CN116271056A

CN116271056A - Cell death inducing agent for cell having BRAF gene mutation, proliferation inhibiting agent for the cell, and pharmaceutical composition for treating disease caused by abnormal proliferation of the cell

Info

Publication number: CN116271056A
Application number: CN202310302851.0A
Authority: CN
Inventors: 新津洋司郎; 西多裕树
Original assignee: Nitto Denko Corp
Current assignee: Nitto Denko Corp
Priority date: 2015-04-16
Filing date: 2016-04-15
Publication date: 2023-06-23
Also published as: WO2016167340A1; RU2017139718A; RU2760835C2; RU2017139718A3

Abstract

The present invention relates to a cell death inducing agent for a cell having a gene mutation, a proliferation inhibiting agent for the cell, and a pharmaceutical composition for treating a disease caused by abnormal proliferation of the cell. The present invention is directed to cells having mutations in the gene that induce cell death or inhibit cell proliferation. The present invention contains a pi-inhibiting drug as an active ingredient.

Description

Cell death inducing agent for cell having BRAF gene mutation, proliferation inhibiting agent for the cell, and pharmaceutical composition for treating disease caused by abnormal proliferation of the cell

The present application is a divisional application of chinese patent application No.201680021988.7 (PCT application No. PCT/JP 2016/062090) having the title of "a cell death inducing agent for a cell having a BRAF gene mutation, a proliferation inhibiting agent for the cell, and a pharmaceutical composition for treating a disease caused by abnormal proliferation of the cell".

Technical Field

The present invention relates to a cell death inducing agent for a cell having a BRAF gene mutation (for example, a cancer cell), a proliferation inhibiting agent for the cell, a pharmaceutical composition for treating a disease caused by abnormal proliferation of the cell, and a method for screening a cell death inducing agent and/or a cell proliferation inhibiting agent.

Background

The BRAF gene is a gene encoding one of serine/threonine kinases constituting the RAS-RAF-MAPK pathway. BRAF gene mutations in various tumors have been reported. For example, a mutation (V600E) in which valine at position 600 is replaced with glutamic acid can be confirmed in various cancer cells. BRAF with the V600E mutation is known to activate downstream signals from time to time, leading to proliferation of cells even without stimulation from outside the cell.

For example, in many cases of colorectal cancer (5 to 15%), melanoma (about 60%), BRAF gene having V600E mutation was confirmed. Mutations in the KRAS gene are frequently confirmed in various cancers such as pancreatic cancer and colorectal cancer. KRAS proteins are G proteins that are locally present inside the cell membrane. KRAS et al RAS form the following cascade: this activates RAF such as CRAF and BRAF, which in turn activates MEK, which activates MAPK (non-patent documents 1 and 2). It is rare that the BRAF mutation and the KRAS mutation are in exclusive relation to each other (non-patent document 3).

Currently, for cancers in which BRAF having the V600E mutation can be identified, treatment with inhibitors against BRAF having the V600E mutation is considered effective. As such inhibitors, vemurafenib (PLX 4032), PLX4720, and the like are known. However, there are cases where cancer cells acquire resistance to the inhibitor due to the sustained administration of the inhibitor, and the therapeutic effect is limited. Thus, there is a need for cell death inducing agents and proliferation inhibiting agents that are more effective against cancer cells having mutations in the BRAF gene, in place of these inhibitors.

glutathione-S-transferase (GST) is one of enzymes catalyzing glutathione conjugation, and is known as an enzyme that conjugates a substance such as a drug with Glutathione (GSH) to form a water-soluble substance. GST is typically classified into 6 isozymes of α, μ, ω, pi, θ and ζ based on the amino acid sequence. Among them, in particular, expression of GST-pi (also called GSTP 1) is elevated in various cancer cells, which has been pointed out to be one of the reasons for resistance against some anticancer agents. In fact, it is known that when GST-pi inhibitor is allowed to act on a cancer cell line that overexpresses GST-pi and exhibits drug resistance, drug resistance is inhibited (non-patent documents 4 to 6). In addition, in recent reports, it has been reported that when an siRNA against GST-pi is allowed to act on an androgen-independent prostate cancer cell line that overexpresses GST-pi, proliferation thereof is inhibited and apoptosis thereof increases (non-patent document 7).

In addition, GST-. Pi.is known to form a complex with c-Jun N-terminal kinase (JNK) to inhibit the activity of JNK (non-patent document 8). Furthermore, GST-pi is known to be involved in S-glutathionylation of a protein involved in cellular stress reaction (non-patent document 9). Furthermore, GST-. Pi.is also known to contribute to the protective effect against Reactive Oxygen Species (ROS) -induced cell death (non-patent document 10). It will be appreciated that GST-pi in GST has various features/functions.

It has been reported that when an siRNA against GST-pi is applied to a cancer cell line having a mutation in KRAS, activation of Akt is inhibited and autophagy is enhanced, but induction of apoptosis is moderate (non-patent document 11). Patent document 1 discloses that apoptosis of cancer cells can be induced by using a drug that inhibits GST-pi and an autophagy inhibitor such as 3-methyladenine as active ingredients. Patent document 2 discloses that, when expression of GST-pi, akt, and the like is simultaneously repressed, cell proliferation can be suppressed, cell death can be induced, and autophagy induced by expression repression of GST-pi can be more significantly inhibited by simultaneously repressing expression of Akt, and the like.

However, there is no knowledge about the relationship between GST-pi expression and cell proliferation or cell death in the above-described cells having a mutation in the BRAF gene, the action of GST-pi related to signal transduction, and the like.

Prior art literature

Patent literature

Patent document 1: WO 2012/176882

Patent document 2: WO2014/098210

Non-patent literature

Non-patent document 1: curr Top Microbiol immunol.2012;355:83-98

Non-patent document 2: curr Opin Pharmacol.20088 Aug;8 (4):419-26.

Non-patent document 3: british Journal of Cancer (2013) 108,1757-1764.

Non-patent document 4: takahashi and Niitsu, gan To Kagaku ryoho.1994;21 (7):945-51

Non-patent document 5: ban et al, cancer Res.1996;56 (15):3577-82

Non-patent document 6: nakajima et al, J Pharmacol Exp Ther.2003;306 (3):861-9

Non-patent document 7: hokaiwado et al, carcinogensis.2008; 29 (6):1134-8

Non-patent document 8: adler et al, EMBO J.1999,18,1321-1334

Non-patent document 9: townsend et al, J.biol. Chem.2009,284,436-445 non-patent document 10: YIn et al, cancer Res.2000, 4053-4057

Non-patent document 11: nishita et al, AACR 102nd Annual Meeting,Abstract No.1065

Disclosure of Invention

Problems to be solved by the invention

Accordingly, an object of the present invention is to provide an agent having a cell death inducing effect and/or a cell proliferation inhibiting effect on a cell having a mutation in a BRAF gene, to provide a pharmaceutical composition for treating a disease caused by abnormal cell proliferation, and to provide a method for screening a cell death inducing agent and/or a cell proliferation inhibiting agent.

Means for solving the problems

In view of the above-described object, the inventors of the present application have conducted intensive studies and as a result have found that, in a cell having a mutation in a BRAF gene, cell proliferation can be effectively inhibited when GST-pi is inhibited, and that, in a cell having a mutation in a BRAF gene that exhibits resistance to a conventionally known BRAF inhibitor, cell proliferation can be effectively inhibited when GST-pi is inhibited, leading to completion of the present invention. The present invention includes the following.

(1) A cell death inducing agent for cells having a mutation in a BRAF gene, which contains a GST-pi inhibiting drug as an active ingredient.

(2) A cell proliferation inhibitor for cells having a mutation in the BRAF gene, which contains a GST-pi-inhibiting agent as an active ingredient.

(3) The agent of (1) or (2), wherein the mutation is a V600E mutation.

(4) The agent according to (1) or (2), wherein the cell having a mutation in the BRAF gene is a cell resistant to a BRAF inhibitor.

(5) The agent according to (1) or (2), wherein the drug is a substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors expressing at least 1 of them.

(6) The agent according to (1) or (2), wherein the cell having a mutation in the BRAF gene is a cancer cell expressing GST-pi at a high level.

(7) A pharmaceutical composition for treating a disease caused by abnormal proliferation of a cell having a mutation in a BRAF gene, comprising the agent of any one of (1) to (6) above.

(8) The pharmaceutical composition of (7), wherein the disease is cancer.

(9) The pharmaceutical composition of (8), wherein the cancer is a cancer that expresses GST-pi at a high level.

(10) The pharmaceutical composition according to (8), wherein the cancer is colorectal cancer or melanoma.

(11) A method of screening for a cell death inducing agent and/or a cell proliferation inhibiting agent for a cell having a mutation in a BRAF gene, the method comprising: the selection of agents that inhibit GST-pi is performed.

(12) The screening method according to (11), comprising the steps of: a step of contacting a test substance with a cancer cell; a step of measuring the GST-pi expression amount in the cells; and a step of selecting the test substance as the GST-pi inhibiting agent when the expression level is reduced as compared with the case of measuring in the absence of the test substance.

The present specification contains the disclosure of Japanese patent application No. 2015-84286 and Japanese patent application No. 2016-78710, which are the basis of the priority of the present application.

Effects of the invention

The cell death inducing agent according to the present invention can induce cell death very efficiently against cells having a mutation in the BRAF gene. Therefore, the cell death inducing agent according to the present invention can exhibit a very high efficacy as a pharmaceutical composition for treating a disease caused by abnormal proliferation of cells having a mutation in the BRAF gene.

In addition, the cell proliferation inhibitor according to the present invention can inhibit cell proliferation very effectively against cells having a mutation in the BRAF gene. Therefore, the cell proliferation inhibitor according to the present invention can exhibit a very high efficacy as a pharmaceutical composition for treating a disease caused by abnormal proliferation of cells having a mutation in the BRAF gene.

In addition, according to the screening method of the present invention, a drug that very efficiently induces cell death and/or inhibits cell proliferation against a cell having a mutation in the BRAF gene can be selected.

Drawings

FIG. 1 is a characteristic diagram showing the results (cell number measurement results, western blotting results) of verifying the effect of inhibiting cell proliferation by GST-pi knock-down in a colorectal cancer cell line having a BRAF gene mutation.

FIG. 2 is a characteristic diagram showing the results (cell number measurement results, western blotting results) of verifying the effect of inhibiting cell proliferation by GST-pi knockout in a melanoma cell line having a BRAF gene mutation.

FIG. 3 is a characteristic diagram showing the results of verifying the effect of GST-pi siRNA on inhibition of cell proliferation in a strain of colorectal cancer cells having a BRAF gene mutation, when the GST-pi siRNA was combined with a BRAF inhibitor.

FIG. 4 is a characteristic diagram showing the results of verifying the effect of inhibiting cell proliferation in a melanoma cell line having a BRAF gene mutation by administering GST-. Pi.siRNA and a BRAF inhibitor in combination.

FIG. 5 is a characteristic diagram showing the results of verifying the effect of inhibiting cell proliferation by GST-pi knock-down in a melanoma cell line having a BRAF gene mutation and having resistance to a BRAF inhibitor.

FIG. 6 is an electrophoretogram showing the results of analysis of GST-pi expression when GST-pi is knocked down in a melanoma cell line having a BRAF gene mutation and having resistance to a BRAF inhibitor by Western blotting.

Detailed Description

The cell death inducing agent and the cell proliferation inhibiting agent according to the present invention contain a GST-pi-inhibiting drug as an active ingredient. The cell death induction agent and the cell proliferation inhibition agent according to the present invention exhibit a cell death induction effect and a cell proliferation inhibition effect on a cell having a mutation in a BRAF gene.

GST-pi, as used in this specification, refers to an enzyme encoded by the GSTP1 gene that catalyzes glutathione conjugation. GST-pi exists in a variety of animals, including humans, and its sequence information is also known (e.g., human: nm_000852 (np_000843), rat: NM-012577 (NP-036709), mouse NM-013541 (NP-038569), etc. the numbers represent the accession numbers of the NCBI database, with the base sequence numbers outside the numbering and the amino acid sequence numbers within brackets. As an example, the nucleotide sequence of the coding region of the human GST-pi gene registered in the database is shown in SEQ ID NO. 1, and the amino acid sequence of the human GST-pi protein encoded by the human GST-pi gene is shown in SEQ ID NO. 2.

As described above, GST-pi can be determined by a specific base sequence and amino acid sequence, but it is also necessary to consider the possibility of occurrence of a mutation (including polymorphism) in the base sequence and amino acid sequence between biological individuals. That is, GST-pi is not limited to a protein having the same sequence as the amino acid sequence registered in the database, but includes a protein having a sequence different from the sequence by 1 or 2 or more (typically 1 or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids and having a function equivalent to GST-pi. GST-pi also includes a sequence encoding a protein having a function equivalent to GST-pi, which is composed of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 97% identity with the above specific nucleotide sequence. The specific function of GST-. Pi.is as described above, and means an enzyme activity for catalyzing conjugation of glutathione.

Unless otherwise indicated, the terms "used in the present specification", "described in the present specification" and the like mean that all the inventions described in the present specification are described as follows. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. All patents, publications, and other publications referred to in this specification are also incorporated in their entirety into this specification.

The "GST-pi-inhibiting agent" used in the present specification is not limited, and includes, for example, an agent that inhibits the production of GST-pi and/or the activity of GST-pi, an agent that promotes the decomposition and/or inactivation of GST-pi, and the like. Examples of the drug for suppressing the production of GST-pi include, but are not limited to, RNAi molecules against DNA encoding GST-pi, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors for expressing the same.

In addition, as a drug for inhibiting GST-pi, any compound that acts against GST-pi can be used. As such a compound, an organic compound (an amino acid, a polypeptide or a derivative thereof, a low molecular compound, a sugar, a high molecular compound, or the like), an inorganic compound, or the like can be used. In addition, such compounds may be any of natural substances and non-natural substances. Examples of the derivative of the polypeptide include a modified polypeptide obtained by adding a modifying group, and a variant polypeptide obtained by changing an amino acid residue. Such a compound may be a single compound, or may be a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a microbial fermentation product, a marine organism extract, a plant extract, or the like. That is, the "GST-pi-inhibiting agent" is not limited to nucleic acids such as RNAi molecules, but includes any compound.

Specifically, examples of the drug that inhibits GST-pi activity include, for example, substances that bind GST-pi, such as glutathione, glutathione analogs (for example, substances described in WO 95/08563, WO 96/40205, WO 99/54346, non-patent document 4, etc.), ketoprofen (non-patent document 2), indomethacin (Hall et al, cancer Res.1989;49 (22): 6265-8), ethacrynic acid, pioglitazone (Tew et al, cancer Res.1988;48 (13): 3622-5), anti-GST-pi antibodies, dominant negative mutants of GST-pi, etc., but are not limited thereto. These drugs may be commercially available or may be appropriately manufactured based on known techniques.

As a drug for inhibiting the production of GST-pi or the activity thereof, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors for expressing the same are preferable from the viewpoints of high specificity and low possibility of side effects.

Inhibition of GST-pi can be determined from the following phenomena: GST-pi expression and activity are inhibited in cells, as compared with the case where GST-pi inhibitor is not allowed to act. The expression of GST-pi can be evaluated by any known method, and examples thereof include, but are not limited to, immunoprecipitation using an anti-GST-pi antibody, EIA, ELISA, IRA, IRMA, western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, hybridization using a nucleic acid specifically hybridizing to a nucleic acid encoding GST-pi or a specific fragment thereof or a transcription product (e.g., mRNA) or a splice product of the nucleic acid, northern blotting, southern blotting, and various PCR methods.

In addition, the activity of GST-pi can be evaluated by analyzing the known activity of GST-pi by any known method, including, for example, binding to a protein such as Raf-1 (particularly phosphorylated Raf-1) or EGFR (particularly phosphorylated EGFR), etc., including, for example, immunoprecipitation, western blotting, mass spectrometry, downdraw method (pull-down), surface Plasmon Resonance (SPR), etc., without limitation.

As used herein, RNAi molecules refer to any molecule that causes RNA interference, and include, without limitation, double-stranded RNAs such as siRNA (small interfering RNA), miRNA (microrna), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeated-related siRNA), modifications thereof, and the like. These RNAi molecules may be commercially available or may be designed and prepared based on known sequence information (i.e., the base sequence shown in SEQ ID NO. 1 and/or the amino acid sequence shown in SEQ ID NO. 2). In addition, in the present specification, antisense nucleic acid includes RNA, DNA, PNA, or a complex thereof.

The DNA/RNA chimeric polynucleotide, when used in the present specification, is not limited, but includes, for example, a double-stranded polynucleotide composed of DNA and RNA for repressing the expression of a target gene as described in Japanese patent application laid-open No. 2003-219893.

When the agent or composition is administered, the amount of the active ingredient contained in the agent or composition of the present invention may be an amount capable of inducing cell death such as apoptosis and/or inhibiting cell proliferation. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount may be known or may be appropriately determined by an in vitro test using cultured cells or the like, a test in a model animal such as a mouse, rat, dog or pig, etc., such a test method being well known to those skilled in the art. Induction of apoptosis can be evaluated by various known methods, for example, detection of phenomena unique to apoptosis such as DNA fragmentation, binding of annexin V to cell membrane, change in mitochondrial membrane potential, activation of caspase, TUNEL staining, and the like. The inhibition of cell proliferation can be evaluated by various known methods, for example, measurement of the number of living cells with time, measurement of the size, volume or weight of a tumor, measurement of DNA synthesis, WST-1 method, brdU (bromodeoxyuridine) method, 3H thymidine incorporation method, and the like. The amount of active ingredient may vary depending on the agent or the mode of administration of the composition. For example, in the case where a plurality of units of the composition are used in 1 administration, the amount of the active ingredient blended in each 1 unit of the composition may be set to one of a plurality of parts of the amount of the active ingredient necessary for 1 administration. The amount to be blended can be appropriately adjusted by those skilled in the art.

In addition, a cell death-inducing agent, a cell proliferation-inhibiting agent, a cell death-inducing composition or a cell proliferation-inhibiting composition can be produced by incorporating a GST-pi-inhibiting drug as an active ingredient.

In addition, a GST-pi-inhibiting agent for cell death induction or cell proliferation inhibition can be provided. In addition, methods of inducing cell death or inhibiting cell proliferation may be provided, which methods comprise administering an effective amount of a GST-pi inhibiting agent.

The method of inducing apoptosis and the like and the method of inhibiting cell proliferation may be either in vitro or in vivo. In addition, for the drug in the method, as described above, an effective amount of the drug may be an amount that induces cell death, or inhibits cell proliferation, in the cells administered. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount may be known or may be appropriately determined by an in vitro test using cultured cells or the like, such test methods being well known to those skilled in the art. Induction of cell death or inhibition of cell proliferation can be evaluated by various known methods including the above-described methods. For such effective amounts, it is not necessary that all cells in the same cell population undergo cell death or proliferation inhibition when the drug is administered to a prescribed cancer cell population. For example, the effective amount may be an amount that inhibits apoptosis or proliferation of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 8% or more, 10% or more, 12% or more, 15% or more, 20% or more, 25% or more of the cells in the cell population.

The cell death inducing agent and the cell proliferation inhibiting agent according to the present invention act on a cell having a mutation in the BRAF gene. The cell having a mutation in the BRAF gene refers to a cell (typically, a cancer cell) that shows abnormal proliferation due to the mutation in the BRAF gene.

It is particularly preferable to apply the cell death inducing agent and the cell proliferation inhibiting agent according to the present invention to a cell expressing GST-pi at a high level (typically, a cancer cell expressing GST-pi at a high level) among cells showing abnormal proliferation due to mutation in BRAF gene. Here, the expression level of GST-. Pi.is a significantly higher expression level of GST-. Pi.than that of a normal cell in a cell in which the BRAF gene has a mutation and shows abnormal cell proliferation. The GST-pi expression level can be measured by a conventional method such as RT-PCR or microarray.

The mutation in the BRAF gene refers to a mutation such as deletion, substitution, addition, or insertion with respect to the amino acid sequence of the wild-type BRAF, or a mutation in the expression control region of the BRAF gene. Here, the mutation in the BRAF gene is a so-called gain-of-function mutation (gain of function mutation). That is, a BRAF gene having a mutation (sometimes also referred to as a mutant BRAF gene) includes a gene encoding a mutant BRAF having enhanced serine/threonine kinase activity due to the mutation. In addition, mutant BRAF genes include genes that contain mutations in the expression control regions and have increased expression levels compared to wild-type BRAF genes. That is, the cell expressing the mutant BRAF gene has the following characteristics: mutant BRAF is expressed, or the expression level of BRAF is increased, whereby a signal transduced downstream from BRAF (for example, a signal transduced to MEK) is constantly sustained.

Examples of the mutant BRAF gene include a gene encoding a mutant BRAF (hereinafter referred to as V600 e) in which valine (which is encoded by codon 600in the wild-type BRAF gene) is substituted for glutamic acid, V600D, V600G, V600K, V600M, V600R, V600L, G469A, G469V, D594N, V insT (insert T), and the like.

The cell death inducing agent or the cell proliferation inhibiting agent of the present invention is effective as a component of a pharmaceutical composition for a disease caused by abnormal proliferation of cancer cells having a mutation in a BRAF gene because it can effectively induce cell death or inhibit cell proliferation even in cancer cells having a mutation in a BRAF gene. In addition, by incorporating a drug that inhibits GST-pi as an active ingredient, a pharmaceutical composition against a disease caused by abnormal proliferation of cancer cells having a mutation in the BRAF gene can be produced. Furthermore, a disease caused by abnormal cell proliferation may be treated/treated, which includes a step of administering the manufactured pharmaceutical composition to a subject in need thereof in an effective amount.

The pharmaceutical composition is effective for the treatment of diseases caused by abnormal proliferation of cancer cells having a mutation in the BRAF gene. Examples of the diseases caused by cancer cells having a mutation in the BRAF gene include, but are not limited to, cancers expressing GST-. Pi.at high levels, and in many cases, cancers having a mutation in the BRAF gene are included in cancers expressing GST-. Pi.at high levels.

Examples of the cancers include, but are not limited to, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, and the like; eye cancer, thyroid cancer (papillary carcinoma), meningioma, brain tumor, pituitary cancer, salivary gland cancer, head and neck cancer, breast cancer, lung cancer (non-small cell lung cancer), esophagus cancer, stomach cancer, duodenum cancer, appendiceal cancer, large intestine cancer, rectal cancer, liver cancer, pancreas cancer, gall bladder cancer, bile duct cancer, anus cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penis cancer, testis cancer, uterine cancer (endometrial cancer), ovarian cancer (ovarian serous cancer), vulval cancer, vaginal cancer, skin cancer (malignant melanoma), and the like, leukemia, malignant lymphoma, and the like. In the present invention, "cancer" includes epithelial malignant tumor and non-epithelial malignant tumor. Cancers in the present invention may exist in any part of the body, for example, brain, head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gall bladder, anus, kidney, ureter, bladder, prostate, penis, testis, uterus, ovary, vulva, vagina, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, vascular system, lymph system such as lymph node, lymph fluid, and the like.

In particular, the cell death inducing agent or cell proliferation inhibiting agent of the present invention is also effective in inducing cell death or inhibiting cell proliferation against cells having acquired resistance to a BRAF inhibitor among cells having a mutation in a BRAF gene. Therefore, the cell death inducing agent or the cell proliferation inhibiting agent of the present invention is effective as a component of a pharmaceutical composition for a disease caused by abnormal proliferation of cancer cells having a mutation in the BRAF gene and having resistance to a BRAF inhibitor. In addition, by incorporating a drug that inhibits GST-pi as an active ingredient, a pharmaceutical composition against a disease caused by abnormal proliferation of cancer cells that have a mutation in the BRAF gene and are resistant to a BRAF inhibitor can be produced. Furthermore, a disease caused by abnormal cell proliferation may be treated/treated, which includes a step of administering the manufactured pharmaceutical composition in an effective amount to a patient in need of the pharmaceutical composition, i.e., a patient having a reduced therapeutic effect of a BRAF inhibitor.

The BRAF inhibitor herein refers to a substance that inhibits signal transduction downstream from BRAF, and in particular includes a substance that specifically inhibits signal transduction by the mutant BRAF having the functional gain mutation (gain of function mutation) described above. As BRAF inhibitors, for example, vemurafenib (PLX 4032), ca s No.:918504-65-1, N- [3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b ] pyridine-3-carbonyl ] -2,4-difluorophenyl ] -1-propanesulfonamide, N- [3- [5- (4-chloropheny l) -1H-pyrrorolo [2,3-b ] pyridine-3-carboyl ] -2,4-difluorophenyl ] propane-1-sulfonamide, PLX4720 (Cas No.:918505-84-7, N- [3- (5-chloro-1H-pyrrolo [2,3-b ] pyridine-3-carbonyl) -2,4-difluorophenyl ] -1-propanesulfonamide, N- [3- (5-chloro-1H-pyrro [2,3-b ] pyridine-3-sulfophenyl) -2,4-difluorophenyl ] propane-1-sulfoamide, etc. are known.

Further, as BRAF inhibitors, there may be mentioned, for example, regorafenib (regorafenib), dasatinib (dasatinib), PLX-8394, beiGene-283, PLX-3603, RG-7304 (CAS No.: 213406-50-9), LY-3009120 (CAS No.: 1454682-72-4), rebastinib (Cas. No.: 1020172-07-9), 1H-pyrazolo [3,4-b ] pyridine-5-carboxamide analog, ASN-003, williams, N- (thiophen-2-yl) benzamide derivatives, DCB-R0237, REDX-04988, EBI-907, EBI-945, cotton cellulose (gossypin), naipole-007, TEW-0201, miRNA-3157 and thiazole derivatives (NMS-P186, NMS-P285, NMS-P383, NMS-P349, NMS-P730 and NMS-P730.

Further, examples of BRAF inhibitors include, in addition to the above-mentioned inhibitors: SB590885 (Cas No.:405554-55-4, N-dimethyl-2- [4- [ (4Z) -4- (1-nitroso-2, 3-indan-5-ylidene) -5- (1H-pyridin-4-ylidene) -1H-imidazol-2-yl ] phenoxy ] ethanamine), B-Raf inhibitor 1 (Cas No.:1093100-40-3,1-N- (4-chlorophenyl) -6-methyl-5-N- [3- (7H-purin-6-yl) pyridin-2-yl ] isoquinoline-1, 5-diamine), B-Raf inhibitor 1 dihydrochloride (Cas No.:1191385-19-9,1-N- (4-chlorophenyl) -6-methyl-5-N- [3- (7H-purin-6-yl) pyridin-2-yl ] isoquinoline-1, 5-diamine; dihydrochloride), dabrafilib (Cas No.:1195765-45-7, N- [3- (7H-chlorophenyl) -6-methyl-5-N- [3- (7-pyridin-6-yl) pyridin-2-yl ] isoquinoline-1, 5-diamine), cas No.: 1191385-9, 1-N- (4-chlorophenyl) -6-methyl-5-N- [3- (7H-imidazol-6-yl) pyridin-2-yl ] isoquinoline-1, 5-diamine, n- [ (2S) -1- [ [4- [3- [ 5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl ] -1-propan-2-yl-pyrazol-4-yl ] pyrimidin-2-yl ] amino ] propan-2-yl ] carbamic acid methyl ester), HG6-64-1 (CasNo.: 1315329-43-1, see WO 2011/090738), PF-04880594 (CasNo.: 1111636-35-1,3- [ [4- [1- (2, 2-difluoroethyl) -3- (1H-pyrrolo [2,3-B ] pyridin-5-yl) pyrazol-4-yl ] pyrimidin-2-yl ] amino ] propionitrile), BRAF inhibitor (CasNo.: 918505-61-0, N- [2, 4-difluoro-3- (5-pyridin-3-yl-1H-pyrrolo [2,3-B ] pyridin-3-carbonyl) phenyl ] -2-propanesulfonamide), B-Raf inhibitor (CasNo.: 1315330-11.), n- [4- [ (4-ethylpiperazin-1-yl) methyl ] -3- (trifluoromethyl) phenyl ] -4-methyl-3- [ (6-methyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy ] benzamide), TAK-632 (Cas No.:1228591-30-7, N- [ 7-cyano-6- [ 4-fluoro-3- [ [2- [3- (trifluoromethyl) phenyl ] acetyl ] amino ] phenoxy ] -1, 3-benzothiazol-2-yl ] cyclopropanecarboxamide), AZ 628 (Cas No.:878739-06-1,3- (2-cyano-propan-2-yl) -N- [ 4-methyl-3- [ (3-methyl-4-oxoquinazolin-6-yl) amino ] phenyl ] benzamide), RAF (Cas No.:927880-90-8, 1-methyl-5- [2- [5- (trifluoromethyl) -1H-imidazol-2-yl ] pyridin-4-yl ] oxy ] N- [4- (trifluoromethyl) phenyl ] benzamide), cas No.:927880-90-8, 1-methyl-5- [4- (trifluoromethyl) phenyl ] N- [4- (35-methyl-2-phenyl ] benzamide, cas No.: 0-35 (Cas No.: 0-35), 1- [3- (6, 7-dimethoxyquinazolin-4-yl) oxyphenyl ] -3- [5- (1, 1-trifluoro-2-methyl-propan-2-yl) -1, 2-oxazol-3-yl ] urea), GDC-0879 (Cas No.:905281-76-7,2- [4- [ (1E) -1-hydroxyimino ] -2, 3-indan-5-yl ] -3-pyridin-4-yl-pyrazol-1-yl ] ethanol), sorafenib tosylate (Cas No.:475207-59-1,4- [4- [ [ 4-chloro-3- (trifluoromethyl) phenyl ] carbamoyl amino ] phenoxy ] -N-methylpyridine-2-carboxamide; 4-methylbenzenesulfonic acid), dabrafenib tosylate (casno.: 1195768-06-9,N- [3- [5- (2-aminopyrimidin-4-yl) -2-tert-butyl-1, 3-thiazol-4-yl ] -2-fluorophenyl ] -2, 6-difluorobenzenesulfonamide; methanesulfonic acid), CEP-32496 hydrochloride (casno.: 1227678-26-3,1- [3- (6, 7-dimethoxyquinazolin-4-yl) oxyphenyl ] -3- [5- (1, 1-trifluoro-2-methyl-propan-2-yl) -1, 2-oxazol-3-yl ] urea; hydrochloride), sorafenib (Cas No.:284461-73-0,4- [4- [ [ 4-chloro-3- (trifluoromethyl) phenyl ] carbamoylamino ] phenoxy ] -N-methylpyridine-2-carboxamide).

The pharmaceutical composition of the present invention may be used in combination with other active ingredients in addition to the GST-pi-inhibiting drug. Here, the term "combination" includes, for example, the following cases: administering the other active ingredients in separate formulations; and administering the other active ingredient in a mixture with at least 1 other drug; etc. When administered as a separate formulation, the formulation containing the other active ingredient may be administered before, simultaneously with, or after the other formulation.

As the other active ingredient, the above BRAF inhibitor can be suitably used. The other active ingredient is an ingredient effective for the treatment of a disease to be treated. For example, in the case where the disease to be treated is cancer, an anticancer agent may be used in combination. Examples of anticancer agents include alkylating agents such as ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, melphalan, and ramustine (ranimustine), antitumor antibiotics such as gemcitabine hydrochloride, enocitabine hydrochloride, sodium stearyl phosphate (cytarabine ocfosfate), cytarabine sulfate, tegafur/uracil (tegafur uracil), tegafur/gemustine/potassium oxazinate complex (tegafur-gimeracil-oteracil potassium combination drugs) (for example, TS-1), metabolic antagonists such as doxifluridine, hydroxyurea, fluorouracil, methotrexate, mercaptopurine, nordaunorubicin hydrochloride, epirubicin hydrochloride, daunorubicin citrate, doxorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, mitomycin C, etoposide, irinotecan hydrochloride, vinblastine, tarabine, fluvoxamine, dacarbazine, dp, and other antitumor antibiotics such as fluvoxamine, and cisplatin sulfate, and other therapeutic agents such as fluvoxamine, and cisplatin, and other therapeutic agents.

In the case where the active ingredient in the various reagents, compositions, methods of treatment and the like of the present invention described in the present specification is a nucleic acid, for example, an RNAi molecule, a ribozyme, an antisense nucleic acid, a DNA/RNA chimeric polynucleotide and the like, the above nucleic acid may be used as a naked nucleic acid directly or may be carried on various vectors. As the vector, any known vector such as a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a viral vector and the like can be used. The vector preferably contains at least a promoter (promoter) which enhances the expression of the carried nucleic acid, in which case the nucleic acid is preferably operably linked to said promoter. By operably linked to a promoter is meant that the nucleic acid and promoter are configured such that the protein encoded by the nucleic acid is properly produced by the action of the promoter. The vector may be a vector capable of replication in a host cell, may be a vector incapable of replication in a host cell, and may be a vector capable of transcription of a gene outside or inside the nucleus of a host cell. In the latter case, the nucleic acid may also be incorporated into the genome of the host cell.

In addition, the active ingredient may be carried on various nonviral lipid or protein carriers. Examples of the carrier include, but are not limited to, cholesterol, liposome, antibody precursor, cyclodextrin nanoparticle, fusion peptide, aptamer, biodegradable polylactic acid copolymer, polymer, etc., whereby the uptake efficiency into cells can be improved (see, for example, pirollo and Chang, cancer res.2008;68 (5): 1247-50, etc.). Cationic liposomes, polymers (e.g., polyethylenimine, etc.), are particularly useful. Further examples of the polymer that can be used as the support include supports described in, for example, US 2008/0207553, US 2008/0312174, and the like.

In the various pharmaceutical compositions of the present invention described in the present specification, the active ingredient may be combined with other optional ingredients as long as the effect of the active ingredient is not impaired. Examples of such optional components include other chemotherapeutic agents, pharmaceutically acceptable carriers, excipients, diluents, and the like. The composition may be coated with a suitable material, for example, an enteric coating or a time-lapse disintegrable material, depending on the route of administration and the form of drug release, or may be incorporated into a suitable drug release device.

The various agents and compositions of the present invention described in the present specification (including various pharmaceutical compositions) can be administered by various routes including oral and non-oral routes (for example, oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, portal intravenous, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, etc., without limitation), and can be prepared into dosage forms suitable for each route of administration. The dosage form and the preparation method may be appropriately any known method (see, for example, standard pharmaceutical science, du-on-side happiness et al, south Jiang Tang, 2003, etc.).

For example, the dosage form suitable for oral administration is not limited, and examples thereof include powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups, and the like, and examples thereof suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, and pre-use formulation injections. Formulations for non-oral administration may be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.

The various agents or compositions of the invention described in this specification (including various pharmaceutical compositions) can be targeted to specific tissues, cells. Targeting can be achieved by any known method. In the case where delivery to cancer is desired, for example, the following methods and the like can be used without limitation: passive targeting is carried out by making the formulation of a size suitable for exerting EPR (high permeability and retention, enhanced permeability and retention) effects, of diameter 50-200 nm (in particular 75-150 nm, etc.); active targeting, which uses ligands such as CD19, HER2, transferrin receptor, folate receptor, VIP receptor, EGFR (Torulin, AAPS J.2007;9 (2): E128-47), RAAG10 (Japanese patent Table 2005-532050), PIPA (Japanese patent Table 2006-506071), KID3 (Japanese patent Table 2007-52997), peptides having RGD motif, NGR motif, F3, lyP-1 (Ruoslahti et al, J Cell biol.2010;188 (6): 759-68) and the like as targeting agents. In addition, retinoids (retinoids) or derivatives thereof are also known to be useful as targeting agents for cancer cells (WO 2008/120815), and thus carriers containing retinoids as targeting agents can also be utilized. In addition to the above documents, the carriers are described in WO 2009/036368, WO 2010/014117, WO 2012/170952, and the like.

The various reagents and compositions of the invention described in the present specification (including various pharmaceutical compositions) may be supplied in any form, and from the viewpoint of storage stability, may be supplied in a form that can be prepared before use, for example, in a form that can be prepared by a doctor and/or pharmacist, nurse, or other auxiliary medical staff at or near a medical site. This form is particularly useful when the reagent or composition of the present invention contains a component such as a lipid, a protein, or a nucleic acid, which is difficult to stably preserve. In this case, the agent or composition of the present invention is provided in the form of 1 or 2 or more containers containing at least 1 of the above-mentioned essential components, and can be formulated before use, for example, within 24 hours, preferably within 3 hours, and more preferably immediately before use. In the preparation, reagents, solvents, dispensing devices, etc. generally available at the preparation site can be used as appropriate.

Thus, the invention also relates to: a kit for formulating a composition comprising 1 or 2 or more containers containing, either alone or in combination, the active ingredients that may be contained in the various agents or compositions of the invention; and, necessary components of various reagents or compositions provided in the form of such kits. The kit of the present invention may contain instructions describing the preparation method, administration method, etc. of the various reagents or compositions of the present invention, for example, instructions, electronic recording media such as a CD, DVD, etc., in addition to the above. In addition, the kit of the present invention may contain all the components of the various reagents or compositions for carrying out the present invention, but not necessarily all the components. Therefore, the kit of the present invention may not contain reagents, solvents, such as sterile water, physiological saline, glucose solution, etc., which are generally available at medical sites, experimental institutions, etc.

The effective amount in the various treatment methods of the present invention described in the present specification is, for example, an amount that alleviates symptoms of a disease or delays or stops the progression of a disease, and is preferably an amount that inhibits or cures a disease. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount can be suitably determined by in vitro tests using cultured cells or the like, tests in model animals such as mice, rats, dogs or pigs, and such test methods are well known to those skilled in the art. In addition, the amount of the drug to be used in the treatment method of the present invention is known to those skilled in the art, or can be appropriately determined by the above-described test or the like.

The specific amount of active ingredient administered in the treatment method of the present invention described in the present specification can be determined in consideration of the following various conditions related to the subject to be treated: for example, the severity of the symptoms, the general health of the subject, age, weight, sex of the subject, diet, period and frequency of administration, the medicines used, responsiveness to treatment, dosage form, and compliance with treatment, etc.

The administration route includes: various routes including both oral and non-oral, e.g., oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, portal intravenous, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, and the like.

The administration frequency may vary depending on the agent used, the nature of the composition, and the conditions of the subject including the above conditions, and may be, for example, 1 time per day (i.e., 2, 3, 4, or 5 times per day), 1 time per day (i.e., 2, 3, 4, 5, 6, 7 days, etc.), 1 time per week, and 1 time per several weeks (i.e., 2, 3, 4, etc.).

As used herein, the term "subject" refers to any biological individual, preferably an animal, more preferably a mammal, even more preferably a human. In the present invention, the subject may be a healthy subject or a subject suffering from a disease. Where it is desired to treat a particular disease, typically it is intended to refer to a subject suffering from, or at risk of suffering from, the disease.

In addition, as used in the present specification, the term "treatment" includes all kinds of preventive and/or therapeutic interventions that are medically allowed for the purpose of cure, temporary relief, prevention, and the like of a disease. For example, the term "treatment" includes medically approved interventions for various purposes including delay or cessation of disease progression, reduction or disappearance of lesions, prevention of disease onset or prevention of recurrence, and the like.

In addition, as described above, the GST-pi-inhibiting drug showed cell death induction and cell proliferation inhibition effects against cells having a mutation in the BRAF gene. Therefore, a cell death inducing agent and/or a cell proliferation inhibiting agent for a cell having a mutation in the BRAF gene can be selected using inhibition of GST-pi as an index. That is, a substance capable of inhibiting GST-pi can be a candidate substance for a cell death inducing agent and/or a cell proliferation inhibiting agent for a cell (typically, a cancer cell) having a mutation in the BRAF gene.

For example, as an example of a cancer cell, a cancer cell having a mutation in the BRAF gene is contacted with a test substance, and the expression level of GST-. Pi.in the cell is measured. The test substance may be selected as a candidate substance for a drug that inhibits GST-pi when the expression level upon contact with the test substance is reduced as compared to the expression level measured in the absence of the test substance.

The substance to be measured is not limited to any particular substance, and may be any substance. The substance to be measured may be a single substance or a mixture containing a plurality of constituent components. The substance to be tested may be a composition containing an unidentified substance such as an extract from a microorganism or a culture medium, or may be a composition containing a known composition at a predetermined composition ratio. The test substance may be any of proteins, nucleic acids, lipids, polysaccharides, organic compounds, and inorganic compounds.

Examples

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to the following examples.

[ example 1 ]

In this example, the effect of inhibiting cell proliferation when a GST-pi-inhibiting drug was applied to a cell having a mutation in the BRAF gene was examined. First, as a cancer cell having a mutation (V600E) in the BRAF gene, a gene containing 5% CO at 37℃was used ₂ 1 colorectal cancer cell line (CACO-2) and 4 melanoma cell lines (A375, SK-MEL-28, A2058 and Malme-3M) were cultured in the air. Regarding the medium used, CACO-2 was MEM+20%, FBS+0.1mM and NEAA, A375 was DMEM+15% and FBS, SK-MEL-28 was EMEM+10% and FBS, A2058 was DMEM+10% and FBS, and Malme-3M was IMDM+20% and FBS, each with antibiotics added.

Next, one day before transfection, each cell was inoculated into a 100mm tissue culture plastic dish using a medium containing no antibiotic, bringing each cell into a 10-20% confluent state. 600pmol of GST-pi siRNA (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, ambion (SEQ ID NO: 3)) was added to 1mL of Opti-MEM I hyposerum medium (GIBCO Co.) and gently mixed. Subsequently, 35. Mu.L of Lipofectamine RNAi MAX (Invitrogen) was diluted in 1mL of Opti-MEM I-reduced serum medium (GIBCO Co.) and gently mixed. After gently mixing the diluted GST-pi siRNA with the diluted Lipofectamine RNAi MAX, incubation was performed for 10 minutes at room temperature. During this time, the medium was replaced with 10mL of Opti-MEM I reduced serum medium. After incubation for 10 min, the complex of GST-pi siRNA and Lipofectamine RNAi MAX was added to the cells at 37℃in the presence of 5% CO ₂ Is incubated in the atmosphere of (2). After 5 hours incubation, 10mL of antibiotic-free medium was replaced. 1 hour after medium exchange, the cells were washed with PBS, stripped using 0.25% pancreatin-EDTA (SIGMA Co.) and suspended in the antibiotic-containing medium. Cells were suspended in 5ml of medium and inoculated into 60mm tissue culture plastic dishes (CACO-2:0.4X10 ⁵ And a375: 1.0X10 ⁵ SK-MEL-28: 0.2X10 ⁵ A2058: 0.8X10 ⁵ Malme-3M: 0.6X10 ⁵ And (c) a).

As control experiments, the same operations were performed using out-of-order siRNA (sisomicle siRNA) (CGAUUCGCUAGACCGGCUUCAUUGCAG, hokkaido System Science co., ltd. (serial No. 4)), or AllStars Negative Control siRNA (allstats negative control siRNA, siControl) (QIAGEN). Following transfection of GST-pi siRNA, the cell numbers were counted on day 0 and day 5, respectively.

In this example, GST-pi expression was confirmed by Western blotting when GST-pi siRNA was allowed to act on CACO-2 cells, A375 cells, SK-MEL-28 cells, A2058 cells and Malme-3M cells, respectively. That is, GST-pi knockdown Western blot analysis was performed using cells harvested on day 3 post-transfection of GST-pi siRNA. First, after washing the collected cells with cold PBS, cold lysate (1% NP-40, 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, complete Mini EDTA-free (Roche Co., ltd.), and PhosSTOP (Roche Co., ltd.) was added thereto, and the cells were cooled with ice and incubated for 30 minutes to make them soluble. Then, the mixture was centrifuged at 15000rpm at 4℃for 15 minutes to obtain a cell extract. The protein amount of the obtained cell extract was measured using a BCA method trace protein concentration measurement kit (Micro BCA Protein Assay Kit) (Thermo SCIENTIFIC). Subsequently, 20. Mu.g of the cell extract was modified under reducing conditions, and SDS-PAGE was performed using Multi Gel II mini/20 (13 w) (Cosmo Bio Co., ltd.) to isolate proteins. After completion of SDS-PAGE, the membrane was transferred to PVDF membrane using a slot transfer device (tank blotting apparatus). The transfer film was incubated in PBS (abbreviated as PBS-T) added with 5% skim milk/0.05% Tween 20 at 4℃for 16 hours, thereby blocking. Then, the reaction was carried out in a membrane-sealing solution (Invitrogen) at 4℃for 16 hours with a diluted anti-GST-pi antibody (MBL). The secondary antibody reaction was performed using a rabbit antibody labeled with horseradish peroxidase (HRP) for 1 hour at room temperature. The signal detection of the band was performed on an X-ray film by using a chemiluminescent method of ECL Western Blocking Detection Reagents (GE Healthcare). For the washes between runs, 4 times each 5 minutes of shaking was performed using PBS-T.

The results for the colorectal cancer cell line are shown in fig. 1, and the results for the melanoma cell line are shown in fig. 2. The results of measuring the number of cells and the results of analysis by Western blotting are shown in fig. 1 and 2. As shown in fig. 1 and 2, in cancer cells having a mutation in the BRAF gene, GST-pi knockdown was performed by using GST-pi siRNA, which is a drug for inhibiting GST-pi, and thus cell proliferation ability of both colorectal cancer cell lines and melanoma cell lines was significantly inhibited. Mutations in the BRAF gene are observed in tumors with high malignancy, and are known as poor prognosis factors for large intestine cancers that cannot be excised among large intestine cancers. Half of melanoma patients have mutations in the BRAF gene, with the highest mortality/malignancy when metastatic capacity is high. The results of this example show that, for such cancers having mutations in the BRAF gene, drugs that inhibit GST-pi are effective for cell proliferation inhibition, and therefore, these drugs are expected to contribute to a novel treatment for refractory cancers.

[ example 2 ]

In this example, the effect of inhibiting cell proliferation when a GST-pi-inhibiting drug and a BRAF inhibitor are allowed to act on a cell having a mutation in the BRAF gene was examined.

First, 1 colorectal cancer cell line (CACO-2) and 2 melanoma cell lines (SK-MEL-28 and A2058) were cultured in the same manner as in example 1, and the cells were brought into a 10-20% confluent state by inoculating them into a 100mm tissue culture plastic dish with a medium containing no antibiotic one day before transfection. 600pmol of GST-pi siRNA (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, ambion (SEQ ID NO: 3)) was added to 1mL of Opti-MEM I hyposerum medium (GIBCO Co.) and gently mixed. Subsequently, 35. Mu.L of Lipofectamine RNAi MAX (Invitrogen) was diluted in 1mL of Opti-MEM I-reduced serum medium (GIBCO Co.) and gently mixed. After gently mixing the diluted GST-pi siRNA with the diluted Lipofectamine RNAi MAX, incubation was performed for 10 minutes at room temperature. During this time, the medium was replaced with 10mL of Opti-MEM I reduced serum medium. After incubation for 10 minutes, the direction isThe complex of GST-pi siRNA and Lipofectamine RNAi MAX was added to the cells at 37℃in the presence of 5% CO ₂ Is incubated in the atmosphere of (2). After 5 hours incubation, 10mL of antibiotic-free medium was replaced. 1 hour after medium exchange, the cells were washed with PBS, stripped using 0.25% pancreatin-EDTA (SIGMA Co.) and suspended in the antibiotic-containing medium. Cells were suspended in 5ml of medium and inoculated into 60mm tissue culture plastic dishes (CACO-2:0.4X10 ⁵ SK-MEL-28: 0.2X10 ⁵ A2058: 0.2X10 ⁵ And (c) a). As a control experiment, allStars Negative Control siRNA (siControl) (QIAGEN) was used for the same operation. From day 1 after transfection, the BRAF inhibitor PLX4720 (Selleck Co.) was added to the medium at 40. Mu.M for CACO-2, 0.7. Mu.M for SK-MEL-28, and 10. Mu.M for A2058, and incubated until day 5. Regarding the control treated with PLX4720, culture was performed with DMSO (and photoplethysmography) added as a solvent. Proliferation assays were performed by counting the number of cells from day 0 to day 5 after transfection.

The results of counting the number of cells for CACO-2 are shown in FIG. 3, and the results of counting the number of cells for SK-MEL-28 and A2058 are shown in FIG. 4. As is clear from FIG. 3, when GST-pi siRNA was used alone to act on a colorectal cancer cell line (CACO-2 line having a mutation in the BRAF gene) and PLX4720 was used together with siControl to act on the colorectal cancer cell line, the effect of inhibiting cell proliferation was remarkable when both GST-pi siRNA and PLX4720 were used together to act on the same cell line. As can be seen from fig. 4, when GST-pi siRNA was used alone to act on a melanoma cell line (SK-MEL-28 and a2058 having a mutation in the BRAF gene), and PLX4720 was used together with siControl to act on the melanoma cell line, the effect of inhibiting cell proliferation was remarkable when the GST-pi siRNA and PLX4720 were used together to act on the same cell line.

[ example 3 ]

In this example, the effect of inhibiting cell proliferation was studied when a drug that inhibits GST-pi was applied to a cell that has a mutation in the BRAF gene and is resistant to a BRAF inhibitor (a BRAF inhibitor-resistant cell).

< measurement of cell number >

The BRAF inhibitor-resistant cells used in this example were prepared as follows. In DMEM medium containing antibiotics and 15% FBS and containing 1 to 5. Mu.M PLX4720 (Selleck Co.) at 37℃in a medium containing 5% CO ₂ The melanoma cell line A375 used in example 1 was incubated for 1 month in the atmosphere. Cell lines that survived 1 month of incubation were used in this example as PLX4720 resistant a375 cells.

In this example, PLX 4720-resistant A375 cells were plated to 0.5X10 s day before transfection with DMEM medium supplemented with 15% FBS without antibiotics ⁶ Each 10ml of the culture medium was inoculated into a 100mm tissue culture plastic dish. 600pmol of GST-pi siRNA (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, ambion (SEQ ID NO: 3)) was added to 1mL of Opti-MEM I hyposerum medium (GIBCO Co.) and gently mixed. Subsequently, 35. Mu.L of Lipofectamine RNAi MAX (Invitrogen) was diluted in 1mL of Opti-MEM I-reduced serum medium (GIBCO Co.) and gently mixed. After gently mixing the diluted GST-pi siRNA with the diluted Lipofectamine RNAi MAX, incubation was performed for 10 minutes at room temperature. During this time, the medium was replaced with 10mL of Opti-MEM I reduced serum medium. After incubation for 10 min, the complex of GST-pi siRNA and Lipofectamine RNAi MAX was added to the cells at 37℃in the presence of 5% CO ₂ Is incubated in the atmosphere of (2). After 5 hours of incubation, 10mL of DMEM medium without antibiotics and supplemented with 15% fbs was replaced. 2 hours after medium exchange, the cells were washed with PBS and then stripped using 0.5% pancreatin-EDTA (SIGMA Co.) and suspended in DMEM medium containing antibiotics and 15% FBS. The cells in suspension were brought to 1.0X10 ⁵ Each 5ml of the culture medium was inoculated into a 60mm tissue culture plastic dish. As a control experiment, the same operation was performed using out-of-order siRNA (CGAUUCGCUAGACCGGCUUCAUUGCAG, hokkaido System Science co., ltd. (sequence number 4)), or AllStars Negative Control siRNA (siControl) (QIAGEN). After transfection of GST-pi siRNA, the cell numbers were counted on

days

0, 1, 2, 3, 4, 5, respectively。

< confirmation of GST-pi expression >)

As described above, GST-pi expression was confirmed by Western blotting when GST-pi siRNA was allowed to act on PLX 4720-resistant A375 cells. That is, western blot analysis of GST-pi knockdown was performed using cells collected at each of the above time points after transfection of GST-pi siRNA.

First, after washing the collected cells with cold PBS, cold lysate (1% NP-40, 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, complete Mini EDTA-free (Roche Co., ltd.), and PhosSTOP (Roche Co., ltd.) was added thereto, and the cells were cooled with ice and incubated for 30 minutes to make them soluble. The cells were centrifuged at 15000rpm at 4℃for 15 minutes to obtain a cell extract. The protein amount of the obtained cell extract was measured using a BCA method trace protein concentration measurement kit (Thermo SCIENTIFIC). Subsequently, 20. Mu.g of the cell extract was modified under reducing conditions, and SDS-PAGE was performed using Multi Gel II mini/20 (13 w) (Cosmo Bio Co., ltd.) to isolate proteins. After completion of SDS-PAGE, the film was transferred to PVDF membrane using a slot transfer device. The transfer film was incubated in PBS (abbreviated as PBS-T) added with 5% skim milk/0.05% Tween 20 at 4℃for 16 hours, thereby blocking. Then, the reaction was carried out in a membrane-sealing solution (Invitrogen) at 4℃for 16 hours with a diluted anti-GST-pi antibody (MBL). The secondary antibody reaction was performed using a rabbit antibody labeled with horseradish peroxidase (HRP) for 1 hour at room temperature. The signal detection of the band was performed on an X-ray film by using a chemiluminescent method of ECL Western Blocking Detection Reagents (GE Healthcare). For the washes between runs, 4 times each 5 minutes of shaking was performed using PBS-T.

< result >

In recent years, it has been elucidated that melanoma cell lines that acquire BRAF inhibitor resistance are associated with recurrence of melanoma. Therefore, to investigate whether GST-pi promoted CRAF dependence of BRAF inhibitor-resistant melanoma, GST-pi was knockdown in PLX 4720-resistant A375. As a result of measurement of cell proliferation, a remarkable proliferation inhibition effect was confirmed (fig. 5). The knockdown of GST-pi was confirmed, and as a result, it was found that GST-pi expression was inhibited on days 2 and 3 (FIG. 6).

This example shows that it is possible to effectively inhibit proliferation of cells having a mutation in the BRAF gene and having resistance to a BRAF inhibitor (BRAF inhibitor-resistant cells). From this result, it is expected to prevent or reduce the recurrence of diseases caused by BRAF inhibitor-resistant cells, such as melanoma.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims

1. The use of a combination of a GST-pi inhibiting drug and a BRAF inhibitor in the manufacture of a cell death inducing agent for cells having a mutation in the BRAF gene,

wherein the GST-pi-inhibiting agent is a substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors expressing at least 1 of them.

2. The use of a combination of a GST-pi inhibiting drug and a BRAF inhibitor in the manufacture of a cell proliferation inhibiting agent for cells having a mutation in the BRAF gene,

3. The use according to claim 1 or 2, wherein the mutation is a V600E mutation.

4. The use according to claim 1 or 2, wherein the cell having a mutation in the BRAF gene is a cell having resistance to a BRAF inhibitor.

5. The use according to claim 1 or 2, wherein the cell having a mutation in the BRAF gene is a cancer cell expressing GST-pi at high levels.

6. The use according to any one of claims 1 to 5, wherein the agent is a pharmaceutical composition for the treatment of a disease caused by abnormal proliferation of cells having a mutation in the BRAF gene.

7. The use according to claim 6, wherein the disease is cancer.

8. The use of claim 7, wherein the cancer is a cancer that expresses GST-pi at high levels.

9. The use according to claim 7, wherein the cancer is colorectal cancer or melanoma.