and the concomitant drug of the present invention is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference. Moreover, you may administer the active ingredient and concomitant drug of this invention to an administration subject as a single formulation containing these. For example, there are multi-drug combination therapy in which a plurality of drugs are combined and infused over 3 to 6 months, and a method of taking an oral preparation for about 2 years.
In addition, preoperative adjuvant therapy such as “chemotherapy” may be performed before surgery to control tumor (cancer) cells that have spread and prevent recurrence due to metastasis or to reduce the scope of surgery. .
Furthermore, postoperative adjuvant therapy such as “chemotherapy” may be performed to suppress the growth of tumor (cancer) cells that have not been removed by local treatment such as surgery or radiation, and to prevent recurrence due to metastasis.
Furthermore, the anticancer drug used in combination may act on normal cells as well as cancer cells, which may appear as a side effect. Typical side effects include nausea, vomiting, loss of appetite, stomatitis, diarrhea or constipation due to disorders of the digestive mucosa, taste abnormalities, decreased white blood cells / red blood cells / platelets / hair loss due to bone marrow disorders, and decreased immunity. Further, side effect reducing drugs for suppressing these can be used in combination. Examples thereof include an antiemetic agent that effectively suppresses nausea (eg, granisetron hydrochloride) and a drug that accelerates recovery from bone marrow injury (eg, erythropoietin, G-CSF, GM-CSF).

The dose of the concomitant drug can be appropriately selected based on the clinically used dose. The mixing ratio of the active ingredient of the present invention and the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like. For example, when the administration subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 100 parts by weight of the preparation containing the active ingredient of the present invention.

EXAMPLES Hereinafter, although an Example and a reference example are hung up and this invention is demonstrated further more concretely, this invention is not limited at all by these.
Alectinib used in each example is hydrochloride.

[Reference Example 1] Evaluation of cell growth inhibitory activity against KIF5B-RET-expressing cells (1) Establishment of KIF5B-RET-expressing cells KIF5B-RET variant 1 (Exon 15 from CDS of KIF5B CDS and Exon 12 to C-terminal of CDS of RET) A lentivirus was prepared using a lentiviral vector pLenti6 (manufactured by Invitrogen) in which a gene fused with the above was incorporated, and the gene was introduced by infecting mouse lymphocyte cells Ba / F3 (manufactured by RIKEN Cell Bank). After the gene introduction, the cells were cultured in DMEM Medium (Sigma-Aldrich) containing 10% FBS (Corning) and 1 ng / mL recombinant mouse IL-3 (Invitrogen) for 24 hours. Thereafter, 7 μg / mL Blastcidine was added to the medium to select transgenic cells. About 1 week later, the culture medium was replaced with DMEM Medium containing 10% FBS to remove IL-3. About 2 weeks later, the expression of phosphorylated RET in cells grown in the absence of IL-3 was detected by Western blotting, and the cell line Ba / F3 KIF5B-RET stably expressing KIF5B-RET using this as an index (WT) was established.

(2) Evaluation of cell growth inhibitory activity using these cells Inoculate Ba / F3 KIF5B-RET (WT) cells in a 96-well plate with DMEM medium containing 10% FBS so that the number of cells is 2,000 cells per well. did. As a control, Ba / F3 cells (parent) without gene transfer were similarly seeded in the presence of IL-3. Vandetanib diluted to 10 mM in dimethyl sulfoxide was diluted with DMEM medium containing 10% FBS to a final concentration of 1 nM to 10 μM and added to a 96-well plate. As a negative control, DMEM medium containing 10% FBS was added.

  After culturing at 37 ° C. in the presence of 5% CO 2 for 3 days, a cell number measurement reagent CellTiter-Glo (R) Luminescent Cell Viability Assay (manufactured by Promega Corporation) was added and stirred, and a luminescence measuring device TriStar LB 941 (manufactured by Berthold) ) Was used to measure the emission intensity. Viability of Ba / F3 KIF5B-RET cells at each concentration of the compound was determined with the measured value in the negative control well as 0% survival rate and the measured value in the well added with DMEM medium containing 10% FBS as 100% survival rate. Was calculated. The result is shown in FIG. Moreover, IC50 value was calculated | required by the logistic regression method from these values.

As a result, Vandetanib exhibited cell growth inhibitory activity with IC50 values of 56 nM (black triangle in the figure) and 2600 nM (white circle in the figure) against Ba / F3 KIF5B-RET (WT) cells and Ba / F3 parent cells, respectively. It was.
From the above results, it was shown that Vandetanib can inhibit cell proliferation with respect to a cell line expressing KIF5B-RET.

[Example 1] Evaluation of cell growth inhibitory activity against KIF5B-RET-expressing cells having Vandetanib resistance mutation (1) Establishment of KIF5B-RET-expressing cells having Vandetanib resistance mutation Vandetanib resistance mutation (RET genes V804L, Y806C, M918T) A lentivirus was prepared using a lentiviral vector pLenti6 (manufactured by Invitrogen) into which KIF5B-RET having the above was incorporated, and the gene was introduced by infecting mouse lymphocyte cells Ba / F3 (manufactured by RIKEN Cell Bank). After the gene introduction, the cells were cultured in DMEM Medium (Sigma-Aldrich) containing 10% FBS (Corning) and 1 ng / mL recombinant mouse IL-3 (Invitrogen) for 24 hours. Thereafter, 7 μg / mL Blastcidine was added to the medium to select transgenic cells. About 1 week later, the culture medium was replaced with DMEM Medium containing 10% FBS to remove IL-3. About 2 weeks later, the expression of phosphorylated RET was detected in cells grown in the absence of IL-3 by Western blotting, and KIF5B-RET having a Vandetanib resistance mutation (secondary mutation) was stably used as an index. Cell lines (Ba / F3 KIF5B-RET V804L, Ba / F3 KIF5B-RET Y806C, Ba / F3 KIF5B-RET M918T) were established.

(2) Evaluation of cell growth inhibitory activity using the cells 2,000 cells per well of Ba / F3 KIF5B-RET V804L, Ba / F3 KIF5B-RET Y806C, Ba / F3 KIF5B-RET M918T in a 96-well plate Inoculated with DMEM medium containing 10% FBS. As a control, Ba / F3 KIF5B-RET (WT) cells were similarly seeded. Vandetanib or Alectinib diluted to 10 mM in dimethyl sulfoxide was diluted with DMEM medium containing 10% FBS to a final concentration of 1 nM to 10 μM and added to a 96-well plate. As a negative control, DMEM medium containing 10% FBS was added.

  After culturing at 37 ° C. in the presence of 5% CO 2 for 3 days, a cell number measurement reagent CellTiter-Glo (R) Luminescent Cell Viability Assay (manufactured by Promega Corporation) was added and stirred, and a luminescence measuring device TriStar LB 941 (manufactured by Berthold) ) Was used to measure the emission intensity. The survival rate of Ba / F3 KIF5B-RET cells at each concentration of the compound was calculated with the measured value in the medium-only well as 0% survival rate and the measured value in the negative control well as 100% survival rate. The result is shown in FIG.

As shown in FIG. 2A, Ba / F3 KIF5B-RET V804L (white square in the figure), Ba / F3 KIF5B-RET Y806C (white triangle in the figure), Ba / F3 KIF5B-RET M918T (in the figure) , White rhombus) showed resistance to Vandetanib compared to Ba / F3 KIF5B-RET (WT) (black circle in the figure), whereas, as shown in FIG. Was not resistant.
From the above results, it is clear that Alectinib inhibits cell proliferation against a cell line expressing KIF5B-RET having a Vandetanib resistance mutation (secondary mutation).

[Example 2] Evaluation of cell growth inhibitory activity against human lung cancer cell line having Vandetanib resistance (1) Establishment of human lung cancer cell line having Vandetanib resistance Human lung cancer cell line LC2 / ad having CCDC6-RET (RIKEN) (Obtained from Research Institute BioResource Center) was seeded with RPMI-1640 / F-12 medium containing 15% FBS so that the cell count was 100,000 cells per 10 cm plastic dish. Vandetanib was added to a final concentration of 30 nM and cultured for about 2 weeks. By replacing the RPMI-1640 / F-12 medium containing 15% FBS, gradually adding high concentrations of Vandetanib and culturing, the Vandetanib resistant strain LC2 / ad VRA that can finally grow in the presence of 1 μM Vandetanib Obtained. Thereafter, the cells were seeded in a 96-well plate by the limiting dilution method, cultured in RPMI-1640 / F-12 medium containing Vandetanib (1 μM) and 15% FBS, and clone LC2 / ad having the V804L mutation and growing in the presence of Vandetanib. VRA-2 was established. When the sequence of the CCDC6-RET gene was confirmed by the Sanger sequencing method, a Vandetanib resistance mutation (V804L, secondary mutation) was found in LC2 / ad VRA-2 cells.

(2) Evaluation of cell growth inhibitory activity using this cell RPMI-1640 / F-12 medium containing 15% FBS in a 96-well plate so that LC2 / ad VRA-2 is 3,000 cells per well Sowing. As a control, LC2 / ad cells were similarly seeded. Vandetanib or Alectinib diluted to 10 mM in dimethyl sulfoxide was diluted with DMEM medium containing 10% FBS to a final concentration of 1 nM to 10 μM and added to a 96-well plate. RPMI-1640 / F-12 medium containing 15% FBS was added as a negative control.

  After culturing at 37 ° C. in the presence of 5% CO 2 for 3 days, a cell number measurement reagent CellTiter-Glo (R) Luminescent Cell Viability Assay (manufactured by Promega Corporation) was added and stirred, and a luminescence measuring device TriStar LB 941 (manufactured by Berthold) ) Was used to measure the emission intensity. The viability of the cells at each concentration of the compound was calculated with the measured value in the medium-only well as the survival rate of 0% and the measured value in the negative control well as the survival rate of 100%. The result is shown in FIG.

As shown in FIG. 3 (A), LC2 / ad VRA-2 (white triangle in the figure) shows resistance to Vandetanib compared to LC / ad2 (black circle in the figure). The values were 3 μM or more and 232 nM, respectively. On the other hand, as shown in FIG. 3B, LC2 / ad VRA-2 showed sensitivity to Alectinib (black diamond in the figure), and the IC50 value was 295 nM.
From the above results, it is clear that Alectinib also inhibits cell proliferation against a human lung cancer cell line expressing CCDC6-RET having a Vandetanib resistance mutation (secondary mutation).

Example 3 Tumor Cell Growth Inhibitory Effect (1) In Vitro Growth Inhibitory Effect on Human Mesothelioma Cell Line Having NCOA4-RET Human Mesothelioma Cell Line EHMES- Having NCOA4-RET in 96 Well Plate 10 (obtained from Ehime University Second Internal Medicine) was seeded in RPMI-1640 medium containing 10% FBS so that the number of cells was 2,000 cells per well. Vandetanib, Alectinib, or Lenvatinib was diluted with dimethyl sulfoxide to a final concentration of 1 nM to 10 μM and added to a 96-well plate. Dimethyl sulfoxide was added as a negative control.

  After culturing at 37 ° C. in the presence of 5% CO 2 for 3 days, a cell number measurement reagent MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium] (manufactured by Sigma) was added and stirred Thereafter, the absorbance intensity was measured using an absorbance measuring apparatus iMark plate reader (manufactured by BIO-RAD). The viability of the cells at each concentration of the compound was calculated with the measured value in the well containing only the medium as the survival rate of 0% and the measured value in the well containing dimethyl sulfoxide as the survival rate of 100%. The result is shown in FIG.

  As is clear from FIG. 4, EHMES-10 showed sensitivity to any of Vandetanib (white circle in the figure), Allectinib (black circle in the figure), and Lenvatinib (white triangle in the figure).

(2) In vivo growth inhibitory effect on human mesothelioma cell line with NCOA4-RET 6 weeks old Male SHO-scid mouse (CLEA Japan) 100 μL PBS (Gibco) ) EHMES-10 cells (1,000,000 cells) suspended therein were transplanted. From the 15th day, PBS or Alectinib was orally administered once a day for 14 days. The solvent (final concentration) for dissolving the compound was 0.02N HCl, 10% dimethyl sulfoxide, 10% Cremophor EL, 15% PEG400, and 15% HPCD (2-hydroxypropyl-β-cyclodextrin). After killing the mouse, the pleural effusion in the thoracic cavity was collected with a 1 mL syringe (Myjector, Terumo), and the amount of pleural effusion was measured with a scale of the syringe. In addition, the tumor in the thoracic cavity was collected and the tumor weight was measured with an analytical electronic balance (HR-202i, manufactured by AND). The results are shown in Table 1.

As shown in Table 1, Alectinib suppressed the amount of pleural effusion and tumor weight by EHMES-10 in a dose-dependent manner.
From the above results, it is clear that Alectinib inhibits the development of a tumor having a RET fusion gene even in the thoracic cavity where pleural mesothelioma originally exists.

  The present invention relates to a RET gene abnormality, particularly a tumor having a secondary mutation in a fusion gene between a RET gene and another gene, and an RET inhibitor other than the aforementioned compound such as alectinib is invalid or resistant to such a RET inhibitor It is useful as a therapeutic or prophylactic agent for tumors. Specific examples of the tumor include leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, etc.), malignant lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma, etc.), brain tumor, neuroblast Tumor, glioma, thyroid cancer, myelodysplastic syndrome, head and neck cancer, esophageal cancer, stomach cancer, colorectal cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, mesothelioma, pancreatic cancer, liver cancer, gallbladder cancer, Examples include skin cancer, malignant melanoma, renal cancer, renal pelvic and ureteral cancer, bladder cancer, uterine cancer, testicular cancer, prostate cancer, and the present invention is useful as a therapeutic or preventive agent for these cancers. It is.

Claims (5)

A therapeutic or prophylactic agent containing a compound represented by formula (I) or a salt thereof, or a solvate thereof as an active ingredient, wherein the tumor is a tumor having a RET gene abnormality, and RET inhibition is excluded from the compound A therapeutic or prophylactic agent for tumors in which the drug is ineffective or resistant to such RET inhibitors, or metastasis of the tumor.

(Wherein R1 represents alkyl having 1 to 6 carbon atoms.) The therapeutic or prophylactic agent according to claim 1, wherein the tumor is a tumor having a fusion gene of a RET gene and another gene and / or a fusion protein of a RET protein and another protein. The tumor is acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, brain tumor, neuroblastoma, glioma, thyroid cancer, myelodysplastic syndrome, Head and neck cancer, esophageal cancer, stomach cancer, colorectal cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, mesothelioma, pancreatic cancer, liver cancer, gallbladder cancer, skin cancer, malignant melanoma, renal cancer, renal pelvic and ureteral cancer The therapeutic or prophylactic agent according to claim 1 or 2, selected from the group consisting of: bladder cancer, uterine cancer, testicular cancer, prostate cancer, and a tumor metastasized from the tumor. The therapeutic or preventive agent according to any one of claims 1 to 3, wherein the tumor is thyroid cancer, lung cancer, or mesothelioma. The therapeutic agent according to any one of claims 1 to 4, wherein the RET inhibitor excluding the compound is vandetanib, lenvatinib, sunitinib, sorafenib, or sorafenib. Preventive agent.

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