JP2017036213A - Cancer stem cell-targeting drug composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a drug that targets a cancer stem cell and thus can prevent recurrence and metastasis so as to completely cure cancer.SOLUTION: A cancer stem cell-targeting drug composition comprises a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, derivatives thereof, and pharmacologically acceptable salts and solvates of the same.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a pharmaceutical composition targeting cancer stem cells.

  In recent years, it has been clarified that a small number of cells having a stem cell-like property called “cancer stem cells” exist in a cell population forming cancer. Cancer stem cells are cells that exist in tumors at the American Association for Cancer Research (AACR), and have both self-replicating ability and the ability to generate various types of cancer cells that make up tumor tissue. (Non-patent Document 1). That is, cancer stem cells produce undifferentiated cancer stem cells by self-replication, while producing non-cancer stem cells different from themselves. In the process, various cancer cells appear due to genetic or epigenetic changes, and are considered to cause intra-tumor heterogeneity. Cancer cell heterogeneity in tumor tissue is clinically important because it can cause the cancer cell population in the tissue to not respond uniformly to treatment.

Cancer stem cells are known to be more resistant to anticancer drugs and radiation therapy than non-cancer stem cells. Reasons for resistance to anti-cancer drugs include high expression of ABC transporters with drug excretion, and drugs that inhibit cell division are not successful because they are in the G0 phase (resting phase) of the cell cycle. And so on.
Cancer stem cells have the ability to self-replicate and have the ability to re-form tumors from a small number of cells, which can cause recurrence from minimal residual disease (MRD). In addition, by obtaining an optimal microenvironment (niche) in a remote organ, tumor tissue can be formed from a small number of cells, so that a metastatic focus can be generated.

  Due to the above properties of cancer stem cells, treatment targeting cancer stem cells is considered important in aiming for radical treatment of cancer.

  By the way, the present inventors have reported that human telomerase reverse transcriptase (hTERT) functions as an RNA-dependent RNA polymerase (RdRP) (Non-patent Document 2). On the other hand, it has been reported that a complex of hTERT, BRG1, and NS / GNL3L (hereinafter referred to as “TBN complex”) is necessary for maintaining the function of cancer stem cells (Non-patent Document 3).

Clarke, M.F.et al .: Cancer Res., 66: 9339-9344, 2006 Maida Y. etal., Nature., 461 (7261): 230-235, 2009 Okamoto N. etal., Proc Natl Amer Sci., 108 (51): 20388-20393, 2011

  An object of the present invention is to provide a medicine that can target cancer stem cells and prevent recurrence and metastasis, and can aim for radical treatment of cancer.

The inventors subsequently found that the TBN complex functions as human RdRP, and therefore hypothesized that RdRP might be involved in maintaining the function of cancer stem cells. If this hypothesis is correct, inhibitors of RdRP may be therapeutics that target cancer stem cells.
Thus, when a specific inhibitor of RdRP was screened from a large number of low molecular weight compounds, VX-222, eribulin, vindesine, β-rubromycin and BIBR1532 were identified. Furthermore, the cancer stem cell type cancer cells were confirmed to be more sensitive to VX-222 than normal cancer cells, and the present invention was completed.
That is, the present invention
[1] Cancer comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof Stem cell targeted drug composition;
[2] The cancer stem cell targeted drug composition according to the above [1], which is administered to a subject confirmed to have cancer stem cells, a subject after cancer treatment, or a subject having cancer metastasis;
[3] Confirmation that the subject has cancer stem cells is performed by examining the expression of a cancer stem cell marker in cells contained in the subject cancer tissue, The cancer stem cells according to [2] above Targeted drug composition;
[4] The cancer stem cell markers are CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, And the cancer stem cell targeted drug composition according to the above [3], selected from the group consisting of N-cadherin;
[5] Cancer comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof Preventive or therapeutic agent for metastasis or recurrence of
[6] The preventive or therapeutic agent for cancer metastasis or recurrence according to [3], which is administered to a subject after cancer treatment;
[7] RNA dependence comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof RNA polymerase inhibitors;
[8] Administering to a subject a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof A method for presenting data for judging whether or not the object is appropriate,
A method comprising examining the expression of a cancer stem cell marker in cells contained in cancer tissue collected from a subject;
[9] The cancer stem cell markers are CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, And the method according to [8] above, which is selected from the group consisting of N-cadherin;
[10] A compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof,
A cancer stem cell marker detection drug,
A cancer stem cell targeted treatment kit comprising:
[11] The cancer stem cell marker detection drug comprises:
Selected from the group consisting of CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N-cadherin An antibody against at least one cancer stem cell marker, or
Selected from the group consisting of CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N-cadherin The cancer stem cell target according to [10], comprising a primer and / or a probe for detecting a mutation in a cancer stem cell marker gene that specifically increases or decreases the expression of at least one cancer stem cell marker. A treatment kit; and [12] a step of incubating a drug candidate compound and an RNA-dependent RNA polymerase in contact with each other;
Selecting a drug candidate compound that inhibits the RNA-dependent RNA polymerase activity;
The present invention relates to a method for screening cancer stem cell targeted drugs.

  According to the present invention, cancer stem cells can be attacked through inhibition of RdRP, so that cancer stem cells resistant to anticancer agents and radiation therapy can also be killed to aim for radical treatment of cancer. . It is also possible to remove recurrence and metastasis by removing minute residual lesions after cancer treatment.

FIG. 1 shows the results of examining the effect of VX-222 on telomerase activity and human RdRP activity. FIG. 2 shows the results of examining the effect of eribulin on telomerase activity and human RdRP activity. FIG. 3 shows the results of examining the effects of vindesine, vincristine, paclitaxel, docetaxel, and etoposide on human RdRP activity. VX-222 is a positive control. FIG. 4 shows the results of examining the effects of β-rubromycin, BIBR1532, and costunolide on human RdRP activity. VX-222 is a positive control. FIG. 5 shows the results of examining the sensitivity of cancer stem cells and normal cancer cells to VX-222.

The cancer stem cell targeted drug composition according to the present invention is a group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof. A compound selected from
As used herein, “cancer stem cell targeted drug” means a drug or reagent having a cell killing effect on cancer stem cells. In this specification, “medicine” is administered to a subject for the purpose of treatment or prevention, and includes drug candidates under development, and “reagent” is not administered to the subject but mainly in vitro in experiments. Means what is used in

  In the present specification, the term “cancer stem cell” is used in its broadest sense, and has a tumor-forming ability with a very small number of cells, a self-replicating ability, and a non-cancer cell-producing ability. A cell characterized by a property such as

Although cancer stem cells have been identified in the following human cancers, “cancer stem cells” in the present specification are not limited to these. It shows with the cancer stem cell marker which can be used for isolation of each cancer stem cell.
(Cancer) (Cancer stem cell marker)
Acute myeloid leukemia CD34 ⁺ / CD38 ⁻
Brain tumor CD133 ⁺
Lung cancer CD133 ⁺
Breast cancer CD44 ⁺ / CD24- ^{/ low} / ESA ⁺ , ALDH1 ⁺
Colorectal cancer CD133 ⁺ , EpCAM ^hi / CD44 ⁺
Pancreatic cancer ESA ⁺ / CD44 ⁺ / CD24 ⁺
Liver cancer CD90 ⁺
Prostate cancer CD44 ⁺ / integrin α ₂ β ₁ ^hi / CD133 ⁺ , Sca-1 ⁺
Head and neck squamous cell carcinoma CD44 ⁺

The cell killing effect on cancer stem cells can be confirmed by a person skilled in the art or a method analogous thereto (for example, a method used in Examples described later).
When a certain drug or reagent has a cell-killing effect on cancer stem cells, it is not limited to a case where it shows a statistically significant cell-killing effect, but may be a degree that a person skilled in the art can judge to have a cell-killing effect. Including.

  VX-222 (CAS Registry Number: 1026785-55-6) is known as a hepatitis C virus polymerase inhibitor (WO2008 / 058393).

Examples of the derivative of VX-222 include, but are not limited to, the following.
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-methoxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl) -amino] -thiophene-2-carboxylic
acid;
5- (3,3-
dimethyl-but-1-ynyl) -3-[(trans-4-methyl-cyclohexanecarbonyl)-(cis-4- [1,2,4] triazol-1-yl-cyclohexyl) -amino] -thiophene-2- carboxylic
acid;
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4- [1,2,4] triazol-1-yl-cyclohexyl)- amino] -thiophene-2-carboxylic
acid;
5- (3,3-dimethyl-but-1-ynyl) -3-[(cis-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl) -amino] -thiophene-2-carboxylic
acid;
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-methyl-cyclohexanecarbonyl)-(1-methyl-piperidin-4-yl) -amino] -thiophene-2-carboxylic
acid; hydrochloride;
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-cis- [1,2,3] triazol-1-yl-cyclohexyl)- amino] -thiophene-2-carboxylic
acid;
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4- [1,2,3] triazol-1-yl-cyclohexyl)- amino] -thiophene-2-carboxylic
acid; and
5- (3,3-dimethyl-but-1-ynyl) -3-[(trans-4-fluoro-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl) -amino] -thiophene-2-carboxylic
acid.

Eribulin (CAS registration number: 253128-41-5) is used as an anticancer agent whose mesylate is indicated for metastatic breast cancer and recurrent breast cancer. It is a macrocyclic ketone synthetic analog of halichondrin B, a natural organic compound derived from sponge, and is represented by the following formula.

Derivatives of eribulin include those described in US Pat. No. 6,214,865, and the eribulin derivatives described in that patent are incorporated herein by reference. Specific examples include those obtained by removing eribulin itself from those represented by the following general formula.

In the formula, A represents a saturated alkyl group having 1 to 6 carbon atoms or an unsaturated alkyl group having 2 to 6 carbon atoms which may have 1 to 10 substituents, and the substituent is a hydroxy group. Group, cyano group, halo group, azide group, oxo group, OR ₁ , SR ₁ , SO ₂ R ₁ , OSO ₂ R ₁ , NR ₂ R ₁ , NR ₂ (CO) R ₁ , NR ₂ (CO) (CO ) R ₁ , NR ₄ (CO) NR ₂ R ₁ , NR ₂ (CO) OR ₁ , (CO) OR ₁ , O (CO) R ₁ , (CO) NR ₂ R ₁ , and O (CO) NR ₂ Selected from the group consisting of R ₁ ;
R ₁ , R ₂ , R ₄ , R ₅ , and R ₆ are each independently H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _6-10 aryl, C _6-10 haloaryl, C _6-10 hydroxyaryl, C _1-3 alkoxy-C ₆ aryl, C _6-10 aryl-C _1-6 alkyl, C _1-6 alkyl-C _6-10 Aryl, C _6-10 haloaryl-C _1-6 alkyl, C _1-6 alkyl-C _6-10 haloaryl, (C _1-3 alkoxy-C ₆ aryl) -C _1-3 alkyl, C _2-9 heterocycle Selected from the group consisting of a group, C _2-9 heterocycle-C _1-6 alkyl, C _2-9 heteroaryl, and C _2-9 heteroaryl-C _1-6 alkyl;
D and D ′ are each independently selected from R ₃ and OR ₃ , wherein R ₃ is selected from the group consisting of H, C _1-3 alkyl, and C _1-3 haloalkyl;
n is 0 or 1;
E is R ₅ or OR ₅ ;
G is selected from the group consisting of O, S, CH ₂ and NR ₆ ;
J and J ′ are each independently selected from the group consisting of H, C _1-6 alkoxy, and C _1-6 alkyl, or J and J ′ together are ═CH ₂ or —O—. (Linear or branched C _1-5 alkylene) -O-;
Q is C _1-3 alkyl;
T is ethylene or ethenylene optionally substituted with (CO) OR ₇ and R ₇ is H or C _1-6 alkyl;
U and U ′ are each independently selected from the group consisting of H, C _1-6 alkoxy, and C _1-6 alkyl, or U and U ′ together are ═CH ₂ or —O—. (Linear or branched C _1-5 alkylene) -O-;
X is H or C _1-6 alkoxy;
Y and Y ′ are each independently H or C _1-6 alkoxy, or Y and Y ′ together are ═CH ₂ or —O— (linear or branched C _1-5 alkylene). -O-;
Z and Z ′ are each independently H or C _1-6 alkoxy, or Z and Z ′ together are ═CH ₂ or —O— (linear or branched C _1-5 alkylene). -O-.

  Vindesine (Vindesine, CAS Registry Number: 53463-48-4) has its sulfate used as an anticancer agent. It is a vinca alkaloid compound synthesized based on vinblastine. Therefore, vindesine derivatives include vinblastine and vinorelbine, which are vinca alkaloid compounds.

β-Rubromycin (CAS Registry Number: 27267-70-5) is a compound that has been reported as a human telomerase inhibitor (Ueno T. et al., Biochemistry, 39: 5995-6002,
2000).

BIBR1532 (CAS Registry Number: 321674-73-1) is also a compound that has been reported as a human telomerase inhibitor (Damm K. et al., EMBO J. 20 (24): 6958-6968,
2001).

  In the present specification, the derivative is not particularly limited as long as the derivative has a cell-killing effect on cancer stem cells.

In the present specification, the pharmacologically acceptable salt is not particularly limited as long as the salt of the compound has a cytocidal effect on cancer stem cells, but may be an organic or inorganic acid or base salt. For example, hydrochloride, sulfate, bisulfate, hydrobromide, hydroiodide, nitrate, perchloric acid, fumarate, maleate, phosphate, superphosphate, isonicotinic acid Salt, glycolate, lactate, salicylate, succinate, gluconate, p-toluenesulfonate, tartrate, pantothenate, ascorbate, acetate, trifluoroacetate, citrate, Examples include mesylate, methanesulfonate, ethanesulfonate, saccharinate, formate, benzoate, malonate, naphthalene-2-sulfonate, and benzenesulfonate. It may be a salt of an amino acid such as L-arginine, L-lysine, and glutamic acid.
Examples of the base salt include alkali metals such as sodium, lithium and potassium, alkaline earth metals such as calcium and magnesium, ammonia, NR ₄ ⁺ (R is an alkyl group having 1 to 4 carbon atoms), aluminum, zinc, Examples include choline and tromethamine salts.

  The solvent used for the solvate in the present specification is not particularly limited as long as the solvate of the compound has a cell-killing effect on cancer stem cells. For example, water, a pharmacologically acceptable organic solvent (ethanol, Hexane, ethyl acetate, etc.).

  When the cancer stem cell targeted drug composition according to the present invention is administered as a medicine, it may be administered to a subject confirmed to have cancer stem cells. Whether or not the subject has cancer stem cells can be confirmed by a person skilled in the art by a known method or a method equivalent thereto, for example, the expression of a cancer stem cell marker in cancer tissue extracted from the subject. It can be examined by measuring.

A cancer stem cell marker is a protein or nucleic acid (mRNA, miRNA, etc.) that is specifically expressed or enhanced in cancer stem cells, and is specifically decreased or expressed in cancer stem cells. No protein or nucleic acid. Measurement of cancer stem cell marker expression may be performed at the protein level or at the nucleic acid level.
Non-limiting examples of cancer stem cell markers include CD34, CD38, CD133, CD44, CD24, ESA, ALDH1 (Aldehyde dehydrogenase 1), EpCAM (Epithelial cell adhesion molecule), CD90, integrin α ₂ β ₁ , Sca-1 (Stem cell antigen-1), hTERT (human telomerase reverse transcriptase), NS (Nucleostemin), BRG1 (Brahma related gene 1), STAT3 (signal transducer and activator of transcription 3), TWIST, SNAIL, and N-cadherin or these The combination of these is mentioned. TWIST, SNAIL, and N-cadherin are known as markers of epithelial mesenchymal transition (EMT). Recent studies suggest that EMT-causing cells acquire stem cell-like functions and produce cancer stem cells. EMT and cancer stem cells are highly related.

Measurement of the expression of a cancer stem cell marker can also be performed by detecting a gene mutation that increases or decreases the expression of the cancer stem cell marker in a cancer stem cell-specific manner. For example, an increase in the expression of a cancer stem cell marker TERT protein is caused by a point mutation of the nucleotide sequence in the promoter region of the TERT gene (C288T and / or C250T; Horn S. et al., Science, 2013, 339 (6122) : 959-961; Huang FW et al., Science, 2013,
339 (6122): 957-959; Killela PJ et al., Proc Natl Acad Sci, 2013, 110 (15): 3021-6026). When there is a mutation that confirms the presence or absence of such a point mutation in the gene and causes an increase or decrease in the expression of a cancer stem cell-specific cancer stem cell marker, the target is the cancer stem cell targeted drug according to the present invention The composition can be administered as a medicament.

  When the cancer stem cell targeted drug composition according to the present invention is administered as a medicine, it may be administered to a subject after cancer treatment. After cancer treatment refers to after radiation therapy, chemotherapy, or surgery. It can be administered at any time after treatment, but for example, if it is administered to a subject in remission after cancer treatment, it attacks the invisible minute residual lesions containing cancer stem cells and causes recurrence or Useful to prevent metastasis.

  When the cancer stem cell targeted drug composition according to the present invention is administered as a medicine, it may be administered to a subject having cancer metastasis. Cancer stem cells are thought to be associated with metastasis, and thus may have the effect of stopping or delaying further progression of metastasis.

  In the present specification, the “subject” is typically a human, but may be a mammal such as a mouse, rat, rabbit, cat, dog, monkey, sheep, cow, or horse.

The present invention includes compounds selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof, It also includes preventive or therapeutic agents for cancer metastasis or recurrence.
The agent for preventing or treating cancer metastasis or recurrence according to the present invention may be administered after cancer treatment, for example, in a remission state. It may also be administered to subjects with cancer metastasis.

  The cancer stem cell targeted drug composition and the preventive or therapeutic agent for cancer metastasis or recurrence according to the present invention are administered to a subject having any cancer as long as cancer stem cells are contained in the cancer tissue. May be. For example, acute or chronic myelogenous leukemia, nervous system type tumors such as astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, schwannoma, neurofibrosarcoma, nerve Blastoma, pituitary tumor (eg, pituitary adenoma), medulloblastoma, melanoma, brain tumor, prostate cancer, head and neck cancer, esophageal cancer, renal cancer, renal cell cancer, pancreas Cancer, breast cancer, lung cancer, colon cancer, colon cancer, stomach cancer, skin cancer, ovarian cancer, bladder cancer, fibrosarcoma, squamous cell carcinoma, neuroectodermal, thyroid tumor, lymphoma, hepatocellular carcinoma, medium Examples include dermatomas and epidermoid carcinomas.

The present invention relates to an RNA comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof. Dependent RNA polymerase inhibitors are also included.
The RdRP inhibitor according to the present invention is used as a pharmaceutical or a reagent in vitro or in vitro.
It can be used in vivo, and can be useful for testing to examine various reactions involving RdRP, and for treating or preventing various diseases involving RdRP.

  Cancer stem cell targeted drug composition, preventive or therapeutic agent for cancer metastasis or recurrence, and RdRP inhibitor according to the present invention include VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and Two or more compounds selected from the group consisting of these pharmacologically acceptable salts and solvates may be contained.

  The cancer stem cell targeted drug composition, preventive or therapeutic agent for cancer metastasis or recurrence, and RdRP inhibitor according to the present invention, as long as it has a cell killing effect on cancer stem cells, Additives can be added as appropriate and the preparation can be formulated by a known method. Examples of pharmaceutical preparations include tablets, capsules, granules, fine granules, powders, solutions, oral preparations such as syrups, chewable and troches, ointments, gels, creams, patches and other external preparations, injections Agents, sublingual agents, inhalants, eye drops, and suppositories. Alternatively, it may be formulated using drug delivery system technology such as liposome preparation, polymer micelle preparation, dendrimer preparation, water-soluble polymer preparation, hydrogel preparation, nanosphere preparation, emulsion preparation and the like. The preparation may be targeted by binding an antibody, or a polymer such as PEG may be bound to control the residence time in blood.

  Additives include excipients, lubricants, disintegrants, binders, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, buffering agents, coloring agents, preservatives, antioxidants. , Stabilizers, absorption promoters and the like.

  Examples of excipients include starch, dextrin, sucrose, lactose, glucose, erythritol, mannitol, sorbitol, maltitol, trehalose, crystalline cellulose, light anhydrous silicic acid, calcium silicate, aluminum silicate, magnesium aluminate metasilicate, Examples include calcium phosphate, calcium hydrogen phosphate, and calcium sulfate. Examples of the lubricant include magnesium stearate, calcium stearate, sodium stearyl fumarate, and talc. Examples of the disintegrant include agar, gelatin, crystalline cellulose, calcium carbonate, sodium hydrogencarbonate, calcium citrate, pectin, dextrin, low-substituted hydroxypropylcellulose, crospovidone, carboxymethylcellulose, and carboxymethylcellulose calcium. Examples of the binder include methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, gum arabic, tragacanth, and gelatin.

  Examples of the emulsifier or surfactant include stearyl triethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, glyceryl monostearate, sucrose fatty acid ester, and glycerin fatty acid ester. Examples of the solubilizer include polyethylene glycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, and sodium citrate. Examples of the suspending agent include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose in addition to the above surfactant. Examples of the buffer include buffer solutions such as phosphate, acetate, and citrate.

  Examples of flavoring agents include mint oil, menthol, lemon oil, ascorbic acid, tartaric acid, malic acid, aspartame, sucralose, acesulfame potassium, and colorants such as iron sesquioxide, yellow iron sesquioxide, carmine, caramel, β -Carotene, titanium oxide, talc, riboflavin sodium phosphate and the like. Examples of preservatives include methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antioxidants such as ascorbic acid, tocopherol, and stabilizers such as ascorbic acid and edetic acid. Examples of sodium, erythosuvate, tocopherol, and absorption promoter include isopropyl myristate, tocopherol, calciferol and the like.

When the cancer stem cell targeted drug composition according to the present invention, the preventive or therapeutic agent for cancer metastasis or recurrence, and the RdRP inhibitor are administered to a subject as a medicament, they are administered in combination with other anticancer agents. Also good.
Non-limiting examples of anticancer agents include oxaliplatin, carboplatin, cisplatin, interferon alpha, interferon beta, interferon gamma, interleukin 2, anastrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, tamoxifen, bicalutamide, Flutamide, prednisolone, newprorelin, letrozole, actinomycin D, aclarubicin, idarubicin, epirubicin, daunorubicin, doxorubicin, pirarubicin, bleomycin, pepromycin, mitomycin C, mitoxantrone, irinotecan, etoposide, sobuzoxane, docetaxane, docetaxe Paclitaxel, vinorelbine, enocitabine, carmofur, gemcitabine, shi Rabin, tegafur, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, dimustine, busulfan, melphalan, ibritumomabutylxetane, imatinib, erlotinib, gefitizib Ozogamicin, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib, rituximab, or any salt thereof.

When the cancer stem cell targeted drug composition, the preventive or therapeutic agent for cancer metastasis or recurrence, and the RdRP inhibitor according to the present invention are administered to a subject as a medicament, the composition is administered in combination with a medicament for reducing its side effects. May be. For example, short-term administration of anti-cancer drugs such as systemic malaise, loss of appetite, vomiting, etc. 5HT ₃ receptor antagonists (ondansetron, granisetron, palonosetron, ramosetron, tropisetron, azasetron, indisetron, etc.), NK ₁ receptor antagonists (aprepitant, etc.), prochlorperazine, metoclopramide, lorazepam, H ₂ blockers, proton pump inhibitors, dopamine receptor antagonists, combinations with administration of butyrophenones and the like.

  When administered in combination with other drugs, the cancer stem cell targeted drug composition according to the present invention, the preventive or therapeutic agent for cancer metastasis or recurrence, and the RdRP inhibitor may be administered simultaneously. , Either may be administered first. In addition, the number of administrations and the administration interval may be different.

  VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof included in the cancer stem cell targeted drug composition, preventive or therapeutic agent for cancer metastasis or recurrence, and RdRP inhibitor according to the present invention, In addition, the amount of these pharmacologically acceptable salts and solvates is determined according to the type of the target disease, the type of active compound, the age, sex, weight, symptom of the subject, presence or absence of combined use with other drugs, etc. It can be determined as appropriate.

The present invention provides a cancer stem cell targeted drug composition according to the present invention, a cancer metastasis or recurrence prevention or treatment agent, or an RdRP inhibitor, a cancer stem cell attack method, cancer recurrence Alternatively, a method for preventing or treating metastasis is also included.
Prior to the treatment method, a step of confirming that the above-described subject has cancer stem cells may be performed.

  The present invention also includes a step of performing chemotherapy, radiation therapy, and surgical treatment, a cancer stem cell targeted drug composition according to the present invention, a preventive or therapeutic agent for cancer metastasis or recurrence, or an RdRP inhibitor. A method of treating cancer comprising the step of administering. By routine administration of a cancer stem cell targeted drug composition or the like after chemotherapy, radiation therapy, or surgical therapy, it is possible to prevent relapse or metastasis targeting an invisible residual tumor.

The present invention also relates to a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof. A method of presenting data for determining whether or not to administer the drug.
A compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof, targets cancer stem cells. Therefore, such data includes data indicating the expression level of a cancer stem cell marker in cells contained in cancer tissue collected from a subject. The act of presenting the data can be performed by, for example, an inspection company. If this data is presented to a doctor or the like, the doctor can determine whether the cancer stem cell according to the present invention is based on the presence or absence of the cancer stem cell in the cancer tissue. The suitability of the targeted drug composition, the preventive or therapeutic agent for cancer metastasis or recurrence, and the RdRP inhibitor can be determined.

The present invention relates to a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof, and cancer. A kit for treatment targeting cancer stem cells, comprising a stem cell marker detection drug, is also included.
With such a kit, the presence or absence of cancer stem cells in the target cancer tissue is evaluated, and if cancer stem cells are present, VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and these drugs Cancer stem cells can be attacked by administration of a compound selected from the group consisting of physically acceptable salts and solvates.
Cancer stem cell marker detection agents include antibodies that specifically recognize cancer stem cell markers and cancer stem cell marker genes (for example, mutations that increase or decrease the expression of cancer stem cell markers specifically for cancer stem cells) For example, a primer that specifically amplifies the region to be contained) and a probe that specifically detects the region. In addition, the kit according to the present invention may include a reagent for labeling an antibody or amplified nucleic acid so as to be detectable, a microtiter plate, a buffer solution, an instruction manual, and the like.

The present invention also includes a screening method for cancer stem cell targeted drugs. As shown in the Examples, as the inventors hypothesized, Vd-222, an RdRP inhibitor, showed a cell killing effect on cancer stem cells. Therefore, specific inhibitors of RdRP can be cancer stem cell targeted drugs.
The screening method according to the present invention includes a step of bringing a drug candidate compound into contact with an RNA-dependent RNA polymerase and incubating, and a step of selecting a drug candidate compound that inhibits RNA-dependent RNA polymerase activity.
The drug candidate compound is not particularly limited, and may be a low molecular compound, a high molecular compound, a nucleic acid, a peptide, a protein, or the like. The step of bringing the drug candidate compound into contact with RdRP and incubating can be performed by those skilled in the art by appropriately selecting conditions according to the type of drug candidate compound. RdRP activity may also be measured according to a method known to those skilled in the art.

  The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to this at all. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

1. Screening for human RdRP inhibitors
1-1. Method for producing M-phase accumulated cells
Day 0: 1 × 10 ⁶ HeLa cells were seeded per 10 cm dish (10 ml of medium).
Day 2: The medium was replaced with a medium containing Thymidine (final concentration 2.5 mM).
Day 3: 24 hours after changing to Thymidine medium, HeLa cells were washed 3 times with 10 ml of PBS, changed to normal medium and cultured.
After culturing in a normal medium for 6 hours, the medium was replaced with a medium containing Nocodazole (final concentration 0.1 μg / ml) and cultured for 14 hours (the medium was adjusted for use).
Day 4: Medium was applied to the dish with a pipette, and the cells were physically removed and collected. The cells were collected in a 50 ml tube (for 5 plates), the number of cells was counted, the medium was removed after centrifugation, and the pellet was stored at −80 ° C.

[Culture medium]
Penicillin (100 U / ml) and streptomycin (100 μg / ml) were added to DMEM Wako 044-29765 (containing 10% IFS).
[Reagent for synchronization]
A 125 mM aqueous solution stock of Thymidine (Nacalai Tesque 07147-61) was prepared, sterilized by filtration through a 0.22 μm filter, and stored at −20 ° C.
Nocodazole (Sigma M1404) 5μg / μl
A DMSO solution was prepared and stored at 4 ° C. protected from light.

1-2. Adjustment of lysate 1-1. The HeLa cells synchronized with M phase were gently washed with 10 ml of PBS (-), centrifuged at 3000 rpm (1,450 xg) at 4 ° C for 5 minutes, and the supernatant was removed (PBS (-) Was ice-cold).
HeLa cells were suspended in lysis buffer A (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.5% NP-40) (1 × 10 ⁷ cells / ml). The buffer was ice-cooled.
Sonication was performed once for 10 seconds and centrifuged at 15,000 rpm (20,400 × g) at 4 ° C. for 15 minutes, and then the lysate was recovered.

1-3. IP-RdRP assay
Add 1 ml of lysate to Pierce Protein A Plus Agarose (Thermo
40 μl (bed volume 20 μl) was added and stirred at 4 ° C. for 30 minutes using a rotator to prepare a preclear. Protein A
Agarose was previously equilibrated with lysis buffer A.
The lysate was recovered by centrifugation at 13,000 xg for 1 minute at 4 ° C. 10 μg anti-hTERT antibody (clone: 10E9-2) and 40 μl (bed volume 20 µ l) Pierce in lysate
Protein A Plus Agarose (Thermo
scientific) was added, and the mixture was reacted overnight at 4 ° C using a rotator.
Centrifugation was performed at 3,300 × g and 4 ° C. for 0.5 min, and the supernatant was removed with an aspirator. The beads (Protein A Plus Agarose) were washed with 1 ml of Wash buffer I (using a rotator for 5 minutes at 4 ° C for 4 times).
Wash buffer I is 1x containing 10% glycerol, 0.1% Triton-X, and 0.06x protease inhibitors (Roche, complete EDTA-free)
acetate buffer.
The beads were washed with AGC solution containing 2 mM CaCl ₂ at 4 ° C. for 5 minutes. The composition of AGC solution is 1x acetate buffer, 10%
Glycerol and 0.02% CHAPS were used.
Micrococcal to remove nucleic acids attached to beads
Nuclease (MNase) treatment was performed.
Bead suspension: 20 μl (bed volume)
MNase (20U / μl, Takara): 1 μl
10xMNase reaction buffer: 8 μl
H ₂ O: 51 μl
The tube was placed on an 8-shaped shaker placed in a 25 ° C incubator and shaken slowly for 15 minutes (25 rpm).
After centrifuging at 3,300 × g and 4 ° C. for 0.5 min and removing the supernatant with aspirate, the beads were washed twice for 5 minutes at 4 ° C. with AGC solution containing 3 mM EGTA.
The beads were washed with 1x acetate buffer containing 0.02% CHAPS at 4 ° C for 5 minutes.

RdRP reaction was performed. The composition of the reagent was as follows.
0.07% CHAPS: 6 μl
HMD solution *: 2 μl
(* 0.2 M HEPES-KOH (pH 7.8), 40 mM MgCl ₂ , and 2 mM DTT)
80 mM rATP: 0.5 μl
8 mM rGTP: 1 μl
0.4 mM rUTP: 1 μl
8 mM rCTP: 1 μl
RNase inhibitor (40U / μl, Toyobo): 0.5 μl
[α- ³² P] UTP, 3000 Ci / mmol: 6 μl
1 μg / μl template RNA: 1 μl
H ₂ O: 1 μl

After preparing the above reagents, the washing buffer was added to the beads (20 μl (bed volume)) removed with aspirate. In the case of adding an inhibitor, after adding a reagent to the beads, 0.4 μl of an inhibitor (10 mM) was added and pipetting was performed.
The tube was placed on an 8-shaped shaker placed in a 32 ° C incubator and shaken slowly (25 rpm), and allowed to react for 2 hours.
5.4 μl of 20 mg / ml Proteinase K (Takara), 2x Proteinase K buffer (20
mM Tris-HCl (pH 7.6), 20 mM EDTA, and 1%
45.4 μl of SDS) was added and reacted at 37 ° C. for 30 minutes.
After adding 109.2 μl of H ₂ O, 200 μl of acid phenol-chloroform (Ambion) was added and centrifuged at 15,000 rpm (21,100 × g) at room temperature for 5 minutes, and then the aqueous layer was transferred to a new tube.
After repeating Phe / Chl treatment again, add 20 μl of 3 M sodium acetate (pH 5.2), 4 μl of GenTLE precipitation carrier (Takara), 250 μl of ethanol, and add 15,000
Centrifugation was performed at rpm (21,100 × g) and 4 ° C. for 20 minutes, and the supernatant was discarded.
The pellet was rinsed with 300 μl of 70% cold ethanol and centrifuged at 15,000 rpm (21,100 × g) at 4 ° C. for 15 minutes. The pellet was dried. The pellet could be stored at -20 ° C for 2 days.

1-4. RNase treatment and electrophoresis
To remove single stranded RNAs due to terminal transferase activity, RNase treatment was performed.
A 20 μl RNA sample was dissolved in H ₂ O as follows.
RNA sample: 20 μl
RNase One (10 U / μl, Promega): 0.2 μl
10x reaction buffer: 20 μl
H ₂ O: 159.8 μl
Incubated at 37 ° C for 2 hours. Furthermore, 2.3 μl of 10% SDS was added and incubated at 37 ° C. for 15 minutes. 2 μl of 20 mg / ml proteinase K (Takara) was added and incubated at 37 ° C. for 15 minutes. After adding 205 μl of acid phenol-chloroform (Ambion) and vortexing at 15,000 rpm (21,100 × g) at room temperature for 5 minutes, the aqueous layer was transferred to a new tube.
20 μl of 3 M sodium acetate (pH 5.2), 4 μl of GenTLE precipitation carrier (Takara) and 250 μl of ethanol were added and centrifuged at 15,000 rpm (21,100 × g) at 4 ° C. for 20 minutes, and the supernatant was discarded.
The pellet was rinsed with 300 μl of 70% cold ethanol and centrifuged at 15,000 rpm (21,100 × g) at 4 ° C. for 15 minutes. Dissolve the pellet with 20 μl H ₂ O, 2x
loading buffer (95% formamide, 20 mM EDTA, and 1%
20 μl of Orange G) was added. After treatment at 95 ° C for 10 minutes, the mixture was rapidly cooled on ice and run on a 7M Urea 10% acrylamide gel.

[reagent]
5x acetate buffer (50 mM HEPES-KOH (pH 7.8), 500 mM potassium acetate, 20
Preparation of mM MgCl ₂ )
In 500 ml:
0.5 M HEPES-KOH (pH 7.8): 50 ml
K-Acetate: 24.5 g
0.5 M MgCl ₂ : 20 ml
The solution was filtered through a 0.2 μm filter and stored at room temperature.
Preparation of 10x MNase reaction buffer
200 mM Tris HCl (pH8.0)
50 mM NaCl
25 mM CaCl ₂
Were mixed.

The results are shown in FIGS. As shown, VX-222, eribulin, and vindesine were identified as compounds that inhibit human RdRP while not affecting telomerase activity. Vincristine, paclitaxel, and docetaxel did not show RdRP inhibitory activity.
Further, as shown in FIG. 4, it was confirmed that β-rubromycin and BIBR1532, which are known as telomerase inhibitors, also inhibit human RdRP. Costunolide, the same telomerase inhibitor, did not show human RdRP inhibitory activity.

2. Action on cancer stem cells Cancer cells and artificial cancer stem cells were seeded in a 96-well plate at 10,000 cells / well. HeLa cells are used as cancer cells, and Nucleostemin is overexpressed as artificial cancer stem cells according to the method of Okamoto et al. (Okamoto N. et al., Proc Natl Amer Sci., 108 (51): 20388-20393). HeLa cells were used.
After 24 hours of culture, VX-222 was added, and after 48 hours of culture, MTT assay was performed to count viable cells.
The results are shown in FIG. Artificial cancer stem cells were confirmed to be more sensitive to VX-222 than normal cancer cells.

Claims (12)

  Cancer stem cell targeting drug comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof Composition.   The cancer stem cell targeted drug composition according to claim 1, which is administered to a subject confirmed to have cancer stem cells, a subject after cancer treatment, or a subject having cancer metastasis.   The cancer stem cell targeted drug composition according to claim 2, wherein confirmation of the subject having cancer stem cells is performed by examining expression of a cancer stem cell marker in cells contained in the subject cancer tissue. . The cancer stem cell markers are CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N- The cancer stem cell targeted drug composition according to claim 3, which is selected from the group consisting of cadherins.   Cancer metastasis or comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof. Preventive or therapeutic agent for recurrence.   The preventive or therapeutic agent for metastasis or recurrence of cancer according to claim 3, which is administered to a subject after cancer treatment.   RNA-dependent RNA polymerase comprising a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof Inhibitor. Suitability of administering to the subject a compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof A method for presenting data for judging
A method comprising examining the expression of a cancer stem cell marker in cells contained in cancer tissue collected from a subject. The cancer stem cell markers are CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N- 9. The method of claim 8, wherein the method is selected from the group consisting of cadherins. A compound selected from the group consisting of VX-222, eribulin, vindesine, β-rubromycin, BIBR1532, and derivatives thereof, and pharmacologically acceptable salts and solvates thereof;
A cancer stem cell marker detection drug,
A cancer stem cell targeted treatment kit comprising: The cancer stem cell marker detection drug is
Selected from the group consisting of CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N-cadherin An antibody against at least one cancer stem cell marker, or
Selected from the group consisting of CD34, CD38, CD133, CD44, CD24, ESA, ALDH1, EpCAM, CD90, integrin α ₂ β ₁ , Sca-1, hTERT, NS, BRG1, STAT3, TWIST, SNAIL, and N-cadherin The cancer stem cell target treatment of Claim 10 containing the primer and / or probe which detect the variation | mutation of the cancer stem cell marker gene which raises or reduces the expression of the at least 1 cancer stem cell marker specifically cancer stem cell specifically For kit. A step of contacting a drug candidate compound with an RNA-dependent RNA polymerase and incubating;
Selecting a drug candidate compound that inhibits the RNA-dependent RNA polymerase activity;
A method for screening cancer stem cell targeted drugs, comprising: