ES2978957T3 - Cell death inducing agent for cells having mutation in the BRAF gene, agent for inhibiting the proliferation of said cells and pharmaceutical composition for treating a patient suffering from abnormal proliferation effects of said cells - Google Patents

Abstract

The present invention induces cell death and/or inhibits cell proliferation of cells that have a mutation in the BRAF gene. The present invention includes, as an active ingredient, a GST-π inhibitor drug. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Cell death inducing agent for cells having mutation in the BRAF gene, agent for inhibiting the proliferation of said cells and pharmaceutical composition for treating a patient suffering from abnormal proliferation effects of said cells

Technical field

The present invention relates to a cell death inducing agent for a cell having a mutation in the BRAF gene (for example, a type of cancer cell), a growth suppressing agent for the same cell and a pharmaceutical composition for the therapy of a disease caused by a growth defect of the same cell, as defined in the claims.

Background of the technique

The BRAF gene is a gene encoding a type of serine threonine kinase that constitutes the RAS-RAF-MAPK pathway. Mutations in the BRAF gene have been reported in various tumors. For example, the mutation in which valine at amino acid 600 is replaced by glutamic acid (V600E) is found in many cancer cells. It is known that BRAF with the V600E mutation always activates downstream signaling and causes cell growth without extracellular stimulation.

For example, the BRAF gene carrying the V600E mutation is found in many colorectal cancers (5-15%) and melanomas (about 60%). Note that mutations in a KRAS gene are found with high frequency in many cancers such as pancreatic cancer and colorectal cancer. The KRAS protein is a G protein present locally on the inner surface of the cell membrane. RAS such as KRAS activates RAF such as CRAF and BRAF, RAF sequentially activates MEK and MEK activates MAPK, thereby forming a cascade (Non-Patent Documents 1 and 2). It is a rare case to contain both BRAF mutation and KRAS mutation since BRA<f>mutation and KRAS mutation are in a mutually exclusive relationship (Non-Patent Document 3).

Currently, for cancers with BRAF that has the V600E mutation, a therapy using an inhibitor against BRAF that has the V600E mutation is considered effective. Vemurafenib (PLX4032) and PLX4720 are known as such inhibitors. However, cancer cells may require resistance to these inhibitors through continuous administration of the same, which limits the effects of the therapy. Therefore, for cancer cells that have a mutation in the BRAF gene, more effective cell death-inducing agents and growth suppressing agents are required to replace these inhibitors.

Glutathione S-transferase (GST), one of the enzymes that catalyzes the conjugation of glutathione, is known as an enzyme that conjugates a substance such as a drug with glutathione (GSH) in an aqueous substance. Based on the amino acid sequence, GST is representatively classified into 6 types of the isozyme, a, |i, o, n, 0, and Q. Of these, the expression of GST-n (glutathione S-transferase pi, also called GSTP1) has been particularly increasing in various cancer cells and is suggested to be a possible factor in resistance to some antineoplastic agents. In fact, it is known that when an antisense DNA or a GST-n inhibitor against GST-n is allowed to act on a drug-resistant cancer cell system that overexpresses GST-n, drug resistance is suppressed (Non-Patent Documents 4 to 6). Furthermore, in a recent report, when an siRNA against GST-n was allowed to act on an androgen-independent prostate cancer cell line overexpressing GST-n, cell growth was suppressed and apoptosis was increased (non-patent document 7).

Additionally, GST-n is known to form a complex with c-Jun N-terminal kinase (JNK) and inhibit JNK activity (Non-Patent Document 8). Furthermore, GST-n is known to be involved in the S-glutathionylation of proteins that are associated with cellular stress responses (Non-Patent Document 9). Furthermore, GST-n is known to contribute to the protective action in reactive oxygen species (ROS)-induced cell death (Non-Patent Document 10). As described above, GST-n among GSTs is understood to have diverse characteristics and functions.

It is reported that when an siRNA against GST-n is allowed to act on a cancer cell system having a KRAS mutation, Akt activation is suppressed and autophagy is increased, but the induction of apoptosis is approximately moderate (Non-Patent Document 11). Patent Document 1 discloses that apoptosis of cancer cells can be induced by using a drug that suppresses GST-n and an autophagy inhibitor such as 3-methyladenine as active components. Patent Document 2 further discloses that when the expression of GST-n and Akt is simultaneously inhibited, cell growth is suppressed and cell death is induced, and autophagy induced by inhibiting GST-n expression by simultaneously inhibiting Akt expression and the like is remarkably suppressed.

However, in the cell that has a mutation in the BRAF gene described above, there is no finding on the relationship between GST-rc expression and cell growth or cell death or the role of GST-rc in signal transduction.

List of references

Patent Bibliography

Patent document 1: document WO2012/176282

Patent document 2: document WO2014/098210

Non-patent bibliography

Non-patent document 1: Curr Top Microbiol Immunol. 2012; 355:83-98

Non-patent document 2: Curr Opin Pharmacol. 2008 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: Banet 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., Carcinogenesis. 2008; 29(6): 1134-8

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

Non-patent document 9: Townsendet al.,J. Biol. Chem. 2009, 284, 436-445

Non-patent document 10: Yinet al., Cancer Res. 200060, 4053-4057

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

Yan-Jie Zhang et al. analyze that mTOR signaling is involved in indomethacin and nimesulide suppression of colorectal cancer cell growth via COX-2-independent pathway, published in Annals of Surgical Oncology, Springer-Verlag, NE, Vol. 18, No. 2, August 28, 2010 (2010-08-28), pages 580–588, XP019879260, ISSN: 1534-4681, DOI: 10.1245/S10434-010-1268-9.

Mertens W.C.et al. review oral indomethacin and ranitidine in advanced melanoma, published in Clinical Onco, W.B. Saunders, Amsterdam, The Netherlands, vol. 8, no. 2, 1 January 1996 (01-01-1996), pages 112-115, XP004929659, ISSN: 0936-6555, DOI: 10.1016/50936-6555(96)80117-8.

Luca, A.D. et al. analyze a novel orally active water-soluble inhibitor of human glutathione transferase that exerts potent and selective antitumor activity against human melanoma xenografts, published in Oncotarget, vol. 6, no. 6, February 2015 (06-02-2015), pages 4126-4143, XP055321169.

Turley, C.K. et al. analyze that GSTP1 suppression induces apoptosis in melanoma cells independently of BRAF mutational status, published in Journal of Surgical Research, vol. 172, no. 2, January 1, 2012 (01-01-2012), page 229, XP055491483, DOI: 10.1016/J.JSS.2011.11.346.

WO 2012/176282 A1 (NITTO DENKO CORP.), filed on December 27, 2012, relates to compositions and methods for modulating the expression of apoa1 and abca1.

WO 2014/098210 A1 (NITTO DENKO CORP.), filed June 26, 2014, relates to an apoptosis-inducing agent.

Tsai, J. et al. discuss the discovery of a selective inhibitor of oncogenic B-raf kinase with potent anti-melanoma activity, published in PNAS, vol. 105, no. 8, 2008, pages 3041-3046, XP055003766.

Summary

The present invention is defined in the claims and provides a pharmaceutical composition for use in a method for the therapy of a cancer caused by a growth defect of a cancer cell having a mutation in a BRAF gene and resistance to a BRAF inhibitor,

the pharmaceutical composition comprising a cell death inducing agent or cell growth suppressing agent for a cell having a mutation in a BRAF gene, said agent comprising a drug that suppresses GST-rc as an active ingredient,

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 one thereof.

Additional aspects of the invention are also defined in the claims.

Technical problem

Under these circumstances, the present invention aims to provide an agent having a cell death-inducing action and/or a cell growth-suppressing action for a cell having a mutation in the BRAF gene, to provide a pharmaceutical composition for the therapy of a disease caused by a cell growth defect, and to provide a method for selecting the cell death-inducing agent and/or the cell growth-suppressing agent.

Pharmaceutical compositions and agents comprised therein are defined in the claims.Solution to the problem

The present inventors carried out extensive studies in view of the above object and found that cell growth is severely suppressed when GST-rc is suppressed in a cell having a mutation in the BRAF gene and cell growth is also severely suppressed when GST-rc is suppressed even in a cell having the mutation in the BRAF gene and is resistant to conventionally known BRAF inhibitors, whereby the present invention was achieved. The present invention includes the following.

Advantageous effects of the invention

The cell death inducing agent of the invention and the compositions comprising the same are defined in the claims.

According to the cell death-inducing agent of the present invention, cell death can be induced very strongly for a cell having a mutation in the BRAF gene. Accordingly, the cell death-inducing agent of the present invention can produce a very high efficacy as a pharmaceutical composition for the therapy of a disease caused by a growth defect of a cell having a mutation in the BRAF gene.

Furthermore, according to the cell growth suppressor agent of the present invention, cell growth can be suppressed very strongly for a cell having a mutation in the BRAF gene. Accordingly, the cell growth suppressor agent of the present invention can produce a very high efficacy as a pharmaceutical composition for the therapy of a disease caused by a growth defect of a cell having a mutation in the BRAF gene.

Furthermore, according to the selection method according to the present disclosure, a drug can be selected to induce cell death and/or suppress cell growth very strongly for a cell having a mutation in the BRAF gene.

Brief description of the drawings

[Figure 1] Figure 1 is a characteristic diagram showing the verification results of the cell growth suppressor effect due to gene attenuation of GST-rc in a colorectal cancer cell line having a mutation in the BRAF gene (cell number counting results, Western blot results).

[Figure 2] Figure 2 are characteristic diagrams showing the verification results of the cell growth suppressor effect due to gene attenuation of GST-rc in a melanoma cell line having a mutation in the BRAF gene (cell number counting results, Western blot results).

[Figure 3] Figure 3 is a characteristic diagram showing the verification results of the cell growth suppression effect when siRNA against GST-rc and a BRAF inhibitor were used in combination in a colorectal cancer cell line carrying a mutation in the BRAF gene.

[Figure 4] Figure 4 are characteristic diagrams showing the verification results of the cell growth suppression effect when siRNA against GST-rc and a BRAF inhibitor were used in combination in a melanoma cell line having a mutation in the BRAF gene.

[Figure 5] Figure 5 is a characteristic diagram showing the verification results of the cell growth suppressor effect due to gene attenuation of GST-rc in a melanoma cell line that has a mutation in the BRAF gene and is resistant to a BRAF inhibitor.

[Figure 6] Figure 6 are electrophoresis photographs showing the results of Western blot analysis of GST-rc expression when GST-rc knockdown was performed in a melanoma cell line that has a mutation in the BRAF gene and is resistant to a BRAF inhibitor.

Description of realizations

The cell death inducing agent and the cell growth suppressing agent, as well as the compositions comprising them, are defined in the claims.

The cell death-inducing agent and the cell growth-suppressing agent of the present invention comprise a drug that suppresses GST-rc as an active ingredient. The cell death-inducing agent and the cell growth-suppressing agent of the present invention demonstrate the cell death-inducing effect and the cell growth-suppressing effect for a cell having a mutation in the BRAF gene.

GST-rc, when used herein, refers to an enzyme that is encoded by the GSTP1 gene and catalyzes the conjugation of glutathione. GST-rc is present in various animals including humans and the sequence information thereof is known (e.g., human: NM_000852 (N_000843), rat: NM_012577 (NP_036709), mouse: NM_013541 (NP_038569), etc. Numbers represent NCBI database accession numbers, numbers outside parentheses are nucleotide sequences, and numbers in parentheses are amino acid sequences.) As an example, the nucleotide sequence in a coding region of the human GST-rc gene recorded in the database is set forth in SEQ ID NO: 1 and the amino acid sequence in the human GST-rc protein encoded by the human GST-rc gene is set forth in SEQ ID NO: 2.

Note that GST-rc can be specified by the specific nucleotide sequence and amino acid sequence as described above, but a mutation in the nucleotide sequence and amino acid sequence (including polymorphisms) possibly caused between biological individuals should be considered. More specifically, GST-rc is not limited to proteins that have the identical sequence to the amino acid sequence recorded in the database, but includes those that have the same function as GST-rc and have a sequence with 1 or 2 or more, usually 1 or several, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids different from the above sequence. Additionally, GST-rc also includes those consisting of a nucleotide sequence that has 70% or more, 80% or more, 90% or more, 95% or more, or 97% or more identity to the above specific nucleotide sequence and that encodes a protein having the same function as GST-rc. Note that the specific function of GST-rc, as described above, refers to the enzymatic activity that catalyzes the conjugation of glutathione.

Unless otherwise defined, all technical terms and scientific terms used in this description have the same meaning as commonly understood by those skilled in the art.

Examples of the “drug that suppresses GST-rc” used in the present disclosure include, but are not limited to, drugs that suppress the production and/or activity of GST-rc and drugs that facilitate the decomposition and/or inactivation of GST-rc. Examples of the drugs that suppress the production of GST-rc include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides against the DNA encoding GST-rc, and vectors expressing the same.

Additionally, for the drug that suppresses GST-rc, any compound that acts on GST-rc can be used. Examples of such compounds that can be used include organic compounds (amino acids, polypeptides or derivatives thereof, low molecular weight compounds, carbohydrates, and high molecular weight compounds) and inorganic compounds. In addition, these compounds may be natural substances or non-natural substances. Examples of the polypeptide derivative include modified polypeptides obtained by adding a modifying group and variant polypeptides obtained by varying an amino acid residue. In addition, these compounds may be a single compound or may be a chemical library, expression products of a gene library, cell extracts, cell culture supernatants, products of fermentation microorganisms, extracts of marine organisms, and plant extracts. More specifically, the “drug that suppresses GST-rc” is not limited to nucleic acids such as RNAi molecules, but includes any compound.

Specific examples of the drug that suppresses the activity of GST-rc include, but are not limited to, substances that conjugate GST-rc such as glutathione, glutathione analogs (e.g., WO 95/08563, WO 96/40205, WO 99/54346 and those described in Non-Patent Document 4, etc.), ketoprofen (Non-Patent Document 2), indomethacin (Hallet al., Cancer Res. 1989; 49(22):6265-8), ethacrynic acid, piriprost (Tewet al., Cancer Res. 1988;48(13):3622-5), anti-GST-rc antibodies and dominant-negative mutants of GST-rc. These drugs are commercially available or can be suitably produced based on known technologies.

The drug that suppresses the production or activity of GST-rc is preferably RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides against the DNA encoding GST-rc or vectors expressing the same due to high specificity and low possibility of side effects.

GST-rc suppression may be determined based on the suppressed expression or activity of GST-rc in a cell compared to the case in which a GST-rc inhibitor was not allowed to act. GST-rc expression may be assessed by any known technique such as, but not limited to, immunoprecipitation method using an anti-GST-rc antibody, EIA, ELISA, IRA, IRMA, Western blot method, immunohistochemical method, immunocytochemical method, flow cytometry method, various hybridization methods, Northern blot method, Southern blot method, and various PCR methods in which a nucleic acid is capable of specifically hybridizing to a nucleic acid encoding GST-rc or a unique fragment thereof or a transcript (e.g., mRNA) or a spliced product of the nucleic acid.

The activity of GST-rc can be assessed by analyzing known activities of GST-rc such as, but not limited to, the ability to bind to proteins such as Raf-1 (particularly phosphorylated Raf-1) and EGFR (particularly phosphorylated EGFR) by any known method such as immunoprecipitation method, Western blot method, mass spectrometry, coimmunoprecipitation ("pulldown") assay or surface plasmon resonance (SPR) method.

The RNAi molecule, when used herein, refers to any molecule that produces RNA interference and includes, but is not limited to, double-stranded RNAs such as siRNA (small interfering RNA), miRNA (micro-RNA), shRNA (short hairpin RNA), dsRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA) and siparRNA (repeat-associated siRNA), and variants thereof. These RNAi molecules are commercially available or can be designed and manufactured based on known sequence information, more specifically the nucleotide sequence as set forth in SEQ ID NO: 1 and/or the amino acid sequence as set forth in SEQ ID NO: 2. Additionally, the antisense nucleic acid, when used herein, includes RNA, DNA, PNA, and complexes thereof.

The DNA/RNA chimeric polynucleotide, when used in the present disclosure, includes, but is not limited to, double-stranded polynucleotides consisting of DNA and RNA that inhibit the expression of a target gene as described in Japanese Patent Publication (Kokai) No. 2003-219893 A.

The amount of an active ingredient to be added to the agent or composition of the present invention may be an amount that induces cell death such as apoptosis and/or suppresses cell growth when the agent or composition is administered. Additionally, an amount that does not cause harmful effects that outweigh the benefits obtained from the administration is preferable. Such an amount is known or suitably determined by an in vitro test using cultured cells or a test in a model animal such as a mouse, rat, dog or pig, and these test methods are well known to those skilled in the art. Induction of apoptosis can be assessed by various known techniques using detection of hallmark phenomena of apoptosis such as DNA fragmentation, binding of annexin V to a cell membrane, changes in mitochondrial membrane potential or activation of caspase or TUNEL staining. Furthermore, the suppression of cell growth can be assessed by various known techniques such as counting the number of viable cells over time, measuring the size, volume and weight of a tumor, measuring the amount of DNA synthesis, WST-1 method, BrdU (bromodeoxyuridine) method or 3H-thymidine incorporation method. The amount of an active ingredient to be added is variable depending on the drug form of the agent and composition. For example, when multiple units of the composition are used for a single administration, the amount of an active ingredient added to a single unit composition may be one of equal multiple units of the amount of the active ingredient required for a single administration. Such amount to be added can be suitably adjusted by those skilled in the art.

Additionally, the addition of the GST-rc suppressing drug as an active ingredient enables the production of the cell death-inducing agent, the cell growth suppressing agent, the cell death-inducing composition or the cell growth suppressing composition.

In addition, the drug that suppresses GST-rc used for inducing cell death or suppressing cell growth may be provided. In addition, the method for inducing cell death or the method for suppressing cell growth comprising administering an effective amount of the drug that suppresses GST-tc may be provided.

It is noted that both of the above methods or uses of inducing cell death such as apoptosis or suppressing cell growth may be in vitro methods or in vivo methods. Additionally, the drug for these methods is as already described above and the effective amount of the drug may be an amount that induces cell death or suppresses cell growth in a cell to which the drug is administered. Additionally, an amount that does not cause harmful effects that outweigh the benefits obtained from the administration is preferable. Such an amount is known or can be suitably determined, for example, by an in vitro test using cultured cells and the like, and the test method is well known to those skilled in the art. The induction of cell death or the suppression of cell growth can be evaluated by various known techniques including those described above. It is not necessary that the above effective amount, when the drug is administered to a specified group of cancer cells, is the amount that always causes cell death or the suppression of the growth of all cells in the group of cells. For example, the above effective amount may be an amount that causes apoptosis or growth suppression 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, or additionally 25% or more of the cells in the above cell group.

The cell death-inducing agent and the cell growth suppressing agent of the present invention act on a cell having a mutation in the BRAF gene. The cell having a mutation in the BRAF gene is a cell that demonstrates a growth defect due to a mutation in the BRAF gene (usually, cancer cells).

Particularly, the cell death inducing agent and the cell growth suppressing agent of the present invention are preferably applied to a cell with high GST-rc expression (usually, a cancer cell with high GST-rc expression) among the cells demonstrating a growth defect due to a mutation in the BRAF gene. The cancer cell with high GST-rc expression used herein means a cell having a significantly higher GST-rc expression level compared with a normal cell among the cells having a mutation in the BRAF gene and demonstrating a cell growth defect. Note that the expression level of GST-rc can be measured according to a routine method such as RT-PCR or microarray.

Mutation in BRAF gene means a mutation such as deletion, substitution, addition, insertion in an amino acid sequence of wild-type BRAF and a mutation in an expression control region of the BRAF gene. Note that the mutation in the BRAF gene herein is a so-called gain-of-function mutation. More specifically, the BRAF gene having a mutation (sometimes referred to as a mutant BRAF gene) includes genes encoding mutant BRAF with increased serine threonine kinase activity caused by the mutation. Additionally, the mutant BRAF gene also includes those having a mutation in an expression control region and an increased expression level compared with the wild-type BRAF gene. More specifically, a cell expressing the mutant BRAF gene has the characteristic of constantly maintaining downstream signaling (e.g., signaling to MEK) from BRAF by expressing mutant BRAF or increasing the expression level of BRAF.

Examples of the mutant BRAF gene include genes encoding mutant BRAF in which the valine encoded by codon 600 in the wild-type BRAF gene is replaced by glutamic acid (denoted as V600E; hereinafter referred to as the same) such as V600D, V600G, V600K, V600M, V600R, V600L, G469A, G469V, D594N and V600insT (insertion of T).

Thus, the cell death inducing agent and the cell growth suppressing agent comprised in the compositions of the present invention and as defined in the claims capable of inducing cell death or suppressing cell growth effectively even for a cancer cell having a mutation in the BRAF gene are effective as components of a pharmaceutical composition for a disease caused by a growth defect of a cancer cell having a mutation in the BRAF gene. Additionally, the formulation of the drug suppressing GST-rc as an active ingredient enables the production of the pharmaceutical composition for a disease caused by the growth defect of a cancer cell having a mutation in the BRAF gene. Furthermore, a disease caused by the cell growth defect can be subjected to treatment and therapy comprising administering an effective amount of the produced pharmaceutical composition to a subject in need thereof.

The pharmaceutical composition as defined in the claims is effective for treating a disease caused by the growth defect of a cancer cell having a mutation in the BRAF gene. The disease caused by a cancer cell having a mutation in the BRAF gene is not limited and includes cancers with high expression of GST-rc, and in many cases, cancer with high expression of GST-rc encompasses cancers having a mutation in the BRAF gene.

Examples include, but are not limited to, sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, and osteosarcoma, carcinomas such as eye cancer, thyroid cancer (papillary cancer), meningeal cancer, brain tumor, pituitary cancer, salivary gland cancer, head and neck cancer, breast cancer, lung cancer (non-small cell cancer), esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer (endometrium), and uterine cancer (endometrium). endometrial), ovarian cancer (serous ovarian cancer), vulvar cancer, vaginal cancer and skin cancer (malignant melanoma), and additionally leukemia and malignant lymphoma. Note that “cancer” in the present invention includes epithelial malignant tumor and non-epithelial malignant tumor. The cancer in the present invention may be present in any part of the body including brain, head and neck, thorax, extremity, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gallbladder, 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, lymphatic system such as lymph node and lymphatic fluid.

Particularly, the cell death-inducing agent and the cell growth suppressing agent comprised in the compositions of the present invention can induce cell death or suppress cell growth effectively even for a cell that has acquired resistance to a BRAF inhibitor among cells having a mutation in the BRAF gene. Accordingly, the cell death-inducing agent or the cell growth suppressing agent comprised in the compositions of the present invention is effective as a component of a pharmaceutical composition for a disease caused by the growth defect of a cancer cell having a mutation in the BRAF gene and resistance to a BRAF inhibitor. Additionally, the formulation of the drug suppressing GST-rc as an active ingredient enables the production of the pharmaceutical composition for a disease caused by the growth defect of a cancer cell having a mutation in the BRAF gene and resistance to a BRAF inhibitor. Furthermore, a disease caused by the cell growth defect can be subjected to treatment and therapy comprising administering an effective amount of the produced pharmaceutical composition to a subject in need thereof, that is, to a patient with a reduced therapeutic effect by a BRAF inhibitor.

The BRAF inhibitor used herein means a substance that inhibits signal transduction from downstream BRAF, particularly the substance that specifically inhibits signal transduction caused by mutant BRAF having the gain-of-function mutation described above. Examples of the known BRAF inhibitor include vemurafenib ((PLX4032), CAS No.: 918504-65-1, N-[3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]propane-1-sulfonamide) and PLX4720 (CAS No.: 918505-84-7, N-[3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl]propane-1-sulfonamide).

Furthermore, examples of BRAF inhibitor include regorafenib, dasatinib, PLX-8394, BeiGene-283, PLX-3603, RG-7304 (CAS No.: 213406-50-9), LY-3009120 (CAS No.: 213406-50-9), CAS: 1454682-72-4), rebastinib (CAS No: 1020172-07 9), 1H-pyrazolo[3,4-b]pyridine-5-carboxamide analogues, ASN-003, vemurafenib prodrug, derivatives of N-(thiophen-2-yl)benzamide, DCB-R0237, REDX-04988, EBI-907, EBI-945, gossypin, nanolipolee-007, TEW-0201, miRNA-3157, and thiazole derivatives (NMS-P186, NMS-P285, NMS-P349, NMS-P383, NMS-P396, and NMS-P730).

Furthermore, examples of BRAF inhibitor include, in addition to the above, SB590885 (CAS No.: 405554-55 4, N,N-dimethyl-2-[4-[(4Z)-4-(1-nitroso -2,3-dihydroinden-5-ylidene)-5-(1H-pyridin-4-ylidene)-1H-imidazole-2-yl]phenoxy]ethanamine), B-Raf inhibitor 1 (CAS No: 1093100-40-3, 1-N-(4-chlorophenyl)-6-methyl-S-N-[3-(7H-purin-6-yl)pyridin-2-yl]isoquinolin-1,5-diamine), dihydrochloride of B-Raf 1 inhibitor (CAS No.: 1191385-19-9, 1-N-(4-chlorophenyl)-6-methyl-5-N-[3-(7H-purin-6-yl)pyridin-2-yl]isoquinolin-1,5-diamine dihydrochloride), dabrafenib (n CAS No: 1195765-45-7, N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl) -2,6-difluorobenzenesulfonamide), LGX818 (CAS No.: 1269440-17-6, N-[(2S)-1-[[4-[3-[5-chloro-2-fluoro-3-(methanesulfonamido)phenyl]-1-propan-2-ylpyrazol-4-yl]pyrimidin-2-yl methyl ]amino]propan-2-yl]carbamate), HG6-64-1 (CAS No.: 1315329-43-1, see WO 2011/090738), PF-04880594 (CAS No. : 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]propanenitrile), BRAF inhibitor (CAS #: 918505-61-0, N-[2,4-difluoro-3-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-carbonyl)phenyl]propane-2-sulfonamide) , B-Raf Inhibitor (CAS No.: 1315330-11-0, 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-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl ]benzamide), RAF265 (CAS No.: 927880-90-8, 1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy- N-[4-(trifluoromethyl)phenyl]benzimidazol-2-amine), CEP-32496 (CAS No.: 1188910-76-0, 1-[3-(6,7-dimethoxyquinazolin-4-yl)oxyphenyl]-3-[5-(1,1,1-trifluoro-2-methylpropan-2-yl)-1, 2-oxazol-3-yl]urea), GDC-0879 (CAS No: 905281-76-7, 2-[4-[(1E)-1-hydroxyimino-2,3-dihydroinden-5-yl ]-3-pyridin-4-ylpyrazol-1-yl]ethanol), sorafenib tosylate (CAS No.: 47520759-1, 4-[4-[[4-chloro-3-( 4-methylbenzenesulfonic acid trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide), mesylate Dabrafenib (CAS No.: 1 195768-06-9, N-(3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4) methanesulfonic acid -yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide), CEP-32496 hydrochloride (cAs#: 1227678-26-3, 1-[3-(6,7-dimethoxyquinazolin-4- yl)oxyphenyl]-3-[5-(1,1,1-trifluoro-2-methylpropan-2-yl)-1,2-oxazol-3-yl]urea) and 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 other than the GST-rc suppressing drug. Combination use herein includes, for example, administration of other active ingredients as independent pharmaceutical preparations and administration of other active ingredients in the form of a combination agent with at least one other drug. When other active ingredients are administered as independent pharmaceutical preparations, a preparation containing other active ingredients may be administered before the other preparations, together with the other preparations, or after the other preparations.

For such other active ingredients, the above BRAF inhibitors may be appropriately used. Additionally, the other active ingredients also include those effective in treating a target disease. For example, when the disease to be treated is cancer, an antineoplastic agent may be used in combination. Examples of the antineoplastic agent include alkylating agents such as ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, melphalan and ranimustine, antimetabolites such as gemcitabine hydrochloride, enocitabine, cytarabine-ocphosphate, cytarabine preparations, tegafur-uracil, tegafur-gimeracil-oteracil potassium combination agents (e.g., TS-1), doxifluridine, hydroxycarbamide, fluorouracil, methotrexate and mercaptopurine, antitumor antibiotics such as idarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, daunorubicin citrate, doxorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, peplomycin sulfate, mitoxantrone hydrochloride and mitomycin C, alkaloids such as etoposide, irinotecan hydrochloride, vinorelbine ditartrate, docetaxel hydrate, paclitaxel, vincristine sulfate, vindesine sulfate and vinblastine sulfate, hormonal therapy agents such as anastrozole, tamoxifen citrate, toremifene citrate, bicalutamide, flutamide and estramustine phosphate, platinum complexes such as carboplatin, cisplatin (CDDP) and nedaplatin, angiogenic inhibitors such as thalidomide, neovastat and bevacizumab and L-asparaginase.

When the active component in each of the agents, compositions and method of treatment of the present invention described herein is a nucleic acid such as an RNAi molecule, a ribozyme, an antisense nucleic acid or a DNA/RNA chimeric polynucleotide, these may be used directly as naked nucleic acid or may also be carried by various vectors. For the vector, any of the known vectors such as plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, virus vectors may be employed. The vector preferably contains at least one promoter which enhances the expression of the nucleic acid to be carried, and the nucleic acid in this case is preferably operably linked to a promoter. Nucleic acid operably linked to a promoter means that the nucleic acid and the promoter are positioned such that the protein encoded by the nucleic acid can be suitably produced by the action of the promoter. The vector may or may not be replicable in a host cell, and the transcription of the gene may take place outside or inside the nucleus of the host cell. In the latter case, the nucleic acid may be incorporated into the genome of the host cell.

Additionally, the active ingredient may also be supported by various non-viral proteinaceous and lipid carriers. Such a carrier is not limited and examples include cholesterols, liposomes, antibody promoters, cyclodextrin nanoparticles, fused peptides, aptamers, biodegradable poly(lactic acid) polymers and copolymers, by which the efficiency of intracellular uptake may be increased (e.g., see Pirollo and Chang, Cancer Res. 2008; 68(5):1247-50, etc.). Cationic polymers and liposomes (e.g., polyethyleneimine, etc.) are particularly useful. Additional examples of the polymer useful as such a carrier include those described in US 2008/0207553 and US 2008/0312174.

For each pharmaceutical composition of the present invention described herein, the active component may be combined with other optional components as long as the effects of the active component are not affected. Examples of the optional component include other chemotherapeutic agents, pharmacologically acceptable carriers, excipients and diluents. Furthermore, depending on the route of administration and the mode of drug release, the above composition may also be coated with a suitable material, for example, an enteric coating or a programmed disintegration material, or incorporated into a suitable drug delivery system.

Each agent and composition (including each pharmaceutical composition) of the present invention described herein can be administered by various routes including both oral and parenteral, and examples of the administration route include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, local, intratumoral, intrarectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary and intrauterine, or may be prepared into a dosage form suitable for each administration route. Any known dosage form and preparation method can be suitably employed (for example, see “Hyojun Yakuzaigaku (“Standard Pharmaceutics” in English), edited by Yoshiteru Watanabe et al., Nankodo, 2003, etc.).

The dosage form suitable for oral administration is not limited and examples include powders, granules, tablets, capsules, solutions, suspensions, emulsions, gels and syrups, and examples of the dosage form suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections and extemporaneously prepared injections. The preparation for parenteral administration may be in the form of an aqueous or non-aqueous isotonic sterile solution or suspension.

Each agent or composition (including each pharmaceutical composition) of the present invention described herein may also be targeted to a specific cell or tissue. Targeting may be achieved by any known technique. When intended to be delivered to a cancer, examples of the techniques that may be used include, but are not limited to, passive targeting in which the preparation is preferably sized to a diameter of 50 to 200 nm, particularly 75 to 150 nm, to produce EPR (enhanced retention and permeability) effects and active targeting in which, as a targeting agent, a ligand such as CD19, HER2, transferrin receptor, folic acid receptor, VIP receptor, EGFR (Torchilin, AAPS J. 2007; 9(2): E128-47), RAAG10 (Japanese Patent Publication (Kohyo) No. 2005-532050), PIPA (Japanese Patent Publication (Kohyo) No. 2006-506071) or KID3 (Japanese Patent Publication (Kohyo) No. 2007-529197), a peptide having a RGD motif or a NGR, f3 or LyP-1 motif (Ruoslahtiet al., J Cell Biol. 2010;188(6):759-68). Furthermore, retinoid and a derivative thereof are also known to be useful as a targeting agent to a cancer cell (WO 2008/120815) and therefore a retinoid-containing carrier can also be used as a targeting agent. Carriers are described in WO2009/036368, WO 2010/014117 and WO 2012/170952 in addition to the above literatures. Each agent or composition (including each pharmaceutical composition) of the present invention described herein may be supplied in any form, and from the viewpoint of storage stability, it may be provided in a form that can be prepared extemporaneously such as the form that can be prepared at or near a medical facility by a doctor and/or a pharmacist, a nurse or other paramedics. Such a form is particularly useful when the agent or composition of the present invention contains components that are difficult to store stably such as lipids, proteins and nucleic acids. In this case, the agent or composition of the present invention is provided in 1 or 2 or more containers containing at least one essential element therefor and is prepared before use, for example, within 24 hours, preferably before 3 hours, more preferably immediately before use. For the preparation, a reagent, a solvent and preparation equipment usually available in a preparation facility can be suitably used.

Also disclosed herein is a composition preparation kit which includes 1 or 2 or more containers containing the active components, which may be contained in each agent or composition of the present invention, individually or in combination, and also essential elements of each agent or composition to be provided in the form of such a kit. The kit may include, in addition to the above, instructions describing the preparation method and the administration method of each agent or composition of the present invention such as written information and a digital storage medium such as a CD or DVD. Additionally, the kit may include all essential elements for completing each agent or composition of the present invention, but it is not always necessary to include all the elements. Therefore, the kit may not include a reagent and a solvent commonly available in medical centers and experimental facilities such as sterile water, physiological saline solution and glucose solution.

The effective amount in each use in a treatment method of the present invention described herein is, for example, an amount that alleviates a symptom of a disease or delays or stops the progress of a disease, preferably an amount that suppresses or recovers a disease. Additionally, an amount that does not cause harmful effects that outweigh the benefits obtained from the administration is preferable. Such an amount is suitably determined by an in vitro test using cultured cells or a test on a model animal such as a mouse, a rat, a dog or a pig, and these test methods are well known to those skilled in the art. The drug dose used in the treatment method of the present invention is known to those skilled in the art or can be suitably determined by the above tests.

The specific dose of active component administered in the use in a treatment method of the present invention described herein may be determined by taking into account various conditions of a subject in need of the treatment, such as the degree of severity of symptoms, general health conditions of the subject, age, body weight and sex of the subject, diet, time and frequency of administration, co-administered drugs, reactivity to therapy, dosage form and compliance with therapy.

The route of administration includes various routes including both oral and parenteral, such as oral, intravenous, intramuscular, subcutaneous, local, intratumoral, intrarectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary and intrauterine.

The frequency of administration varies depending on the property of the agent or composition used and the conditions of the subject including the above, but may be, for example, multiple times a day (more specifically, 2 times, 3 times, 4 times, or 5 times or more a day), once a day, every few days (more specifically, every 2, 3, 4, 5, 6, or 7 days), every week, every several weeks (more specifically, every 2, 3, or 4 weeks).

The term "subject" used in the present description means any biological individual, preferably animals, further preferably mammals, further preferably human individuals. In the present invention, the subject may be healthy or affected by a disease, but when a treatment for a specific disease is envisaged, the subject normally means a subject who is affected by, or is susceptible to being affected by, the disease.

Additionally, the term “treatment,” when used herein, encompasses all medically acceptable classes of preventive and/or therapeutic intervention for the purpose of recovery, temporary remission, or prevention of a disease. For example, the term “treatment” encompasses medically acceptable interventions for a variety of purposes including slowing or stopping the progression of a disease, remitting or making a lesion disappear, preventing the onset, or preventing recurrence.

The drug that suppresses GST-rc demonstrates the induction of cell death and/or the suppression of cell growth for a cell having a mutation in the BRAF gene as described above. Therefore, with the suppression of GST-rc as an indicator, the cell death-inducing agent and/or the cell growth-suppressing agent can be selected for a cell having a mutation in the BRAF gene. More specifically, a substance capable of suppressing GST-rc may be a candidate substance for the cell death-inducing agent and/or the cell growth-suppressing agent for a cell having a mutation in the BRAF gene (usually, cancer cells).

For example, a test substance is contacted with a cancer cell having a mutation in the BRAF gene, as an example of the cancer cell, to measure the expression level of GST-rc in the cell. When the expression level in the case of contact with the test substance is reduced compared with the expression level measured in the absence of the test substance, the test substance can be selected as a candidate substance for the drug that suppresses GST-rc.

The test substance herein is not limited and may be any substance. The test substance may be a single substance or a mixture consisting of a plurality of constituent components. The test substance may have a composition containing unidentified substances such as an extract of a microorganism or a culture broth or a composition containing known compositions in a predetermined composition ratio. Additionally, the test substance may be any of proteins, nucleic acids, lipids, polysaccharides, organic compounds and inorganic compounds.

Examples

Hereinafter, the present invention is further described in detail with reference to examples, but the technical scope of the present invention is not limited thereto.

[Example 1]

In the present example, the cell growth suppressor effect was studied when the GST-rc-suppressing drug was allowed to act on a cell having a mutation in the BRAF gene. First, as a cancer cell having a mutation (V600E) in the BRAF gene, 1 species of a colorectal cancer cell line (CACO-2) and 4 species of melanoma cell lines (A375, SK-MEL-28, A2058, Malme-3M) were cultured at 37 °C under an atmosphere containing 5% CO2. The media used were MEM+20%, FBS+0.1 mM and NEAA for CACO-2, DMEM+15% and FBS for A375, EMEM+10% and FBS for SK-MEL-28, DMEM+10% and FBS for A2058 and IMDM+20% and FBS for Malme-3M, to all of which an antibiotic was added.

The day before transfection, each cell was inoculated onto a 100 mm plastic tissue culture dish using antibiotic-free medium such that 10-20% confluence was achieved. 600 pmol of GST-rc siRNA (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, Ambion (SEQ ID NO: 3)) was added to 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently shaken. Subsequently, 35 µl of Lipofectamine RNAi MAX (Invitrogen) was diluted with 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently shaken. The diluted GST-rc siRNA and diluted Lipofectamine RNAi MAX were gently shaken and then incubated at room temperature for 10 minutes. Meanwhile, the medium was replaced with 10 mL of Opti-MEM I reduced serum medium. After 10 min incubation, the complex of siRNA against GST-rc and Lipofectamine RNAi MAX was added to the cell and incubated at 37 °C under an atmosphere containing 5% CO2. After 5 h incubation, the medium was replaced with 10 mL of antibiotic-free medium. The medium was washed with PBS 1 h after replacement, cells were detached using 0.25% trypsin-EDTA (SIGMA) and suspended in antibiotic-containing medium. Cells were suspended in 5 ml of medium and inoculated onto a 60 mm plastic histoculture plate (CACO-2: 0.4 * 105 cells, A375: 1.0 * 105 cells, SK-MEL-28: 0.2 * 105 cells, A2058: 0.8 * 105 cells, Malme-3M: 0.6 * 105 cells).

The same operation was carried out as control experiments using random siRNA (CGAUUCGCUAGACCGGCUUCAUUGCAG, Hokkaido System Science Co., Ltd. (SEQ ID NO: 4)) or AllStars negative control siRNA (siControl) (QIAGEN). After siRNA transfection against GST-rc, the cell numbers were counted on day 0 and day 5, respectively.

Additionally, in the present example, the expression of GST-rc was confirmed by Western blotting respectively when siRNA against GST-rc was allowed to act on CACO-2 cells, A375 cells, SK-MEL-28 cells, A2058 cells and Malme-3M cells. More specifically, using the cell collected 3 days after the transfection of siRNA against GST-rc, Western blotting analysis on the knockdown of GST-rc was carried out. The collected cells were first washed with cold PBS, to which was added cold lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, EDTA-free Complete Mini (Roche) and PhosSTOP (Roche)), cooled on ice and incubated for 30 min for solubilization. Subsequently, centrifugation was carried out at 4 °C, 15000 rpm for 15 minutes, thereby obtaining a cell extract. Proteins were quantitatively determined for the obtained cell extract using the Micro BCA Protein Assay Kit (Thermo SCIENTIFIC). Next, 20 |ig of the cell extract was denatured under reducing conditions, and SDS-PAGE was performed using MULTIGEL II mini 4/20 (13 W) (Cosmo Bio) to separate the proteins. After the completion of SDS-PAGE, the proteins were transcribed onto a PVD membrane using a tank transfer apparatus. The transfer membrane was incubated in PBS with 5% skim milk/0.05% Tween 20 (abbreviated as PBS-T) at 4 °C for 16 hours to perform blocking. Subsequently, the membrane was reacted with anti-GST-rc antibody (MBL) diluted with membrane blocking solution (Invitrogen) at 4 °C for 16 h. Reaction with secondary antibody was carried out at room temperature for 1 h using horseradish peroxidase (HRP)-labeled rabbit antibody. Band signal detection on X-ray film was carried out by chemiluminescence method using ECL Western blot detection reagents (GE Healthcare). Washing was carried out between each operation by shaking for 5 min, 4 times using PBS-T.

The results on the colorectal cancer cell line are shown in Figure 1, and the results on the melanoma cell line are shown in Figure 2. Note that Figures 1 and 2 together show the results of cell number counting and the results of Western blot analysis. As shown in Figures 1 and 2, in both the colorectal cancer cell line and the melanoma cell line, the cell growth ability was markedly suppressed due to the gene attenuation of GST-rc by siRNA against GST-rc, which is the drug that suppresses GST-rc, in cancer cells carrying a mutation in the BRAF gene. The mutation in the b Ra F gene was found in high-grade tumors and is known as a poor prognostic factor in unresectable colorectal cancers. Half of melanoma patients have mutations in the BRAF gene, and when metastatic potential is high, lethality and malignancy are the highest. The results of the present study suggest that the drug that suppresses GST-rc is effective in suppressing cell growth of cancer cells that have a mutation in the BRAF gene, thereby raising expectations for a new therapy in these treatment-resistant cancers.

[Example 2]

In the present example, the cell growth suppressor effect was studied when the drug that suppresses GST-rc and a BRAF inhibitor were allowed to act on a cell that has a mutation in the BRAF gene.

First, 1 kind of colorectal cancer cell line (CACO-2) and 2 kind of melanoma cell lines (SK-MEL-28 and A2058) were respectively cultured in the same manner as in Example 1, and the day before transfection, they were inoculated into a 100-mm plastic tissue culture dish using antibiotic-free medium so that 10 to 20% confluence was achieved. 600 pmol of siRNA against GST-rc (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, Ambion (SEQ ID NO: 3)) was added to 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently shaken. Subsequently, 35 µl of Lipofectamine RNAi MAX (Invitrogen) was diluted with 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently vortexed. The diluted GST-rc siRNA and diluted Lipofectamine RNAi MAX were gently vortexed and then incubated at room temperature for 10 minutes. Meanwhile, the medium was replaced with 10 ml of Opti-MEM I reduced serum medium. After the 10-minute incubation, the complex of GST-rc siRNA and Lipofectamine RNAi MAX was added to the cell and incubated at 37 °C under an atmosphere containing 5% CO2. After the 5-hour incubation, the medium was replaced with 10 ml of antibiotic-free medium. The medium was washed with PBS 1 h after replacement, cells were detached using 0.25% trypsin-EDTA (SIGMA) and suspended in antibiotic-containing medium. Cells were suspended in 5 ml of medium and inoculated onto a 60 mm plastic tissue culture plate (CACO-2: 0.4 × 105 cells, SK-MEL-28: 0.2 × 105 cells, and A2058: 0.8 × 105 cells). The same operation was performed as a control experiment using AllStars negative control siRNA (siControl) (QIAGEN). On day 1 after transfection, PLX4720 (Selleck), a BRAF inhibitor, was added to the medium to achieve 40 |iM for CACO-2, 0.7 |iM for SK-MEL-28, and 10 |iM for A2058, and the culture was continued until day 5. The PLX4720-treated control was cultured by adding DMSO solvent (Wako Pure Chemical Industries, Ltd.). Growth assay was performed by counting the cell number from day 0 to day 5 after transfection.

Figure 3 shows the results on cell number counting of CACO-2, and Figure 4 shows the results on cell number counting of SK-MEL-28 and A2058. As evident from Figure 3, it was suggested that compared with the case where GST-rc siRNA was allowed to act alone on colorectal cancer cell line (CACO-2 line carrying a mutation in BRAF gene) or the case where PLX4720 was allowed to act on colorectal cancer cell line together with siControl, the cell growth suppressive effect was considerable in the case where GST-rc siRNA and PLX4720 were allowed to act in combination on such a cell line. Furthermore, as evident from Figure 4, it was suggested that compared with the case where GST-rc siRNA was allowed to act individually on melanoma cell lines (SK-MEL-28 and A2058 which have a mutation in BRAF gene) or the case where PLX4720 was allowed to act on melanoma cell lines together with siControl, the cell growth suppressive effect was considerable in the case where GST-rc siRNA and PLX4720 were allowed to act in combination on such melanoma cell lines.

[Example 3]

In the present example, the cell growth suppressor effect was studied when the drug that suppresses GST-rc was allowed to act on a cell that has a mutation in the BRAF gene and resistance to a BRAF inhibitor (BRAF inhibitor-resistant cell).

<Cell Number Count>

The BRAF inhibitor-resistant cell used in the present example was produced as follows. The melanoma cell line A375 used in Example 1 was incubated in DMEM medium containing antibiotic to which 1 to 5 µM PLX4720 (Selleck) and 15% FBS were added at 37 °C under an atmosphere containing 5% CO2 for one month. The cell line that survived after 1 month incubation was determined as PLX4720-resistant A375 cells and was used in the present example.

In the present example, the day before transfection, PLX4720-resistant A375 cell was inoculated into a 100 mm plastic tissue culture dish using antibiotic-free DMEM medium containing 15% FBS such that 0.5 * 106 cells/10 ml was achieved. 600 pmol of siRNA against GST-rc (GGGAGGCAAGACCUUCAUUTT, siRNA ID #2385, Ambion (SEQ ID NO: 3)) was added to 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently vortexed. Subsequently, 35 µl of Lipofectamine RNAi MAX (Invitrogen) was diluted with 1 ml of Opti-MEM I reduced serum medium (GIBCO) and gently vortexed. The diluted GST-rc siRNA and diluted Lipofectamine RNAi MAX were gently shaken and then incubated at room temperature for 10 minutes. Meanwhile, the medium was replaced with 10 mL of Opti-MEM I reduced serum medium. After 10-min incubation, the complex of GST-rc siRNA and Lipofectamine RNAi MAX was added to the cell and incubated at 37 °C under an atmosphere containing 5% CO2. After 5-h incubation, the medium was replaced with 10 mL of antibiotic-free DMEM medium containing 15% FBS. The medium was washed with PBS 2 hours after replacement, cells were detached using 0.5% trypsin-EDTA (SIGMA) and suspended in DMEM medium containing antibiotic and 15% FBS. Cells were inoculated in suspension onto a 60-mm plastic tissue culture dish so that 1.0 * 105 cells/5 ml were achieved. The same operation was carried out as control experiments using random siRNA (CGAUUCGCUAGACCGGCUUCAUUGCAG, Hokkaido System Science Co., Ltd. (SEQ ID NO: 4)) or AllStars negative control siRNA (QIAGEN). After transfection of siRNA against GST-rc, the cell numbers were counted on days 0, 1, 2, 3, 4, and 5, respectively.

<Confirmation of GST-rc expression>

Expression of GST-rc was confirmed by Western blotting when GST-rc siRNA was allowed to act on PLX4720-resistant A375 cell as described above. More specifically, using cells harvested at each of the above time points after GST-rc siRNA transfection, Western blotting analysis of GST-rc knockdown was performed.

The harvested cells were first washed with cold PBS, to which was added cold lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, EDTA-free complete Mini (Roche), and PhosSTOP (Roche)), cooled on ice, and incubated for 30 min for solubilization. Centrifugation was carried out at 4 °C, 15000 rpm for 15 min, thereby obtaining a cell extract. Proteins for the obtained cell extract were quantitatively determined using Micro BCA Protein Assay Kit (Thermo SCIENTIFIC). Then, 20 µg of the cell extract was denatured under reducing conditions, and SDS-PAGE was performed using MULTIGEL II mini 4/20 (13 W) (Cosmo Bio) to separate proteins. After the completion of SDS-PAGE, proteins were transcribed onto a PVDF membrane using a tank blotting apparatus. The blotting membrane was incubated in PBS containing 5% skim milk/0.05% Tween 20 (abbreviated as PBS-T) at 4°C for 16 h to perform blocking. Subsequently, the membrane was reacted with an anti-GST-rc antibody (MBL) diluted with membrane blocking solution (Invitrogen) at 4°C for 16 h. The secondary antibody reaction was carried out at room temperature for 1 h using a horseradish peroxidase (HRP)-labeled rabbit antibody. Band signal detection on X-ray film was carried out by the chemiluminescence method using ECL Western blotting detection reagents (GE Healthcare). Washing was carried out between each of the operations by shaking for 5 minutes, 4 times using PBS-T.

<Results>

In recent years, it has been understood that melanoma cell line that acquired resistance to BRAF inhibitor is involved in melanoma recurrence. Therefore, GST-rc was subjected to gene knockdown using PLX4720-resistant A375 cells to investigate whether GST-rc facilitates CRAF dependence in BRAF inhibitor-resistant melanoma. Cell growth measurement revealed the remarkable growth suppressor effect (Figure 5). When gene knockdown of GST-rc was confirmed, GST-rc expression was suppressed on day 2 and day 3 (Figure 6).

The present example suggests that the growth of cells having a mutation in the BRAF gene and resistance to a BRAF inhibitor (BRAF inhibitor-resistant cell) was effectively suppressed. Based on the results, the effect of preventing or reducing diseases in which BRAF inhibitor-resistant cell is a factor, for example, melanoma recurrence, can be expected.

Claims (4)

i. A pharmaceutical composition for use in a method for the therapy of a cancer caused by a growth defect of a cancer cell having a mutation in a BRAF gene and resistance to a BRAF inhibitor, the pharmaceutical composition comprising a cell death-inducing agent or cell growth suppressing agent for a cell having a mutation in a BRAF gene, said agent comprising a drug that suppresses GST-rc as an active ingredient, 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 one thereof. 2. Pharmaceutical composition for use according to claim 1, wherein the mutation is V600E mutation. 3. Pharmaceutical composition for use according to any one of claims 1-2, wherein the cancer is a cancer with high expression of GST-rc. 4. Pharmaceutical composition for use according to any one of claims 1-2, wherein the cancer is colorectal cancer or melanoma.