WO2010001989A1

WO2010001989A1 - Agent for reducing cancer stem cell and/or cancer progenitor cell, and agent for preventing recurrence and/or metastasis of cancer

Info

Publication number: WO2010001989A1
Application number: PCT/JP2009/062183
Authority: WO
Inventors: 宏昌宮地; 孝聡魚地; 裕美横山; 秀幸小野寺
Original assignee: 協和発酵キリン株式会社
Priority date: 2008-07-03
Filing date: 2009-07-03
Publication date: 2010-01-07

Abstract

Disclosed are: an agent for reducing a cancer stem cell and/or a cancer progenitor cell, which comprises an inhibitor of a heat-shock protein-90 (Hsp90) family protein as an active ingredient; and others. The inhibitor of a Hsp90 family protein is a benzoyl compound represented by formula (I) [wherein n represents an integer of 1 to 5; R¹ represents CONR⁷R⁸ (wherein R⁷ and R⁸ independently represent a hydrogen atom, a substituted or unsubstituted lower alkyl, or the like), or the like; R² represents a substituted or unsubstituted aryl, or the like; R³ and R⁵ independently represent a hydrogen atom, a substituted or unsubstituted lower alkyl, or the like; R⁴ represents a hydrogen atom, or the like; and R⁶ represents a hydrogen atom, a halogen, a substituted or unsubstituted lower alkyl, or the like] or a pharmacologically acceptable salt thereof.

Description

Cancer stem cell and / or cancer progenitor cell reducing agent and cancer recurrence and / or metastasis preventive agent

The present invention relates to an agent for reducing cancer stem cells and / or cancer progenitor cells, which contains a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient. The present invention also relates to a preventive agent for cancer recurrence and / or metastasis, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.

Cancer is the number one cause of death in Japan in recent years, with more than 300,000 deaths each year. Despite significant advances in molecular analysis and understanding of cancer, and advances in cancer detection and treatment, cancer mortality is still high and still effective against many cancers. No therapeutic drug or treatment has been found.
Existing treatments for cancer, such as surgery, radiation therapy, chemotherapy, hormonal therapy, and immunotherapy, have some therapeutic effects on cancer, but on the appearance of recurrent cancer cells and cancer metastasis. The effect is limited. There is a need to develop preventive agents for cancer recurrence and metastasis, or new therapeutic agents for treating recurrent or metastatic cancer.

In various cancers, it is known that there is heterogeneity in cancer cells constituting the lesion [Cancer Research, 44, 2259-2265 (1984)], Cancer Have been thought to originate from a single cell [Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America), 58, 1468-1471 (1967); Blood Cells, 5, 261-282 (1979)]. An assay using colony-forming ability in soft agar and colony-forming ability in spleen as an index has shown that cells with colony-forming ability are present only in trace amounts in the cancer cell population [Science, 197, 461-463 (1977); Nature, 199, 79-80 (1963)]. It was also shown that a small number of cells having tumorigenic activity (tumor forming ability) by transplantation in vivo [Cancer, Vol. 14, 971-978 (1961); Nature (Nature) , 367, 645-648 (1994)]. These results indicate that, in the cancer cell population, the majority of cancer cells have a remarkably low ability to form tumors, and only some cells or their populations that are present in trace amounts have a high ability to form tumors. It was done. Two models have been proposed to explain this: a stochastic model and a hierarchical model [Nature, 414, 105-111 (2001) ]. The probabilistic model is that all cancer cells in a tumor have tumorigenic activity, but such ability is activated in some cells infrequently and asynchronously. On the other hand, in the hierarchical model, only a very small population of cells within a tumor exhibits a strong proliferation ability and a high tumorigenic activity, and further generates various progeny cells to form a hierarchical structure.

In recent years, the identification and separation of cells that exist in a very small amount in a heterogeneous cancer cell population has led to the development of various antibodies for detecting cell surface markers, and the development and separation of flow cytometers for analyzing and separating cells. The development of immunodeficient mice, such as non-obese diabetic / combined immunodeficiency (NOD / SCID) mice, suitable for transplantation and analysis of cancer cells, among which many findings support the hierarchical model It has been reported. That is, the existence of cancer stem cells as a small amount of cell population with strong proliferation ability and high tumorigenic activity consistent with the hierarchical model has been clarified [Nature Reviews Cancer, Volume 5, 311-321 (2005)].

For example, in human acute myeloid leukemia, cancer stem cells have been shown to be enriched in CD34 positive / CD38 negative cell populations that are present only in trace amounts, similar to normal hematopoietic stem cells. Showed tumorigenic activity and secondary transplantation was also possible [Nature Medicine, Vol. 3, 730-737 (1997)]. In this CD34 positive / CD38 negative cell population, unlike normal hematopoietic stem cells, c-kit expression was lost [Experimental Hematology, 28, 660-671 (2000) ]]. In human multiple myeloma, it was shown that cancer stem cells having tumorigenic activity in NOD / SCID mice were concentrated in a small number of CD138 negative fractions [Blood, Vol. 103, 2332]. -2336 (2004)].

The existence of cancer stem cells has been reported not only in blood cancer cells but also in some solid cancers. For example, in human breast cancer, the CD44 positive / CD24 negative (or CD24 low expression) cell fraction present infrequently, and the CD44 positive / CD24 negative (or CD24 low expression) and epithelial-specific antigen: ESA) positive cell fraction was shown to be enriched for cancer stem cells [US Pat. No. 6,985,522; Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America), 100, 3983-3988 (2003)]. In human glioblastoma and human medulloblastoma, cancer stem cells were enriched in a small number of CD133 positive cell fractions, and cancer stem cells were shown to have tumorigenic activity in NOD / SCID mice [Nature, No. 432, 396-401 (2004)]. Furthermore, in human prostate cancer, it has been suggested that cancer stem cells exist in the fraction of CD44 positive / α2β1 highly expressed / CD133 positive cells, which is a very small cell population [Cancer Research, 65 Volume 10946-10951 (2005)].

Furthermore, in human lung cancer tissue, it was shown that cancer stem cells exist in the side population (SP) fraction, which is a very small cell population [Cancer Research, Vol. 67 4827-4833 (2007)].
In addition, human breast cancer stem cells, human colon cancer stem cells, human lung cancer stem cells, human glioblastoma and medulloblastoma cancer stem cells, human melanoma cancer stem cells, etc., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) In addition, it has been reported that a serum-free medium supplemented with platelet-derived growth factor (PDGF) or the like forms a floating cell mass (sphere), and is maintained and concentrated [Nature, Vol. 432, 396-401 (2004); Proceedings of the National Academy of Sciences of the United States of the United States of Sciences of the United States of Sciences of the United States of Sciences of the United States of Science America), 101, 3781-3786 (2004); Oncogene, 23, 9392-9400 (2004); Cancer Research, 65, 9328-9337 (2005); Can -Cancer Research, 65, 5506-5511 (2005); Nature, 445, 111-115 (2007); Cell Death and Differentiation (Cell) Death and Differentiation), Vol. 15, pp. 504-514 (2008)].

Cancer stem cells are characterized by the ability of at least one of the following (1) to (3) [Cancer Research, 66, 9339-9344 (2006); The New England Journal of Medicine, 355, 1253-1261 (2006)].
(1) Cancer stem cells have a self-replicating ability. Self-replication is not synonymous with cell proliferation. Self-replicating ability refers to the ability to produce at least one daughter cell that retains the same ability and degree of differentiation as the parent cell in the cell lineage by symmetric or asymmetric division.
(2) Cancer stem cells can be differentiated into multiple types of cancer cells constituting a tumor via cancer precursor cells and the like. A plurality of types of cancer cells differentiated from cancer stem cells have a hierarchical structure starting from cancer stem cells in the cell lineage, as in the case of normal stem cells. Tumors having various characteristics are formed by gradually producing various types of cancer cells from cancer stem cells.
(3) Cancer stem cells possess high tumorigenic activity. Cancer stem cells allow excessive proliferation of cancer cell populations by repeating self-renewal and differentiation through symmetric or asymmetric division.

Cancer stem cells having such characteristics are attracting attention as new target cells for cancer radical therapy and methods for suppressing recurrence including cancer metastasis and regrowth [Cancer Research, Vol. 66 1883-1890 (2006)].
In recent years, leukemia cancer stem cell research has progressed significantly, and molecules involved in reactive oxygen production, such as Bcl-2, Hsp90, and farnesyltransferase, have been identified as molecular targets in leukemia cancer stem cells [Blood, No. 2] 105, 4163-4169 (2005); Blood, 110, 4427-4435 (2007); Blood, 110, 678-685 (2007); Cell Cycle, Volume 6, pages 2227-2231 (2007); Cancer Cell, Volume 10, pages 375-388 (2006); Leukemia, Volume 19, 1184 -1191 (2005)].

On the other hand, for solid cancer, it has been confirmed that cancer stem cells exist in various tumors, but it is not easy to obtain from clinical specimens like leukemia cancer stem cells, and cancer stem cells except for some tumors. Therefore, it is difficult to construct a method for evaluating anti-cancer stem cell activity. Therefore, it is not clear whether molecular targets that have been suggested to be effective in leukemia stem cells are also effective in solid cancer stem cells.

Hsp90α, Hsp90β, Grp94, Hsp75 / TRAP1, etc. have been identified as Heat shock protein 90 (Hsp90) family proteins, one of the molecular chaperones [Pharmacology & Therapeutics, 79, 129-168 (1998); Molecular Endocrinology, 13, 1435-1448 (1999)]. Hsp90 family proteins form specific complexes with molecules involved in cell proliferation and canceration, and are clearly involved in cell cycle, proliferation, cell survival, cell immortalization, angiogenesis, metastasis invasion, etc. Has been. Hsp90 family protein inhibitors are considered to be useful as therapeutic agents for diseases involving Hsp90 family proteins or proteins to which Hsp90 family proteins bind (Hsp90 client protein), such as cancer therapeutic agents (for example, non-patent literature) 1 and 2).

Geldanamycin and Radicicol, which inhibit the functions of Hsp90 family proteins, have been shown to bind to and inhibit the ATP / ADP binding region located at the N-terminus of Hsp90 family proteins. (For example, see Non-Patent Documents 3 to 5). Benzases such as geldanamycin, herbimycin A, 17-allylamino-17-demethoxygeldanamycin (17-AAG), 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) Noid ansamycin-based compounds (see, for example, Non-Patent Document 1 and Patent Document 1) and radicicol derivatives (for example, see Non-Patent Document 6) have been reported to exhibit antitumor effects.

In addition, it has been reported that coumarin compounds such as Novobiocin show the same effect as the low molecular weight compound that binds to the N-terminus by binding to the C-terminal region of Hsp90 (for example, non-biobiotin). (See Patent Document 7).
Furthermore, as Hsp90 family protein inhibitors, benzophenone derivatives (see Patent Documents 2 and 3), biphenyl derivatives (see Patent Document 4), rebrastatin (Reblastatin), EH21A2 and related compounds (for example, Non-Patent Document 8, Patent Document 5) Reference), purine derivatives (see Patent Document 6), pyrazole derivatives (see Patent Document 7), and the like have been reported.

On the other hand, IPI-504, a geldanamycin derivative, has been reported to show a selective inhibitory effect on mouse chronic leukemia cancer stem cells (Non-patent Document 9). However, the function and importance of Hsp90 in human cancer stem cells including leukemia and solid cancer remain unclear.

International Publication No. 02/079167 Pamphlet International Publication No. 2005/000778 Pamphlet International Publication No. 2006/088193 Pamphlet International Publication No. 2005/063222 Pamphlet US Patent Application Publication No. 2005/0026894 International Publication No. 02/036075 Pamphlet International Publication No. 03/055860 Pamphlet

An object of the present invention is to provide a cancer stem cell and / or cancer progenitor cell reducing agent containing a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient. Another object of the present invention is to provide a preventive agent for cancer recurrence and / or metastasis, which contains a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.

The present invention relates to the following (1) to (51).
(1) A cancer stem cell and / or cancer progenitor cell reducing agent comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
(2) Heat shock protein 90 (Hsp90) family protein inhibitors are represented by formula (I)

[Wherein n represents an integer of 1 to 5,
R ¹ is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted heterocyclic alkyl, substituted or unsubstituted Aryl, CONR ⁷ R ⁸ (wherein R ⁷ and R ⁸ are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkanoyl, substituted or Represents an unsubstituted aryl, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclic alkyl or a substituted or unsubstituted aroyl, or a nitrogen where R ⁷ and R ⁸ are adjacent Together with the atoms to form a substituted or unsubstituted heterocyclic group) or NR ⁹ R ¹⁰ (where R ⁹ and R ¹⁰ represents the same as R ⁷ and R ⁸ above,
R ² represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group,
R ³ and R ⁵ are the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Represents aralkyl or substituted or unsubstituted aroyl,
R ⁴ represents a hydrogen atom, hydroxy or halogen,
R ⁶ is a hydrogen atom, halogen, cyano, nitro, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cyclo Alkyl, amino, lower alkylamino, di-lower alkylamino, carboxy, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryloxy, substituted or unsubstituted aryl, substituted or unsubstituted A benzoyl compound represented by the above-mentioned or a pharmacologically acceptable salt thereof: or a pharmacologically acceptable salt thereof, which represents a substituted heterocyclic group, a substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclic alkyl. .

(3) The reducing agent according to (2), wherein R ² is phenyl substituted with 1 to 3 substituents or phenyl.
(4) The reducing agent according to (2), wherein R ² is a substituted or unsubstituted aromatic heterocyclic group.
(5) The reducing agent according to any one of (2) to (4), wherein R ³ , R ⁴ and R ⁵ are hydrogen atoms.
(6) The reducing agent according to any one of (2) to (5), wherein R ¹ is CONR ⁷ R ⁸ (wherein R ⁷ and R ⁸ are as defined above).
(7) R ¹ is CONR ^7a R ^8a (wherein R ^7a and R ^8a are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, or a substituted or unsubstituted heterocyclic alkyl) (2) The reducing agent according to any one of (5).

(8) The above (2) to (5) wherein R ¹ is CONR ^7b R ^8b (wherein R ^7b and R ^8b together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group). ) The reducing agent according to any one of
(9) The reducing agent according to any one of (2) to (8), wherein R ⁶ is a hydrogen atom, lower alkyl, halogen or aryl.
(10) The reducing agent according to any one of (2) to (8), wherein R ⁶ is lower alkyl.
(11) The reducing agent according to any one of (2) to (8), wherein R ⁶ is ethyl.
(12) Heat shock protein 90 (Hsp90) family protein inhibitor is 2- {2-ethyl-3,5-dihydroxy-6- [3-methoxy-4- (2-morpholinoethoxy) benzoyl] phenyl} -N, N-bis (2-methoxyethyl) acetamide or 2- [2-ethyl-3,5-dihydroxy-6- (4-methoxybenzoyl) phenyl] -N- (2-hydroxyethyl) -N- (2-methoxy The reducing agent according to (1) above, which is ethyl) acetamide or a pharmacologically acceptable salt thereof.
(13) The heat shock protein 90 (Hsp90) family protein inhibitor is represented by the formula (II)

{Where n1 represents an integer from 0 to 10,
R ¹¹ is a hydrogen atom, hydroxy, cyano, carboxy, nitro, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or Unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aroyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted heterocyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted Arylsulfonyl, substituted or unsubstituted heterocyclic group, CONR ¹⁷ R ¹⁸ (wherein R ¹⁷ and R ¹⁸ are the same or different, a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, Substituted or unsubstituted lower alkanoyl, substituted or Represents unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclic alkyl or substituted or unsubstituted aroyl, or nitrogen in which R ¹⁷ and R ¹⁸ are adjacent NR ¹⁹ R ²⁰ [wherein R ¹⁹ and R ²⁰ are the same or different, and represent a hydrogen atom, a substituted or unsubstituted lower alkylsulfonyl, and a substituted or unsubstituted heterocyclic group together with an atom] Substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl, substituted or unsubstituted substituted heterocyclealkyl, during aroyl substituted or unsubstituted, or CONR ²¹ R ²² (wherein, the R ²¹ and R ²² each R ¹⁷ and R ¹⁸ Or represents a a) synonymous, or R ¹⁹ and R ²⁰ form a substituted or unsubstituted heterocyclic group together with the adjacent nitrogen atom] or OR ²³ (wherein, R ²³ is a substituted or unsubstituted Lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl or substituted or unsubstituted heterocyclic ring Represents alkyl)
R ¹² represents a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heterocyclic group;
R ¹³ and R ¹⁵ are the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Lower alkylsulfonyl, substituted or unsubstituted arylsulfonyl, carbamoyl, sulfamoyl, substituted or unsubstituted lower alkylaminocarbonyl, substituted or unsubstituted di-lower alkylaminocarbonyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted Represents substituted heterocyclic carbonyl, substituted or unsubstituted aralkyl or substituted or unsubstituted aroyl;
R ¹⁴ and R ¹⁶ are the same or different and are a hydrogen atom, hydroxy, halogen, cyano, nitro, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted Lower alkoxy, substituted or unsubstituted cycloalkyl, amino, lower alkylamino, di-lower alkylamino, carboxy, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aryloxy, substituted or unsubstituted aryl, substituted Or a substituted or unsubstituted lower alkanoyl, a substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclic alkyl}, or a pharmaceutically acceptable salt thereof The reducing agent according to (1), wherein

(14) R ¹¹ is hydroxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkoxycarbonyl, substituted Or an unsubstituted heterocyclic group, substituted or unsubstituted aryl, CONR ¹⁷ R ¹⁸ (wherein R ¹⁷ and R ¹⁸ are as defined above) or NR ¹⁹ R ²⁰ (wherein R ¹⁹ and R ²⁰ , respectively) Are the same as defined above, respectively.
(15) The reducing agent according to the above (13) or (14), wherein R ¹² is substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group.
(16) The reducing agent according to the above (13) or (14), wherein R ¹² is substituted or unsubstituted phenyl.
(17) The reducing agent according to the above (13) or (14), wherein R ¹² is substituted or unsubstituted furyl.
(18) The reducing agent according to any one of (13) to (17), wherein R ¹³ , R ¹⁴ and R ¹⁵ are hydrogen atoms.
(19) The reducing agent according to any one of (13) to (18), wherein R ¹⁶ is a hydrogen atom, halogen, or substituted or unsubstituted lower alkyl.
(20) Heat shock protein 90 (Hsp90) family protein inhibitor is 17-allylamino-17-demethoxygeldanamycin (17-AAG) or 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) ) Or a pharmacologically acceptable salt thereof.

(21) The reducing agent according to any one of (1) to (20), wherein the cancer stem cells and / or cancer precursor cells are cells having tumorigenic activity.
(22) A cancer cell stem cell and / or cancer progenitor cell having tumorigenic activity, a cell population present in a tumor primary lesion, a cell population present in a tumor recurrence lesion, and a micrometastasis composed of one or more cells Cell population present in metastatic tumor nests, cell population present in bone marrow, side population (Side population) cell population, quiescent cell population, G0 cell population, average content of RNA content in all cancer cell populations A relatively low cell population, a cell population whose pyroin Y uptake is relatively lower than the average uptake in all cancer cell populations, a CD34 positive cell population, a CD38 negative cell population, a CD138 negative cell population, a CD44 positive cell Population, cell population whose CD24 expression level is relatively lower than the average expression level in all cancer cell populations, epithelial-specific antigen (ESA) positive cell population, sphere cell population, CD24 positive cell population At least one cell population selected from a population, a CD166 positive cell population, an α2β1 integrin positive cell population, a CD133 positive cell population, a CD90 positive cell population, a CD55 positive cell population, and a cell population having high aldehyde dehydrogenase (ALDH) activity The reducing agent according to any one of (1) to (20), which is a cell belonging to 1.

(23) The reducing agent according to any one of (1) to (20), wherein the cancer stem cells and / or cancer precursor cells are cells belonging to a side population cell population having tumorigenic activity.
(24) The reducing agent according to any one of (1) to (23), wherein the cancer is cancer caused by a hematopoietic tumor or solid cancer.
(25) The reducing agent according to any one of (1) to (23), wherein the cancer is lung cancer.
(26) The reducing agent according to the above (25), wherein the lung cancer is small cell lung cancer.
(27) The reducing agent according to the above (25), wherein the lung cancer is non-small cell lung cancer.
(28) The reducing agent according to any one of (1) to (23), wherein the cancer is breast cancer.
(29) The reducing agent according to any one of the above (1) to (28), which is administered simultaneously or sequentially with at least one compound.
(30) The reducing agent according to the above (29), wherein at least one compound is an antitumor agent.
(31) A preventive agent for cancer recurrence and / or metastasis, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
(32) The heat shock protein 90 (Hsp90) family protein inhibitor is represented by the formula (I)

(Wherein, n, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above) or a pharmacologically acceptable salt thereof (31) The prophylactic agent according to the above.
(33) A heat shock protein 90 (Hsp90) family protein inhibitor is represented by the formula (II)

Wherein n1, R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ are as defined above, or a pharmacologically acceptable salt thereof (31) The prophylactic agent according to the above.
(34) The prophylactic agent according to any one of (31) to (33), wherein the cancer stem cell and / or cancer precursor cell is a cell having tumorigenic activity.
(35) A cell population present in a tumor primary lesion, a cell population present in a tumor recurrence lesion, and a micrometastasis composed of one or more cells in which cancer stem cells and / or cancer precursor cells have tumorigenic activity Cell population present in metastatic tumor nests, cell population present in bone marrow, side population (Side population) cell population, quiescent cell population, G0 cell population, average content of RNA content in all cancer cell populations A relatively low cell population, a cell population whose pyroin Y uptake is relatively lower than the average uptake in all cancer cell populations, a CD34 positive cell population, a CD38 negative cell population, a CD138 negative cell population, a CD44 positive cell Population, cell population whose CD24 expression level is relatively lower than the average expression level in all cancer cell populations, epithelial-specific antigen (ESA) positive cell population, sphere cell population, CD24 positive cell population At least one cell population selected from a population, a CD166 positive cell population, an α2β1 integrin positive cell population, a CD133 positive cell population, a CD90 positive cell population, a CD55 positive cell population, and a cell population having high aldehyde dehydrogenase (ALDH) activity The prophylactic agent according to any one of the above (31) to (33), which is a cell belonging to.

(36) The prophylactic agent according to any of (31) to (33) above, wherein the cancer stem cells and / or cancer progenitor cells are cells belonging to a side population (Side population) cell population having tumorigenic activity.
(37) The prophylactic agent according to any one of (31) to (36), wherein the cancer is cancer caused by a hematopoietic tumor or solid cancer.
(38) The preventive agent according to any of (31) to (36), wherein the cancer is lung cancer.
(39) The preventive agent according to the above (38), wherein the lung cancer is small cell lung cancer.
(40) The preventive agent according to (38), wherein the lung cancer is non-small cell lung cancer.
(41) The prophylactic agent according to any of (31) to (36), wherein the cancer is breast cancer.
(42) The prophylactic agent according to any of (31) to (41) above, which is administered simultaneously or sequentially with at least one compound.
(43) The prophylactic agent according to the above (42), wherein at least one compound is an antitumor agent.

(44) A cancer stem cell and / or cancer progenitor cell reducing agent in a patient having cancer stem cells and / or cancer precursor cells, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
(45) A preventive agent for recurrence and / or metastasis of cancer in a patient having cancer stem cells and / or cancer precursor cells, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
(46) A method for reducing cancer stem cells and / or cancer precursor cells, comprising a step of administering an effective amount of a heat shock protein 90 (Hsp90) family protein inhibitor.
(47) A method for preventing cancer recurrence and / or metastasis, comprising a step of administering an effective amount of a heat shock protein 90 (Hsp90) family protein inhibitor.
(48) A heat shock protein 90 (Hsp90) family protein inhibitor for use in reducing cancer stem cells and / or cancer precursor cells.
(49) A heat shock protein 90 (Hsp90) family protein inhibitor for use in preventing cancer recurrence and / or metastasis.
(50) Use of a heat shock protein 90 (Hsp90) family protein inhibitor for the production of a cancer stem cell and / or cancer progenitor cell reducing agent.
(51) Use of a heat shock protein 90 (Hsp90) family protein inhibitor for the manufacture of an agent for preventing cancer recurrence and / or metastasis.

The present invention provides an agent for reducing cancer stem cells and / or cancer progenitor cells, which contains a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient. The present invention also provides a preventive agent for cancer recurrence and / or metastasis, which contains a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.

Hereinafter, the compounds represented by formulas (I) and (II) are referred to as compounds (I) and (II). The same applies to the compounds of other formula numbers.
In the definition of each group of formula (I) and (II):
The lower alkyl part of lower alkyl, lower alkoxy, lower alkoxycarbonyl, lower alkylaminocarbonyl, di-lower alkylaminocarbonyl, lower alkylsulfonyl, lower alkylamino and di-lower alkylamino includes, for example, linear or branched carbon number Examples thereof include 1 to 8 alkyls, specifically, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl and the like. The two lower alkyl moieties in di-lower alkylamino and di-lower alkylaminocarbonyl may be the same or different.

Examples of the lower alkenyl include linear or branched alkenyl having 2 to 8 carbon atoms, specifically vinyl, allyl, 1-propenyl, methacryl, crotyl, 1-butenyl, 3-butenyl, 2- Examples include pentenyl, 4-pentenyl, 2-hexenyl, 5-hexenyl, 2-heptenyl, 2-octenyl and the like.
Examples of lower alkynyl include linear or branched alkynyl having 2 to 8 carbon atoms, and specific examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and the like.

Examples of lower alkanoyl include linear or branched alkanoyl having 1 to 7 carbon atoms, and specifically include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl and the like. It is done.
Examples of cycloalkyl include cycloalkyl having 3 to 8 carbon atoms, and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

The aryl moiety of aryl, arylsulfonyl, aryloxy and aroyl includes, for example, monocyclic, bicyclic or tricyclic aryl having 6 to 14 carbon atoms, specifically phenyl, indenyl, naphthyl, anthryl. Etc.
Examples of aralkyl include aralkyl having 7 to 15 carbon atoms, and specific examples include benzyl, phenethyl, benzhydryl, naphthylmethyl and the like.

As the aromatic heterocyclic group, for example, a 5-membered or 6-membered monocyclic aromatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, and a 3- to 8-membered ring are condensed. A bicyclic or tricyclic condensed aromatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, specifically, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl Quinolyl, isoquinolyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, thienyl, furyl, thiazolyl, oxazolyl, indolyl, indazolyl, benzoimidazolyl, azotriazolyl, azothryl , Purinyl, benzodioxo Alkylsulfonyl and the like.

Examples of the heterocyclic group part of the heterocyclic group, heterocyclic carbonyl and heterocyclic alkyl include alicyclic heterocyclic groups in addition to the groups mentioned in the definition of the aromatic heterocyclic group. Examples of the alicyclic heterocyclic group include a 5-membered or 6-membered monocyclic alicyclic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and a 3- to 8-membered ring. And a condensed alicyclic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and the like, specifically, pyrrolidinyl, piperidino, Piperidyl, piperazinyl, morpholino, morpholinyl, thiomorpholino, thiomorpholinyl, homopiperidino, homopiperidyl, homopiperazinyl, tetrahydropyridinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, tetrahydropyranyl, dihydrobenzofuranyl, oxopiperazinyl, 2- Examples include oxopyrrolidinyl, oxolanyl, dioxolanyl and the like.

Examples of the heterocyclic group formed together with the adjacent nitrogen atom include a 5-membered or 6-membered monocyclic heterocyclic group containing at least one nitrogen atom (the monocyclic heterocyclic group includes other Or a condensed heterocyclic group containing at least one nitrogen atom, which is a bicyclic or tricyclic condensed 3- to 8-membered ring. The cyclic heterocyclic group may contain other nitrogen atom, oxygen atom or sulfur atom), specifically pyrrolidinyl, piperidino, piperazinyl, morpholino, thiomorpholino, homopiperidino, homopiperazinyl, tetrahydropyridyl , Tetrahydroquinolyl, tetrahydroisoquinolyl, oxopiperazinyl, 2-oxopyrrolidinyl and the like.

The alkylene part of the heterocyclic alkyl has the same meaning as that obtained by removing one hydrogen atom from the definition of the lower alkyl.
Halogen means each atom of fluorine, chlorine, bromine and iodine.
Substituents (A) in substituted lower alkyl, substituted lower alkoxy, substituted lower alkoxycarbonyl, substituted dilower alkylaminocarbonyl, substituted lower alkylaminocarbonyl, substituted lower alkylsulfonyl, substituted lower alkenyl and substituted lower alkynyl are the same or different. For example, substituted, unsubstituted hydroxy, oxo, cyano, nitro, carboxy, amino, halogen, substituted or unsubstituted lower alkoxy, cycloalkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkylamino, di-lower alkylamino, And hydroxy lower alkylaminocarbonyl. The substitution position of the substituent is not particularly limited. Here, halogen, lower alkoxy, cycloalkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkylamino and di-lower alkylamino are as defined above. The alkylene part of hydroxy lower alkylaminocarbonyl has the same definition as that obtained by removing one hydrogen atom from the definition of lower alkyl. Substituents in the substituted lower alkoxy are the same or different and include, for example, hydroxy having 1 to 3 substituents, halogen, etc., and the halogen is as defined above.

Substituted lower alkanoyl, substituted cycloalkyl, substituted aryl, substituted arylsulfonyl, substituted aryloxy, substituted aralkyl, substituted aroyl, substituted heterocyclic alkyl, substituted heterocyclic group, substituted heterocyclic carbonyl, substituted phenyl, substituted furyl, substituted aromatic hetero The substituents (B) in the substituted heterocyclic group formed together with the ring group and the adjacent nitrogen atom are the same or different, for example, hydroxy having 1 to 3 substituents, halogen, nitro, cyano, amino, Carboxy, carbamoyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, lower alkylaminocarbonyl, di-lower alkylaminocarbonyl, substituted or unsubstituted lower alkoxy, aralkyloxy, lower alkylsulfonyl, lower alkylsulfanyl, Low grade Alkylthio, aryloxy, cycloalkyl, lower alkoxycarbonyl, lower alkylamino, di-lower alkylamino, lower alkanoyl, heterocyclic group, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic alkyloxy, substituted or unsubstituted Heterocyclic carbonylalkyloxy and the like. The substitution position of the substituent is not particularly limited. Here, halogen, lower alkyl, lower alkenyl, lower alkylaminocarbonyl, di-lower alkylaminocarbonyl, aryloxy, lower alkoxy, cycloalkyl, lower alkoxycarbonyl, lower alkylamino, di-lower alkylamino, lower alkanoyl, heterocyclic group and Aryl is as defined above, and the lower alkyl moiety of lower alkylsulfonyl, lower alkylsulfanyl and lower alkylthio is as defined above, and the aralkyl moiety of aralkyloxy is as defined above for aralkyl. And the heterocyclic group moiety and alkylene of the heterocyclic carbonylalkyloxy are the same as those obtained by removing one hydrogen atom from the definitions of the heterocyclic group and the lower alkyl, respectively. Substituents in substituted lower alkyl, substituted lower alkenyl, substituted lower alkoxy and substituted aryl are the same or different, for example, hydroxy having 1 to 3 substituents, carboxy, lower alkanoyl, halogen, lower alkoxy, cyano, lower alkylamino, Di-lower alkylamino and the like, and the halogen, lower alkanoyl, lower alkoxy, lower alkylamino and di-lower alkylamino are as defined above. Examples of the substituents in the substituted heterocyclic alkyloxy and the substituted heterocyclic carbonylalkyloxy are the same or different and include, for example, hydroxy having 1 to 3 substituents, halogen, lower alkyl, lower alkoxy, heterocyclic group, etc. The halogen, lower alkyl, lower alkoxy and heterocyclic groups shown are the same as defined above.

The pharmacologically acceptable salts of Compound (I), Compound (II), 17-AAG and 17-DMAG are, for example, pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts And amino acid addition salts.
Examples of pharmacologically acceptable acid addition salts of compound (I), compound (II), 17-AAG and 17-DMAG include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, acetic acid, etc. Organic salts such as salt, maleate, fumarate and citrate are listed. Examples of pharmacologically acceptable metal salts include alkali metal salts such as sodium salt and potassium salt, magnesium salt and calcium salt. Examples include alkaline earth metal salts such as salts, aluminum salts, and zinc salts. Examples of pharmacologically acceptable ammonium salts include salts such as ammonium and tetramethylammonium, which are pharmacologically acceptable. Examples of organic amine addition salts include addition salts such as morpholine and piperidine, and examples of pharmacologically acceptable amino acid addition salts include glycine, phenylalanine, lysine, and aspartic acid. Addition salts such as laginic acid and glutamic acid can be mentioned.

When it is desired to obtain salts of Compound (I), Compound (II), 17-AAG and 17-DMAG, Compound (I), Compound (II), 17-AAG and 17-DMAG are obtained in the form of salt. In some cases, it can be purified as it is, and when it is obtained in free form, dissolve or suspend Compound (I), Compound (II), 17-AAG or 17-DMAG in an appropriate solvent, and add acid or base. What is necessary is just to form a salt.
Compounds (I) and (II) may have isomers such as positional isomers, geometric isomers or optical isomers, but possible isomers and mixtures of these isomers in any ratio are Hsp90 family protein inhibitor It can be used as an agent.

Further, Compound (I), Compound (II), 17-AAG and 17-DMAG, and pharmacologically acceptable salts thereof may exist in the form of adducts with water or various solvents. These adducts can also be used as Hsp90 family protein inhibitors.
Hsp90 family protein inhibition refers to inhibiting the binding of the Hsp90 family protein and the Hsp90 client protein (Hsp90 client protein) or inhibiting the functions of the Hsp90 family protein such as inhibiting the ATPase activity. means.

Examples of Hsp90 family proteins include Hsp90α protein, Hsp90β protein, grp94, hsp75 / TRAP1 and the like.
The protein to which the Hsp90 family protein binds may be any protein to which the Hsp90 family protein binds.For example, EGFR, Erb-B2, Bcr-Abl, src, raf-1, AKT, Flt-3, PLK, Wee1 , FAK, cMET, hTERT, HIF1-α, mutant p53, estrogen receptor, androgen receptor, etc. [Expert Opinion on Biological Therapy, Volume 2, 3- 24, (2002)].

Hsp90 family protein inhibitors include low molecular weight compounds that act on Hsp90 family gene products including Hsp90 family proteins or messenger RNA (mRNA), short double-stranded RNA (small RNA) (siRNA), antibodies, antibody fragments, etc. In addition, microRNA (miRNA) involved in the transcriptional control or translational regulation of the Hsp90 gene is also included. Preferably, a low molecular weight compound is used.

Examples of low molecular weight compounds of Hsp90 family protein inhibitors include compound (I), compound (II), geldanamycin, 17-AAG, 17-DMAG, herbimycin A, radicicol, lebrastatin, EH21A2, novobiocin and these compounds. Derivatives, purine derivatives (for example, compounds described in WO 02/036075 pamphlet), pyrazole derivatives (for example, compounds described in WO 03/055860 pamphlet), and pharmacologically acceptable salts thereof , IPI-504, NVP-AUY922, STA-9090, AT13387, SNX-5422, BIIB021 and the like. Preferred examples include compound (I), compound (II), 17-AAG and 17-DMAG, and pharmacologically acceptable salts thereof.

Moreover, the reducing agent and the preventive agent of the present invention can be used in combination with at least one compound or a cancer treatment method. Examples of the compound used in combination include an antitumor agent and a protein or low molecular weight compound other than the antitumor agent.
Examples of antitumor agents include protein drugs, chemotherapeutic agents, hormone therapy agents, molecular targeted drugs, differentiation inducers, bone resorption inhibitors, nucleic acid drugs (siRNA, antisense oligos), etc. Compounds. In addition, irradiation (radiotherapy) can be performed before or after administration of the reducing agent or prophylactic agent of the present invention.

Examples of radiation in radiation therapy include negative electrons, positrons, protons, fast neutrons, negative π mesons, heavy ions, charged particles, X-rays, γ-rays, radio waves, infrared rays, ultraviolet rays, and visible light.
Examples of protein pharmaceuticals include cytokines and antibodies.
Examples of cytokines include interleukin-2 (IL-2), IFN-α, IFN-γ, GM-CSF, G-CSF, TNF-α, and IL-1β.

Examples of antibodies include anti-EGFR antibody {cetuximab (Erbitux)}, anti-ErbB2 antibody {trastuzumab (Herceptin)}, anti-VEGF antibody {bevacizumab (Avastin) ], Anti-CD20 antibody {rituximab (rituximab) [rituximab (Rituxan)]}, anti-CD33 antibody {gemtuzumab ozogamicin (Mylotarg)}, anti-CD52 antibody {alemtuzumab (campus) [Alemtuzumab (Campath)]}, anti-TRAIL antibody and the like.

Examples of chemotherapeutic agents include tubulin agonists, DNA agonists, antimetabolites and the like.
Examples of tubulin agonists include vinblastine, vindesine, vincristine, vinorelbine, paclitaxel (taxol), docetaxel (taxotere) and the like.

Examples of DNA agonists include, for example, chlorambucil, cyclophosphamide, melpharan, cisplatin, carboplatin, carbplatin, dacarbazine (DTIC), oxalo Oxaloplatin, bleomycin, doxorubicin (adriamycin) [doxorubicin lipoox (doxil)], idarubicin, mitomycin, antoxantrone , Etoposide, camptothecin, CPT-11,10-hydroxy-7-ethyl-camptothecin (SN38), irinotecan, topotecan, 5-azacytidine, decitabine ) And the like.

Examples of antimetabolites include, for example, 5-fluorouracil, fludarabine, hydroxyurea, cytarabine, methotrexate, capecitabine, gemcitabine [gemcitabine (gemzar)], tegafur / uracil compounding agent (UFT), clofarabine, nelarabine and the like.

Examples of hormone therapy agents include antiandrogens, antiestrogens, androgens, estrogen, LH-RH agonists (chemical castrations), progestins, aromatase inhibitors, steroid sulfatase inhibitors, and the like.
Examples of hormone therapy agents include, for example, leuprolide, goserelin, megestrol, tamoxifen, ICI182780, toremifene, fadrozole, letrozole, flutamide (Flutamide), bicalutamide, testolactone, mitotane, prednisolone, dexamethasone and the like.

Examples of molecular target drugs include Bcr-Abl inhibitors, EGFR inhibitors, JAK inhibitors, multikinase inhibitors, kinesin Eg5 inhibitors, Flt-3 inhibitors, mTOR inhibitors, proteasome inhibitors, HDAC inhibitors, DNA Examples include methylation inhibitors, farnesyltransferase inhibitors, Bcl-2 inhibitors, Aurora inhibitors, Abl kinase inhibitors, VEGFR inhibitors, FGFR inhibitors, PDGFR inhibitors, ephrin inhibitors, and the like.

Examples of molecular targeted drugs include, for example, gefitinib (Iressa) [gefitinib (Iressa)], erlotinib タル (Tarceva), lapatinib (Tykerb) [lapatinib (Tykerb), HKI-272, BIBW-2992, BMS -599626], Imatinib (Gleevec) [imatinib (Gleevec), STI571], Dasatinib (Sprycel) [BMS-354825], Nilotinib (Tasigna) [nilotinib (Tasigna), AMN107], Sunitinib (Sutent) sunitinib (SUTENT), SU11248], Sorafenib (Nexabar) [BAY43-9006], CHIR-258, vatalanib (PTK-787), R-1155777 (tipifarnib, zarnestra), rapamycin, temsirolimus [temsirolimus temsirolimus, CCI-779], Bortezomib (Velcade) [bortezomib (Velcade), PS-341], PR-171, NPI-0052, Borinostat (Zolinza) [vorinostat (Zolinza), suberanilohydroxamic ac id, SAHA], valproic acid, MS-275, asparaginase, asparaginase (pegaspargase (Oncaspar)) and the like.

Examples of Flt-3 inhibitors include CEP-701, PKC412, MLN518, CHIR-258, indazole derivatives (for example, compounds described in International Publication No. 2005/012257 or International Publication No. 2005/012258), pyrimidines. Derivatives (for example, compounds described in International Publication No. 2005/095382 pamphlet), isoindolinone phthalimide derivatives (for example, compounds described in International Publication No. 2005/095341 pamphlet) and the like.

Examples of the differentiation inducer include, for example, all-trans retinoic acid, arsenous acid, thalidomide, lenalidomide, bexarotene (targretin) and the like.
Examples of the bone resorption inhibitor include bisphosphonate (zoledronic acid, Zometa).

The above compounds may not give sufficient therapeutic or preventive effects when administered alone, or may have side effects when administered at high doses. However, a high therapeutic and / or prophylactic effect can be obtained by combining the above compound and the reducing agent or the preventing agent of the present invention. Furthermore, since a high therapeutic effect is obtained by the combination of the reducing agent or prophylactic agent of the present invention and the above compound, the number of administrations and the dosage of the above compound can be reduced. Therefore, in addition to a sufficient therapeutic effect, side effects can be reduced.

Compound (I) or a pharmacologically acceptable salt thereof used in the present invention can be synthesized, for example, by the method described in International Publication No. 2005/000778.
Compound (II) or a pharmacologically acceptable salt thereof used in the present invention can be synthesized, for example, by the method described in International Publication No. 2005/063222.
Specific examples of Hsp90 family protein inhibitors used in the present invention include geldanamycin, 17-DMAG, radicicol, lebrastatin, EH21A2, novobiocin, PU24FCl, which are obtained as commercially available products or known methods Can be synthesized or fermented.

Specific examples of the Hsp90 family protein inhibitors used in the present invention are also shown in the following Tables 1-1, 1-2, 2-1 and 2-2, but the present invention is not limited thereto. In the following table, Ph represents phenyl.
Compounds 1 to 23 listed in Tables 1-1 and 1-2 can be synthesized by the method described in the pamphlet of International Publication No. 2005/000778, and Compounds 24-44 described in Tables 2-1 and 2-2 are published internationally. It can be synthesized by the method described in the 2005/063222 pamphlet.

In the present invention, the cancer stem cell refers to a cell having at least one of the following capabilities (1) to (3).
(1) Self-replicating ability. Self-replication is not synonymous with cell proliferation. Self-replicating ability refers to the ability to produce at least one daughter cell that retains the same ability and degree of differentiation as the parent cell in the cell lineage by symmetric or asymmetric division.
(2) It has the ability to differentiate into a plurality of types of cancer cells constituting a tumor via cancer precursor cells and the like. A plurality of types of cancer cells differentiated from cancer stem cells have a hierarchical structure starting from cancer stem cells in the cell lineage, as in the case of normal stem cells. Tumors having various characteristics are formed by gradually producing various types of cancer cells from cancer stem cells.
(3) High tumorigenicity (tumor-forming activity). Cancer stem cells allow excessive proliferation of cancer cell populations by repeating self-replication and differentiation through symmetrical or asymmetric division.

In the present invention, a progenitor cell is positioned between a stem cell and a terminally differentiated cell in the cell lineage. Since cell differentiation proceeds by changes in the phenotype of successive cells, a method for strictly distinguishing stem cells from progenitor cells has not been clarified. The cancer progenitor cell in the present invention means a cell that is close to the cancer stem cell in the differentiation lineage and is present downstream of the cancer stem cell, and the cancer stem cell of the present invention includes a mixed population of cancer stem cell and cancer precursor cell. It is.

The cancer stem cells of the present invention are derived from normal stem cells, normal progenitor cells or normal differentiated cells of any tissue, and include tumor primary lesions, tumor recurrence lesions, and metastatic tumor lesions including micrometastasis composed of one or more cells, bone marrow Present in peripheral blood and the like.
The cancer stem cells in the present invention include cancerous stem cells and cells having tumorigenic activity. The cancer stem cell may be any cell as long as it has any of the above properties (1) to (3), but preferably a cancer stem cell of blood cancer, a cancer stem cell of various solid cancers, etc. More preferable are cancer stem cells of various solid cancers.

Examples of cancer stem cells of hematological cancer include cancer stem cells such as myeloma and lymphoma. Specifically, acute leukemia, myelodysplastic syndrome, chronic leukemia, chronic myeloproliferative disease, multiple myeloma, malignant lymphoma, etc. Cancer stem cells.
Examples of cancer stem cells for acute leukemia include cancer stem cells such as acute myeloid leukemia and cancer stem cells such as acute lymphocytic leukemia. Cancer stem cells for acute myeloid leukemia include acute undifferentiated myeloid leukemia and acute undifferentiated type. Examples include cancer stem cells such as myeloblastic leukemia, acute differentiated myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, erythroleukemia, acute megakaryoblastic leukemia It is done. Cancer stem cells of acute lymphoblastic leukemia include precursor lymphoblastic leukemia / lymphoma, precursor T cell acute lymphoblastic leukemia, juvenile B cell acute lymphoblastic leukemia, and mature B cell acute lymphoblastic leukemia. Cancer stem cells such as

Examples of cancer stem cells of chronic leukemia include cancer stem cells such as chronic myeloid leukemia and cancer stem cells such as chronic lymphocytic leukemia. Cancer stem cells of chronic lymphocytic leukemia include B cell type chronic lymphocytic leukemia, T cell type. Examples include cancer stem cells such as chronic lymphocytic leukemia and small cell lymphoma.
Examples of cancer stem cells of malignant lymphoma include cancer stem cells such as Hodgkin lymphoma (Hodgkin's disease), non-Hodgkin lymphoma (non-Hodgkin's disease), brain lymphoma (primary malignant lymphoma of the central nervous system), and skin lymphoma.

Examples of cancer stem cells of Hodgkin lymphoma include cancer stem cells such as nodular lymphocyte-dominated Hodgkin lymphoma and classic Hodgkin lymphoma (nodule sclerosis type, mixed cell type, lymphocyte rich type, lymphocyte depletion type) and the like.
Examples of cancer stem cells of non-Hodgkin lymphoma include cancer stem cells such as NK / T cell lymphoma and B cell lymphoma. Examples of cancer stem cells of NK / T cell lymphoma include precursor T lymphoblast type lymphoma / leukemia, Examples include cancer stem cells such as T-cell lymphoblastic lymphoma, mature T-cell tumor, extranodal NK / T-cell lymphoma / nasal type, and blastic NK cell lymphoma. Cancer stem cells of mature T cell tumors include T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, enteropathic intestinal T cell lymphoma, hepatosplenic γδ T cell lymphoma, vascular immunoblast type T cell Examples include cancer stem cells such as lymphoma, peripheral T-cell lymphoma, undifferentiated large cell lymphoma, and adult T-cell leukemia / lymphoma (ATLL).

Cancer stem cells of B cell lymphoma include follicular lymphoma, MALT lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, B cell lymphoblastic lymphoma, Burkitt lymphoma / leukemia, mantle cell Examples include cancer stem cells such as lymphoma.
Cancer stem cells of cutaneous lymphoma include mycosis fungoides, lymphoid papulosis, Sezary syndrome, cutaneous CD30 positive undifferentiated large cell lymphoma, cutaneous B cell lymphoma, subcutaneous panniculitis-like T cell lymphoma, subcutaneous cellulitis-like Examples include cancer stem cells such as T cell lymphoma, cutaneous γδ type T cell lymphoma, cutaneous NK / T cell lymphoma, cutaneous CD8 type lymphoma.

The solid cancer stem cells include sarcomas and carcinoma stem cells, specifically fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, Lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, bone marrow cancer, bronchogenic carcinoma, renal cell carcinoma, ureteral cancer, liver cancer, bile duct cancer, Choriocarcinoma, seminoma, fetal cancer, Wilms tumor, cervical cancer, endometrial cancer, testicular cancer, lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, myeloblastoma, craniopharyngeal cancer , Laryngeal cancer, tongue cancer, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, black Tumor, peritoneal dissemination, teratoma, neuroblastoma, medulloblastoma, and cancer stem cells such as retinoblastoma. Examples of cancer stem cells of lung cancer include small cell lung cancer and non-small cell lung cancer cancer stem cells, preferably non-small cell lung cancer cancer stem cells.

Examples of cancer stem cells in the present invention include cancer stem cells of blood cancer that can be identified by cancer stem cell markers, cancer stem cells of various solid cancers that can be identified by cancer stem cell markers, and preferably cancers of blood cancer having tumorigenic activity Examples include stem cells and cancer stem cells of various solid cancers having tumorigenic activity, and more preferable examples include cancer stem cells of various solid cancers having tumorigenic activity.
Examples of hematologic cancer stem cells include CD34-positive and CD38-negative cells in acute myeloid leukemia, and CD138-negative cells in multiple myeloma.

Cancer stem cells of solid cancer include (1) CD44 positive and CD24 negative (or low CD24 expression) cells in breast cancer, CD44 positive and CD24 negative (or low CD24 expression), and epithelial-specific antigen (ESA) ) Positive cells, sphere cells, etc. (2) CD44 positive and CD24 positive cells, CD133 positive cells, sphere cells, etc. in brain tumors and neuroblastomas, (3) CD44 positive and CD166 positive cells in colorectal cancer, CD133 positive cells, sphere cells, etc. (4) CD44 positive, α2β1 integrin highly expressing and CD133 positive cells in prostate cancer, (5) CD44 positive, CD24 positive cells, CD44 positive, CD24 positive and ESA positive in pancreatic cancer Cells, CD133-positive cells, etc. (6) CD44-positive cells in head and neck cancers, (7) Side population (SP) cells in liver cancer, CD44-positive and CD90-positive cells, CD133-positive cells And (8) side population (SP) cells, CD133 positive cells, sphere cells and the like in various solid cancers such as lung cancer.

Cancer stem cells in the present invention include cells that can be detected or identified by a flow cytometer using an antibody specific for the marker possessed by these, immunohistochemical staining, etc., for example, CD34, CD38, CD138, CD44, CD24, Examples include cancer stem cells in which cancer stem cell markers such as ESA, CD133, CD166, CD90, and α2β1 have the characteristics described above. In addition, the cancer stem cell in the present invention may be any method as long as it specifically binds to the cancer stem cell and can detect or identify the cell, such as biotin, fluorescent dye, fluorescent bead or fluorescent protein. In addition, cells that can be detected or identified by methods using aptamers, antisense oligo RNAs, antisense oligo DNAs, and the like that are labeled in this manner are also included.

Examples of SP cells included in various solid cancers include cells having characteristics such as high Hoechst33342 excretion ability, high drug excretion ability, high drug resistance, and many cells in the stationary phase, such as flow. Cells that can be fractionated by these features by cytometer are included.
Examples of the SP cell detection method include a method using staining with Hoechst33342.

Examples of markers serving as an index of drug excretion ability include ABCP1, ABCB5, ABCG2 gene products, which are ATP-binding cassette (ABC) transporter superfamily gene products.
As an index for detecting cells in the stationary phase of the cell cycle, G0 / G1 cell population ratio, pyronin Y (pyroninY) staining, etc. in a cell cycle measurement method using Propidium iodide (PI) staining, which is a nucleic acid binding dye, is used. Examples include the G0 cell population ratio in the RNA content measurement method.

Some SP cell identification markers include high Aldehyde Dehydrogenase (ALDH) activity and a high CD55 expression level.
As cancer stem cells, quiescent cell population, G0 phase cell population, cells whose RNA content is relatively lower than the average content in all cancer cell populations (RNAlow cell) population, and pyronin Y uptake total cancer cell population Cell population relatively lower than the average uptake amount in cells, CD55 positive cell population, cell population whose CD55 expression level is higher than the average expression level in all cancer cell populations, cell population with high aldehyde dehydrogenase (ALDH) activity And cancer stem cells belonging to a cell population having ALDH activity that is relatively higher than the average activity in the whole cancer cell population.

In addition, SP cells are detected by staining with Hoechst33342, various detections using markers representing the characteristics of the SP cells, characteristics of cancer stem cells of the blood cancer and solid cancer (CD34, CD38, CD138, CD44, CD24, ESA, Various detections using cancer stem cell markers (CD133, CD166, CD90, α2β1, etc.) can also be performed in combination. The SP cells also include cancer stem cells contained in various solid cancers that can be identified or separated by these detection methods.

Furthermore, the cancer stem cell in the present invention includes a cancer stem cell expressing an Hsp90 family protein and a protein (Hsp90 client protein) that binds to the Hsp90 family protein.
The reducing agent of the present invention prevents cancer recurrence during or after treatment such as surgical treatment such as resection to remove the lesion, radiation therapy, chemotherapy, adjuvant chemotherapy before or after surgery, hormone therapy, immunotherapy, etc. And / or cancer metastasis prevention, primary cancer treatment and / or prevention, cancer eradication treatment and the like can be used for any cancer treatment and / or prevention, and the preventive agent of the present invention is Prevention of recurrence of cancer and / or metastasis of cancer during or after treatment such as surgery, radiation therapy, chemotherapy, adjuvant chemotherapy before or after surgery, hormonal therapy, immunotherapy, etc. It can be used for prevention of cancer, such as prevention and cancer eradication treatment. Examples of the reducing agent of the present invention include cancers caused by hematopoietic tumors (for example, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma, etc.), breast cancer, endometrial cancer. Cancer treatment with cervical cancer, prostate cancer, bladder cancer, kidney cancer, stomach cancer, esophageal cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, oral neck cancer, osteosarcoma, melanoma or brain tumor and / or Or it can be used for prevention. Among them, it is preferably used for the treatment and / or prevention of lung cancer such as breast cancer, small cell lung cancer, non-small cell lung cancer and the like. Furthermore, it can be used for the treatment and / or prevention of cancer containing the cancer stem cells. Moreover, the reducing agent and the preventive agent of the present invention are preferably used for the prevention of the cancer described above, and more preferably used for the prevention of recurrence and / or metastasis of the cancer. Moreover, it is also preferably used for the eradication treatment of the cancer.

The effects of the reducing agent and the preventive agent of the present invention, for example, the effect of reducing the number of cancer stem cells, the effect of not increasing the number of cancer stem cells, the anti-cell effect against cancer stem cells, the growth inhibitory effect against cancer stem cells, the cell killing effect against cancer stem cells, For example, it can be examined by measuring the anti-cell activity of in vitro. Examples of the method for measuring in vitro anti-cell activity include a method using a cell fraction containing cancer cells or cancer stem cells, a method using a cancer cell line containing cancer stem cells, and the like.

Cells including cancer stem cells derived from patients with blood cancer or solid cancer, cancer cell lines include ES-2, SK-OV-3, Caov-3, OV-90, TOV-112D, OVCAR-3, A549, H460 , SK-LU-1, Calu-6, H358, H596, A427, Calu-1, Calu-3, H292, H1299, H128, H345, FaDu, H23, H526, H209, H69, SHP77, K562, HL60, U937 , KG-1, KG-1a, THP-1, Meg-01, CMK, MV-4-11, HCC1143, HCC1395, HCC1937, MCF-7, MDA-MB-231, MDA-MB-415, MDA-MB -468, SW620, COLO201, COLO320DM, DLD-1, COLO205, SW1116, LS180, LS174T, SW480, HCT-15, HT29, HCT116, A172, SNB-19, SK-N-FI, M059K, M059J, Capan-1 And various cells such as Capan-2, CFPAC-1, BxPC-3, MIA-2Paca-2, PANC-1, AsPC-1, HPA. These cancer cell lines are available from public cell banks such as American Type Collection (ATCC), Japanese Collection, Research, Bioresources (JCRB), Deutsche, Sammlung, von, Mikroorganismen, und Zellkulturen, GmbH (DSMZ), or as commercial products. Available.

In addition, cancer stem cells derived from cancer cell lines include (1) CD34 positive / CD38 negative cells in cell lines derived from acute myeloid leukemia, (2) CD138 negative cells in multiple myeloma cell lines, etc. ( 3) CD44 positive / CD24 negative (or CD24 low expression) cells, CD44 positive / CD24 negative (or CD24 low expression) and epithelial-specific antigen (ESA) positive cells, spheres (4) CD44-positive and CD24-positive cells, CD133-positive cells, sphere cells, etc. in brain tumor-derived and neuroblastoma-derived cell lines, (5) CD44-positive and CD166-positive in colon cancer-derived cell lines Cells, CD133-positive cells, sphere cells, etc. (6) CD44-positive, α2β1 integrin highly expressing and CD133-positive cells in prostate cancer-derived cell lines, (7) CD44-positive and CD24-positive cells in pancreatic cancer-derived cell lines , CD44 yang And CD24-positive and ESA-positive cells, CD133-positive cells, etc. (8) CD44-positive cells in head and neck cancer-derived cell lines, (9) Side population (SP) cells in liver cancer-derived cell lines, CD44-positive and CD90-positive cells, CD133-positive cells, etc. (10) Side population (SP) cells, CD133-positive cells, sphere cells, etc. in various solid cancer-derived cell lines such as lung cancer-derived cell lines Can be mentioned.

Furthermore, as cancer stem cells, for example, (1) stem cells derived from normal tissues such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells, germline stem cells, embryonic stem cells, various stem cells such as induced pluripotent stem cells, and (2) hematopoietic stem cells. Progenitor cells, progenitor cells derived from various tissues such as neural progenitor cells, (3) epithelial cells, vascular endothelial cells, fibroblasts, myocytes, bone cells, normal cells derived from various tissues such as hepatocytes, (4) epithelium Cancer cells derived from various tissues such as cancer, adenocarcinoma, sarcoma, glioma, etc. (5) PI3K, STAT3, βCatenin, Notch, Hedgehog, Wnt, hTERT, Bmi1 to various cells of various tissue-derived cancer cell lines , Evi1, TEL / AML1, Meis1, HoxA9, HoxD13, HoxB3, HoxB8, HoxA10, MLL-ENZ, MLL-AF9, AML-ETO, MOZ-TIF2, CDX2, Bcr-Abl, BCL6, maf-B, FGFR, c -oncogenes such as maf, SCL, Hox11, LMO2, LMO1, E2a-Pbx1, TEL-Abl, myc, ENS-WT1, k-ras, Bcrp1, ras, src, jun, met, fos, ret, EML4-ALK Guidance , N-ethyl-N-nitrosourea, N-Methyl-N-nitrosourea, 3,4-benzopyrene, 3-methylcholanthrene, 2-acetylaminofluorene, 7,12-dimethylbenz [a] anthracene, N-nitroso-N-buthylurea, 7 , 8,12-trimethylbenz (a) anthracene and other treatments with chemical carcinogens, heavy particle rays, X-rays, gamma rays, ultraviolet rays, cells that can be induced by irradiation with microwaves, microwaves, PTEN from each of these cells, p53, Examples also include cells that can be induced by deletion, mutation, or inactivation of tumor suppressor genes such as Rb, BRCA1, BRCA2, WT1, and VHL.

A method using a cell fraction containing cancer stem cells (intracellular mitochondrial dehydrogenase activity measurement system) will be described below.
Cell staining is performed using various cancer stem cell marker antibodies, Hoechst33342, and the like, and the cells are fractionated using a flow cytometer, a magnetic bead, etc., and the cancer cells and the cancer stem cell fraction are fractionated.

Various cancer cell fractions and cancer stem cell fractions are seeded, for example, in a 96-well plate and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C.) for 0 to 168 hours, preferably 24 to 168 hours. To do. An Hsp90 family protein inhibitor adjusted to each concentration is added and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C.) for 0 to 168 hours, preferably 24 to 168 hours. After completion of the culture, for example, CellTiter-Glo (registered trademark), 3- (4,5-dimetylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), 2,3-Bis (2-methoxy-4-) nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide (XTT), 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H- tetrazolium (MTS), 2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium (WST-1), 2- (2-methoxy-4-nitrophenyl) ) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8), preferably WST-8, and add color according to the manual attached to each reagent . The absorbance is measured, a colorimetric test is performed, and the number of remaining cells is detected.

In the cancer stem cell fraction, the number of cells remaining after treatment with an Hsp90 family protein inhibitor at a final concentration of 1 fmol / L to 100 mmol / L, preferably a final concentration of 1 pmol / L to 100 μmol / L, is the negative control group (Hsp90 family Hsp90 family protein inhibitors are judged to be effective against cancer stem cells.
In addition, cells after treatment with an Hsp90 family protein inhibitor at a final concentration of 1 fmol / L to 100 mmol / L, preferably a final concentration of 1 pmol / L to 100 μmol / L, in both the cancer cell fraction and the cancer stem cell fraction. Hsp90 family protein inhibitor concentration that shows 0.1 to 99.9%, preferably 20% to 80% cell survival rate compared to the negative control group (no Hsp90 family protein inhibitor added) in each group Is calculated. Compare the Hsp90 family protein inhibitor concentration that shows the same cell survival rate in both fractions, and if the Hsp90 family protein inhibitor concentration in the cancer stem cell fraction is equal to or smaller than that in the cancer cell fraction, The sensitivity to the Hsp90 family protein inhibitor is equivalent in the cancer cell fraction and the cancer stem cell fraction, or the cancer stem cell fraction is higher. That is, when the Hsp90 family protein inhibitor concentration in the cancer stem cell fraction is smaller than that in the cancer cell fraction, the Hsp90 family protein inhibitor has stronger anti-cell activity against cancer stem cells than cancer cells Is shown. This indicates that the effect can be expected from a lower dose when targeting cancer stem cells.

However, in the case of the above method, since some of the cancer stem cells differentiate into cancer cells via cancer progenitor cells during the cell culture period after cell sorting, the measured sensitivity difference is substantially small. It is necessary to consider the possibility of being done.
Next, a method using a cancer cell line containing cancer stem cells will be described.
Various cancer cells including cultured cancer cell lines are seeded in a cell culture flask and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C.) for 0 to 168 hours, preferably 24 to 168 hours. Add Hsp90 family protein inhibitors to a final concentration of 1 fmol / L to 100 mmol / L, preferably 1 pmol / L to 100 μmol / L, and add 1 in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C). Incubate for ~ 168 hours, preferably 24-168 hours. After completion of the culture, cell staining is performed using various cancer stem cell marker antibodies such as Hoechst33342 and the ratio of cancer cells and cancer stem cell fractions is calculated using a flow cytometer or the like.

Calculate the ratio of the fraction of cancer cells and cancer stem cells per 1 to 1,000,000 cells, preferably 100 to 100,000 cells after completion of the culture, negative control group (no Hsp90 family protein inhibitor added) and Hsp90 family protein inhibitor Comparisons are made between treatment groups. When the ratio of the cancer stem cell fraction of the Hsp90 family protein inhibitor treatment group is equal to or less than the ratio of the cancer stem cell fraction of the negative control group, the Hsp90 family protein inhibitor is effective against cancer cells and cancer stem cells. Therefore, it is judged that they have the same effectiveness or are highly effective against cancer stem cells. That is, when the ratio of the cancer stem cell fraction of the Hsp90 family protein inhibitor treatment group is small, it is determined that the Hsp90 family protein inhibitor has stronger anti-cell activity against cancer stem cells than cancer cells.

In addition, as a control treatment group, the same test using the above-mentioned various antitumor agents was performed, and 1 to 1,000,000 cells, preferably 100 to 100,000 cells per cell and cancer stem cell fraction after completion of the culture. The ratio is calculated, and the comparison is performed between the control treatment group and the Hsp90 family protein inhibitor treatment group. When the ratio of the cancer stem cell fraction of the Hsp90 family protein inhibitor treatment group is equal to or less than the ratio of the cancer stem cell fraction of the control treatment group, the Hsp90 family protein inhibitor is equivalent to the comparison control drug. Or is more effective than a comparative control drug. That is, when the ratio of the cancer stem cell fraction of the Hsp90 family protein inhibitor treatment group is small, it is determined that the Hsp90 family protein inhibitor has stronger anti-cell activity against cancer stem cells than various antitumor agents.

The effects of the reducing agent and the preventive agent of the present invention can also be examined by measuring ex vivo tumorigenic activity.
Examples of animal models include immunodeficient animals such as nude rats, nude mice, SCID mice, NOD / SCID mice, and NOG mice, mice having normal immune systems, mammals such as rats, rabbits, dogs, monkeys, and cancer tissues. Mammalian cancer cells such as humans, mice, rats, rabbits, dogs, monkeys, etc., preferably human cancer cells, including, for example, subcutaneous, intradermal, intraperitoneal, intramammary, subrenal, Examples thereof include xenotransplantation or allograft model in which transplantation is performed in various sites such as in the pancreas, under the colon, under the caecum, under various organs, intrapulmonary, intrabronchial, intramedullary, intravenous, etc., preferably subcutaneously.

Various cancer cells including cultured cancer cell lines are seeded in a cell culture flask and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C.) for 0 to 168 hours, preferably 24 to 168 hours. Add Hsp90 family protein inhibitors to a final concentration of 1 fmol / L to 100 mmol / L, preferably 1 pmol / L to 100 μmol / L, and add 1 in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C). Incubate for ~ 168 hours, preferably 24-168 hours. After completion of the culture, viable cells are adjusted to 1 to 10,000,000 cells, preferably 10 to 100,000 cells, and transplanted to a host animal in the presence or absence of 1 to 99.9%, preferably 10 to 50% matrigel. . Tumor engraftment rate of the negative control group (no Hsp90 family protein inhibitor added) and Hsp90 family protein inhibitor treatment group, period required for tumor engraftment, tumor volume (eg, tumor major axis (mm) x tumor minor axis ( mm) × Tumor minor axis (mm) / 2) and the like can be compared to evaluate the effect of the Hsp90 family protein inhibitor.

In the Hsp90 family protein inhibitor treated group compared to the negative control group, when at least one or more of the effects of decreased tumor engraftment rate, prolonged period necessary for tumor engraftment and decreased tumor volume is observed, Hsp90 family protein inhibitors are considered to have a high inhibitory effect on tumorigenic activity. That is, the Hsp90 family protein inhibitor decreases the content of cancer stem cells in living transplanted cells and is shown to be more effective against cancer stem cells than cancer cells.

In addition, by using the animal model, the effect of the Hsp90 family protein inhibitor was compared by comparing the effect of the control treatment group using the various antitumor agents with the effect of the Hsp90 family protein inhibitor treatment group. It can also be evaluated. That is, the tumor engraftment rate of each group, the period necessary for tumor engraftment, the tumor volume, etc. are measured, and compared with the control treatment group, the decrease in the tumor engraftment rate of the Hsp90 family protein inhibitor treatment group, When at least one of the effects of prolonging the time required for tumor engraftment and reducing the tumor volume is observed, the effectiveness of Hsp90 family protein inhibitors on cancer stem cells and the effect on tumorigenic activity of various antitumor agents It is judged to be higher than the one.

Further, using the animal model, the effect of the single treatment group of the various antitumor agents, the effect of the single treatment group of the Hsp90 family protein inhibitor, and the combined treatment group of the various antitumor agents and the Hsp90 family protein inhibitor By comparing the effect, the effect of the Hsp90 family protein inhibitor can also be evaluated. That is, the tumor engraftment rate of each group, the period necessary for tumor engraftment, the tumor volume, etc. are measured, and compared with each single treatment group, the tumor engraftment rate decreases in the combination treatment group, and is necessary for tumor engraftment. If at least one or more effects of prolonging the period and decreasing the tumor volume are observed, it is judged that the Hsp90 family protein inhibitor has a combined effect with various antitumor agents.

The effect of the reducing agent of the present invention can also be examined by measuring in vivo antitumor activity using an animal model. In addition, the preventive effect of the preventive agent of the present invention on recurrence or metastasis can be evaluated using an animal model.
Examples of animal models include immunodeficient animals such as nude rats, nude mice, SCID mice, NOD / SCID mice, and NOG mice, mice having normal immune systems, mammals such as rats, rabbits, dogs, monkeys, and cancer tissues. Mammalian cancer cells such as humans, mice, rats, rabbits, dogs, monkeys, etc., preferably human cancer cells, including, for example, subcutaneous, intradermal, intraperitoneal, intramammary, subrenal, Examples thereof include xenotransplantation or allograft model in which transplantation is performed in various sites such as in the pancreas, under the colon, under the caecum, under various organs, intrapulmonary, intrabronchial, intramedullary, intravenous, etc., preferably subcutaneously.

Examples of the administration method of the Hsp90 family protein inhibitor to the animal model include oral administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, intramedullary administration, to the tumor site and its surroundings. Local administration etc. are mentioned.
Various cancer cells including cultured cancer cell lines are seeded in a cell culture flask and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air, 37 ° C.) for 1 to 168 hours, preferably 24 to 168 hours. After completion of the culture, viable cells are adjusted to 1 to 10,000,000 cells, preferably 10 to 1,000,000 cells, and transplanted to the host animal in the presence or absence of 1 to 99.9%, preferably 10 to 50% Matrigel. The host animal is administered 1 ng / kg to 1,000 mg / kg, preferably 1 μg / kg to 500 mg / kg, of the Hsp90 family protein inhibitor before and / or after transplantation.

Tumor volume (for example, tumor major axis (mm) x tumor minor axis (mm) x tumor minor axis (mm) / 2) and Hsp90 family protein inhibitor administered group of negative control group (Hsp90 family protein inhibitor not administered) By comparing the tumor volumes of the Hsp90 family protein inhibitors, the antitumor effect of Hsp90 family protein inhibitors can be evaluated. In addition, the antitumor effect of an Hsp90 family protein inhibitor can be evaluated by comparing the number of metastatic tumors, the type and number of organs to which metastasis occurs, the tumor volume of metastatic tumors, and the like.

Compared to the negative control group, at least the decrease in tumor volume (decrease in tumor growth activity) in the Hsp90 family protein inhibitor administration group, the number of tumor metastases, the type and number of metastasis organs, or the decrease in tumor volume of metastatic tumors, etc. If one or more effects are observed, it is determined that the Hsp90 family protein inhibitor has an anti-tumor effect.
In addition, the antitumor effect of the control group administered with at least one of the various antitumor agents at 1 ng / kg to 1,000 mg / kg, preferably 1 μg / kg to 500 mg / kg, using the animal model And the anti-tumor effect of the Hsp90 family protein inhibitor administration group, the effect of the Hsp90 family protein inhibitor can be evaluated.

Compared with the control group, the tumor volume decreased (decreased tumor growth activity) in the Hsp90 family protein inhibitor group, or when the tumor shrinked, the tumor volume returned to the size at the start of the study. If at least one effect such as an extension of the time required for the treatment is observed, the Hsp90 family protein inhibitor is judged to have a higher antitumor effect than various antitumor agents.
Furthermore, using the animal model, the effect of the single administration group of the various antitumor agents, the effect of the single administration group of the Hsp90 family protein inhibitor, and the combined administration group of the various antitumor agents and the Hsp90 family protein inhibitor By comparing the effect, the effect of the Hsp90 family protein inhibitor can also be evaluated. That is, compared with the antitumor effect of the group administered with various antitumor agents alone or the group administered with Hsp90 family protein inhibitor alone, the tumor volume decreased (decreased tumor growth activity) or the tumor was reduced in the combination administration group If at least one effect such as an increase in the time required for the tumor volume to return to the initial size of the study is observed, it is determined that there is a combined effect of an Hsp90 family protein inhibitor and an antitumor agent. Therefore, it is judged that the effect of the combined use of an Hsp90 family protein inhibitor and an antitumor agent is high.

In addition, excision of the formed tumor using the animal model, administration of the various antitumor agents alone, or combined administration of various antitumor agents and Hsp90 family protein inhibitors, followed by Hsp90 family protein inhibition By subsequently administering the agent, the effect of the Hsp90 family protein inhibitor in the recurrence model can also be evaluated. That is, the Hsp90 family protein inhibitor continued in the tumor excision group, the group administered with various antitumor agents alone, or the group administered with various antitumor agents and Hsp90 family protein inhibitors as compared to the group that did not receive further drug treatment. In the administration group, when at least one effect such as a decrease in tumor volume (decrease in tumor growth activity), complete disappearance of tumor and its continuation is observed, Hsp90 family protein inhibitors are effective in preventing / suppressing recurrence. It is judged that there is.

In the animal model, in order to measure the effect of the reducing agent and the preventive agent of the present invention, a part of an animal body tissue, for example, free cancer cells (Isolated tumor cell, or ITC) in the peripheral blood or bone marrow, tumor mass, etc. It is also possible to use. A part of these body tissues can be obtained by a method of collecting from peripheral blood, bone marrow, tumor lesion, tumor metastasis, or the like, for example, blood sampling, bone marrow puncture, or biopsy. A part of the body tissue is collected before and after administration of the Hsp90 family protein inhibitor, and the above various cancer stem cell marker antibodies, flow cytometers using Hoechst33342, etc., evaluation of sphere formation ability, immunohistochemical staining, etc. are used. Thus, the abundance of cancer stem cells contained in a part of body tissue can be calculated. If the prevalence of cancer stem cells in some body tissues after administration of an Hsp90 family protein inhibitor is lower than the prevalence of cancer stem cells in some body tissues before administration of an Hsp90 family protein inhibitor, the Hsp90 family protein inhibitor is It is judged to have the ability to reduce stem cells. That is, it is judged that the Hsp90 family protein inhibitor is effective for the treatment and / or prevention of cancer.

The effects of the reducing agent and the preventive agent of the present invention can also be examined by measuring clinical effects on patients or clinical specimens of cancer patients.
As clinical specimens, free cancer cells (Isolated tumor cells, ITC) and tumor masses in peripheral blood or bone marrow, for example, are used. These clinical specimens can be obtained by a method of collecting from peripheral blood, bone marrow, tumor lesion, tumor metastasis, or the like, for example, a blood collection method, a bone marrow puncture method, a biopsy method, or the like. Clinical specimens were collected before and after administration of Hsp90 family protein inhibitors, and clinically determined by using the above-mentioned various cancer stem cell marker antibodies, flow cytometers using Hoechst33342, evaluation of sphere formation, immunohistochemical staining, etc. The abundance ratio of cancer stem cells in the specimen can be calculated. The ability of Hsp90 family protein inhibitors to reduce cancer stem cells when the prevalence of cancer stem cells in clinical specimens after administration of Hsp90 family protein inhibitors is lower than the prevalence of cancer stem cells in clinical specimens before administration of the Hsp90 family protein inhibitors It is judged that there is. That is, it is judged that the Hsp90 family protein inhibitor is effective for the treatment and / or prevention of cancer.

In patients treated with Hsp90 family protein inhibitors, the cancer recurrence rate, such as increased cancer healing rate, increased tumor shrinkage rate, regrowth rate or metastasis rate, was lower than in the untreated patient group treated with Hsp90 family protein inhibitors. If a clinical effect associated with a decrease in at least one cancer stem cell, such as an extended period until cancer recurrence, an increase in survival rate, or an increase in disease-free survival rate, is observed, Hsp90 family protein inhibitors can be used for cancer therapy and / or Or it is judged that it is effective as a preventive agent.

In addition, in patients who receive Hsp90 family protein inhibitors, the cancer cure rate is higher than in patients who are receiving other cancer treatments such as anti-cancer therapy, radiation therapy, surgery, hormone therapy, and immunotherapy. To decrease at least one cancer stem cell, such as increase in tumor shrinkage rate, decrease in cancer recurrence rate such as regrowth rate or metastasis rate, extension of period until cancer recurrence, increase in survival rate, increase in disease-free survival rate, etc. If the accompanying clinical effect is observed, it is judged that the Hsp90 family protein inhibitor is more effective for the treatment and / or prevention of cancer than other cancer treatment methods.

In addition, in Hsp90 family protein inhibitors and other anti-cancer therapies such as combination therapy for cancer therapy such as radiotherapy, surgery, hormone therapy, immunotherapy, Hsp90 family protein inhibitors alone or other Cancer such as increased cancer cure rate, increased tumor shrinkage rate, reproliferation rate or metastasis rate, compared to the group of patients who receive cancer treatment alone, such as anticancer therapy, radiation therapy, surgery, hormone therapy, immunotherapy, etc. Hsp90 family protein inhibitors can be used if a clinical effect associated with a decrease in at least one cancer stem cell is observed, such as a decrease in recurrence rate, an extended period until cancer recurrence, an increase in survival rate, or an increase in disease-free survival rate. It is judged that the combined effect with anti-cancer therapy, for example, cancer therapy such as radiotherapy, surgery, hormone therapy, immunotherapy, etc. is high.

Compound (I), Compound (II), 17-AAG and 17-DMAG or pharmacologically acceptable salts thereof can be administered alone as they are, but are usually provided as various pharmaceutical preparations. Is desirable.
The reducing agent and the preventive agent of the present invention are Hsp90 family protein inhibitors such as Compound (I), Compound (II), 17-AAG and 17-DMAG or pharmacologically acceptable salts thereof as active ingredients. Or as a mixture with any other therapeutically active ingredient. In addition, these pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.

These preparations are pharmaceutically acceptable diluents, excipients, disintegrants, lubricants, binders, surfactants, water, physiological saline, vegetable oils, solubilizers, in addition to the active ingredients. It can be prepared by a conventional method using an isotonic agent, a preservative, an antioxidant and the like.
In preparing tablets, for example, excipients such as lactose, disintegrants such as starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, surfactants such as fatty acid esters, plasticizers such as glycerin Etc. may be used according to a conventional method.

In preparing the injection, water, physiological saline, vegetable oil, solvent, solubilizer, tonicity agent, preservative, antioxidant and the like may be used in a conventional manner.
Compound (I), Compound (II), 17-AAG and 17-DMAG or pharmacologically acceptable salts thereof are usually used orally or parenterally as injections when used for the above purpose. The effective dose and frequency of administration vary depending on the administration form, patient age, body weight, symptoms, etc., but it is usually preferable to administer 0.01 to 20 mg / kg per day.

Next, the pharmacological action of the Hsp90 family protein inhibitor will be described more specifically with test examples. In Test Examples 1 to 7, the test compound used was hydrochloride of compound 22, compound 23, compound 41, geldanamycin, radicicol, 17-DMAG, rebrastatin, EH21A2, PU24FCl and novobiocin. Moreover, the Hsp90 family protein inhibitor and the existing antitumor agent used in this test example were obtained as commercial products, or were produced by fermentation or synthesized by a known method.

Test example 1: Analysis of drug sensitivity of cancer stem cells using side population (SP) cell fraction A549 cell population with cancer stem cell activity is enriched in the SP cell fraction of A549, a human non-small cell lung cancer strain [Cancer Research, Vol. 67, 4827-4833 (2007)]. Using a flow cytometer, the SP cell fraction present in A549 after addition of a test compound (geldanamycin) or an existing antitumor agent was analyzed by a literature method [Cancer Research, Vol. 67, 4827-4833. Page (2007)].

As existing antitumor agents, 5-fluorouracil (5-FU) (Sigma), Mitomycin (Kyowa Hakko Kogyo), etoposide (VP-16) (Sigma), cisplatin ( Cisplatin (Sigma), methotrexate (Sigma), paclitaxel (Sigma), docetaxel (Aventis) were used.

For culture of A549 (ATCC), F12K medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin solution (Invitrogen) was used. Cell culture flask (Nalgen-Nunk) was seeded with 5 μL of A549 cell solution adjusted to 10 million cells / mL with culture medium per 1mL of culture medium, and 5% CO2 for 48 hours at 37 ° C. The cells were cultured in an incubator (95% air). Add 3 μL each of the test compound (geldanamycin, Sigma) diluted with cell culture medium or a solution containing an existing antitumor agent (final concentration 0.1 to 3.0 μmol / L), and again 5 The cells were cultured at 37 ° C. for 48 hours in a% carbon dioxide incubator.

A549 was recovered from the culture vessel using 0.05% trypsin / 0.53 mmol / L EDTA · 4Na solution (Invitrogen), suspended in DMEM medium (Invitrogen) containing 2% fetal calf serum (Invitrogen), and the cells An adjustment solution (1 million mL of 1 million cells / mL) was prepared. Hoechst33342 (Invitrogen) was added to the cell preparation so as to have a final concentration of 5 μg / mL, and the cells were allowed to stand for 90 minutes at 37 ° C. with stirring at intervals of 15 minutes to perform cell staining.

After the staining reaction, the cells are washed with a Phosphate-buffered saline (PBS) solution (Invitrogen) containing 5% fetal bovine serum (Invitrogen) to give Propidium Iodide (Invitrogen) at a final concentration of 1 μg / mL. Thus, dead cells were stained and stored in ice. SP cell content was analyzed using a flow cytometer [BD FACSAria, Becton Dickinson (BD)].

The SP cell content ratio of the compound addition group (the test compound or the existing antitumor agent addition group) is the SP cell content obtained by culturing the untreated group to which the test compound and the antitumor agent are not added in the same manner as the compound addition group. The rate (2.4%) was measured in the same manner as in the compound addition group, and calculated according to the following formula.
SP cell content ratio = SP cell content ratio in the compound addition group / SP cell content ratio in the untreated group

In addition, as an experiment for confirming the SP cell fraction, verapamil, which is an inhibitor of ABC transporter, was subjected to staining reaction with Hoechst33342 on the test compound and the cell preparation without treatment with the existing antitumor agent, and before cell washing. The test of the verapamil treatment group to which (Sigma) was added to a final concentration of 50 μmol / L was performed in the same manner as described above. In this treatment group, disappearance of the SP cell fraction by verapamil was confirmed.

Table 3 shows the effects of test compounds and existing antitumor agents on the SP cell fraction.
In the group containing 5-fluorouracil, the SP cell content was 6.3%, and a 2.63-fold increase in SP cell fraction (SP cell content ratio: 2.63) was confirmed. In any of the anti-tumor agent-added groups, there was almost no decrease in the SP cell fraction compared to the untreated group, the SP cell content ratio was 0.83 or more, and the sensitivity of cancer stem cells and cancer cells to anti-tumor agents was equivalent. Some have been shown to be less sensitive to cancer stem cells. On the other hand, in the test compound addition group, a marked decrease in the SP cell fraction was confirmed, and the SP cell content ratio was 0.09.

From the above, it was clarified that the test compound showed a remarkable effect of decreasing the SP cell fraction and was more sensitive than the cancer cell. This result suggests that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells.

Test Example 2: Analysis of concentration dependence of test compound on cancer stem cells using side population (SP) cell fraction Test compound (Geldana) on SP cell fraction of A549 using the same test method as in Test Example 1. The concentration-dependent reduction effect of (mycin) was analyzed.
3 μL each of solutions containing test compounds (Sigma) prepared by serial dilution in cell culture medium were added to the medium containing A549 cultured for 48 hours (final concentration: 0.003 to 3.0 μmol / L) And cultured for 48 hours. After analyzing the SP cell content using the method described in Test Example 1, the SP cell content ratio was calculated.

Table 4 shows the effect of the test compound at each final concentration on the SP cell fraction.
The test compound decreased the SP cell fraction in a concentration-dependent manner and showed a concentration-dependent decrease in the SP cell content ratio.
These results suggest that Hsp90 family protein inhibitors have concentration-dependent anti-cell activity against cancer stem cells.

Test Example 3: Analysis of the effects of Hsp90 family protein inhibitors on cancer stem cells using side population (SP) cell fraction (1)
Using the same test method as in Test Example 1, the effect of various test compounds on the SP cell fraction of A549 was analyzed.
Various test compounds (geldanamycin (Sigma), 17-DMAG (compound described in WO 02/079167) pamphlet), PU24FCl [2-fluoro-9-pent] -4-ynyl-8- (2-chloro-3,4,5-trimethoxy-benzyl) -9H-purine-6-ylamine] (compound described in WO 02/036075 pamphlet), EH21A2 [Bioorganic 3 μL each of the solution containing the compound described in Bioorganic & Medicinal Chemistry Letters, Vol. 18, 1577-1580 (2008)]} was added to the medium containing A549 for 72 hours. (Final concentration: 1.0 to 10 μmol / L) and cultured for 24 hours. After analyzing the SP cell content using the method described in Test Example 1, the SP cell content ratio of each test compound was calculated.

The effects of each test compound at each final concentration on the SP cell fraction are shown in Table 5.
In any of the test compound addition groups, a decrease in the SP cell fraction was confirmed, showing a marked decrease in the SP cell content ratio, clearly showing that the SP cell fraction was more effective than the cancer cell. became.
These results also suggest that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells.

Test Example 4: Analysis of effects of Hsp90 family protein inhibitors on cancer stem cells using side population (SP) cell fraction (2)
Using the same test method as in Test Example 1, the effect of various test compounds on the SP cell fraction of A549 was analyzed.
Various test compounds diluted and prepared in cell culture medium [hydrochloride of compound 22, compound 23, compound 41, geldanamycin (Sigma), radicicol (Calbiochem), rebrastatin [bioorganic and A549 obtained by culturing 3 μL each of solutions containing Compound of Compound No. 18 or 1577-1580 (2008)] or Novobiocin (Calbiochem) for 48 hours, each containing Bioorganic & Medicinal Chemistry Letters, Vol. 18, 1577-1580 (2008) The medium was added to the medium (final concentration: 3.0 μmol / L) and cultured for 48 hours. After analyzing the SP cell content using the method described in Test Example 1, the SP cell content ratio of each test compound was calculated.

Table 6 shows the effect of each test compound on the SP cell fraction.
In any of the test compound addition groups, a decrease in the SP cell fraction was confirmed, showing a marked decrease in the SP cell content ratio, clearly showing that the SP cell fraction was more effective than the cancer cell. became.
These results also suggest that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells.

Test Example 5: Effect on tumorigenic activity using ex vivo evaluation method When the SP cell fraction present in A549 is removed, the tumorigenic activity of A549 decreases [Cancer Research, Vol. 67, 4827- 4833 (2007)]. The tumorigenic activity of A549 was measured by an ex vivo evaluation model using A549 in which the abundance ratio of the SP cell fraction was changed by treatment with a test compound (geldanamycin) or an existing antitumor agent (5-fluorouracil).

For culture of A549 (ATCC), F12K medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin solution (Invitrogen) was used. Cell culture flask (Nalgen-Nunk) was seeded with 5 μL of A549 cell solution adjusted to 10 million cells / mL with culture medium per 1mL of culture medium, and 5% CO2 for 48 hours at 37 ° C. The cells were cultured in an incubator (95% air). Add 3 μL each of the solutions containing test compound (Sigma) or fluorouracil (Sigma) prepared by diluting in cell culture medium (final concentration 3.0 or 30.0 μmol / L), and again 5% CO2 The cells were cultured for 48 hours at 37 ° C. in an incubator.

A549 was recovered from the culture vessel using 0.05% trypsin / 0.53 mmol / L EDTA · 4Na solution (Invitrogen), suspended in Phosphate-buffered saline (PBS) solution (Invitrogen), and cell preparation solution (1000 cells) 1 mL / mL was prepared. The cell preparation and Matrigel (BD) were mixed at a volume ratio of 1: 1 and stored in ice until transplantation. On the day before cell transplantation, cells were transplanted to the number of 100 cells per mouse subcutaneously in the ventral part of nude mice (SLC) to which 100 μg of Asialo GM1 antibody (WAKO) was intraperitoneally administered. Tumor engraftment was determined once a week for 10 weeks for nude mice transplanted with cells. Further, after culturing the non-treated group to which no test compound or antitumor agent was added in the same manner as the compound-added group, the obtained A549 was transplanted into nude mice, and tumor engraftment was determined.

Table 7 shows the number of tumor engraftments at each final concentration of the test compound and the existing antitumor agent.
In the untreated group and the anti-tumor agent-treated group, tumor engraftment with high tumorigenic activity was observed in 2 out of 2 cases, whereas in the test compound-treated group, tumorigenicity was accompanied by a decrease in the SP cell fraction. Suppression of tumor engraftment due to decreased or lost activity was observed.
Based on the above, it was found that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells, and are more effective against cancer stem cells than cancer cells, reducing the tumorigenic activity of tumors. did. That is, it was suggested that Hsp90 family protein inhibitors are effective against cancer recurrence and metastasis.

Test Example 6: tumorigenic activity of A549 Removal of the SP cell fraction present in effect A549 of Hsp90 family protein inhibitor against tumorigenic activity using the ex vivo evaluation method is reduced [Cancer Research (Cancer Research), 67, 4827-4833 (2007)]. The tumorigenic activity of A549 was measured by an ex vivo evaluation model using A549 in which the abundance ratio of the SP cell fraction was changed by treatment with a test compound (hydrochloride of compound 22, PU24FCl, rebrastatin, or novobiocin).

For culture of A549 (ATCC), F12K medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin solution (Invitrogen) was used. Cell culture flask (Nalgen-Nunk) was seeded with 5 μL of A549 cell solution adjusted to 10 million cells / mL with culture medium per 1mL of culture medium, and 5% CO2 for 48 hours at 37 ° C. The cells were cultured in an incubator (95% air). 3 µL each of solutions containing test compounds (Sigma) diluted and prepared in cell culture medium were added (final concentration: 3.0 µmol / L), and again in a 5% CO2 incubator at 37 ° C at 48 ° C. Incubate for hours.

A549 was recovered from the culture vessel using 0.05% trypsin / 0.53 mmol / L EDTA · 4Na solution (Invitrogen), suspended in Phosphate-buffered saline (PBS) solution (Invitrogen), and cell preparation solution (1000 cells) 1 mL / mL was prepared. The cell preparation and Matrigel (BD) were mixed at a volume ratio of 1: 1 and stored in ice until transplantation. On the day before cell transplantation, cells were transplanted to the number of 100 cells per mouse subcutaneously in the ventral part of nude mice (SLC) to which 100 μg of Asialo GM1 antibody (WAKO) was intraperitoneally administered. Tumor engraftment was determined once a week for 10 weeks for nude mice transplanted with cells. In addition, after culturing the untreated group to which no test compound was added in the same manner as the compound-added group, the obtained A549 was transplanted into nude mice, and tumor survival was determined.

Table 8 shows the number of tumors engrafted in the test compound-treated A549 transplant group.
In the untreated group, tumor engraftment with high tumorigenic activity was observed in 8 out of 8 cases, but in the test compound treated group, due to the decrease or loss of tumorigenic activity accompanying the decrease in SP cell fraction Inhibition of tumor engraftment was observed.
Based on the above, it was found that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells, and are more effective against cancer stem cells than cancer cells, reducing the tumorigenic activity of tumors. did. That is, it was suggested that Hsp90 family protein inhibitors are effective against cancer recurrence and metastasis.

Test Example 7: Analysis of the effects of Hsp90 family protein inhibitors on cancer stem cells using side population (SP) cell fraction (3)
A cell population having cancer stem cell activity is concentrated in the SP cell fraction of MCF-7, a human breast cancer strain [Cancer Research, 65, 6207-6219 (2005)]. Using a flow cytometer, SP cell fraction present in MCF-7 after addition of various test compounds (hydrochloride of compound 22, compound 23, compound 41, geldanamycin, radicicol, or PU24FCl) [Cancer Research, 65, 6207-6219 (2005)].

MCF-7 (ATCC) was cultured with 10% fetal bovine serum (Invitrogen), 1% MEM non-essential amino acid solution (Invitrogen), 1% sodium pyruvate solution (Invitrogen), and 1% penicillin A MEM medium (Invitrogen) containing a streptomycin solution (Invitrogen) was used. Cell culture flasks (Nalgen-Nunk) were seeded with 10 μL of MCF-7 adjusted to 10 million cells / mL with a culture medium per 1 mL of culture medium and incubated at 37 ° C for 48 hours at 5% CO2 incubator ( The cells were cultured in (95% air). A solution containing a test compound diluted and prepared in a cell culture medium was added in an amount of 3 μL each (final concentration: 3.0 μmol / L), and cultured again at 37 ° C. for 48 hours in a 5% carbon dioxide incubator.

MCF-7 was collected from the culture vessel using 0.05% trypsin / 0.53 mmol / L EDTA / 4Na solution (Invitrogen) and suspended in DMEM medium (Invitrogen) containing 2% fetal calf serum (Invitrogen). A cell preparation solution (1 million cells / mL) was prepared. Hoechst33342 (Invitrogen) was added to this cell preparation so as to have a final concentration of 5 μg / mL, and then the cells were allowed to stand for 90 minutes while stirring at 37 ° C. at intervals of 15 minutes to perform cell staining.

The SP cell content ratio in the compound addition group (test compound) is the same as the compound addition group in the SP cell content ratio (4.1%) obtained by culturing the untreated group without addition of the test compound in the same manner as the compound addition group. And calculated according to the following formula.

SP cell content ratio = SP cell content ratio in the compound addition group / SP cell content ratio in the untreated group

In addition, as an experiment to confirm the SP cell fraction, verapamil (Sigma), an inhibitor of ABC transporter, was added to the cell preparation without treatment with test compound after staining with Hoechst33342 and before cell washing. The verapamil treatment group added to a final concentration of 50 μmol / L was tested in the same manner as described above. In this treatment group, disappearance of the SP cell fraction by verapamil was confirmed.

The effect of each test compound on the SP cell fraction is shown in Table 9.
In any of the test compound addition groups, a decrease in the SP cell fraction was confirmed, showing a marked decrease in the SP cell content ratio, clearly showing that the SP cell fraction was more effective than the cancer cell. became.
These results also suggest that Hsp90 family protein inhibitors have high anti-cell activity against cancer stem cells.

Formulation Example 1 (tablet)
A tablet having the following composition is prepared by a conventional method.
Compound 1 5 mg
Lactose 60 mg
Potato starch 30 mg
Polyvinyl alcohol 2 mg
Magnesium stearate 1 mg
Tar pigment Trace amount 98 mg

Formulation Example 2 (Injection)
An injection having the following composition is prepared by a conventional method.
Compound 17 2 mg
D-mannitol 10 mg
Hydrochloric acid aqueous solution appropriate amount Sodium hydroxide aqueous solution appropriate amount
Distilled water for injection qs 2.0 mL

Claims

A cancer stem cell and / or cancer progenitor cell reducing agent comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
Heat shock protein 90 (Hsp90) family protein inhibitors are represented by formula (I)

[Wherein n represents an integer of 1 to 5,
R 1 is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted heterocyclic alkyl, substituted or unsubstituted Aryl, CONR 7 R 8 (wherein R 7 and R 8 are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkanoyl, substituted or Represents an unsubstituted aryl, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclic alkyl or a substituted or unsubstituted aroyl, or a nitrogen where R 7 and R 8 are adjacent Together with the atoms to form a substituted or unsubstituted heterocyclic group) or NR 9 R 10 (where R 9 and R 10 represents the same as R 7 and R 8 above,
R 2 represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group,
R 3 and R 5 are the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Represents aralkyl or substituted or unsubstituted aroyl,
R 4 represents a hydrogen atom, hydroxy or halogen,
R 6 is a hydrogen atom, halogen, cyano, nitro, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cyclo Alkyl, amino, lower alkylamino, di-lower alkylamino, carboxy, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryloxy, substituted or unsubstituted aryl, substituted or unsubstituted The reducing agent according to claim 1, which is a benzoyl compound represented by a substituted heterocyclic group, a substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclic alkyl, or a pharmaceutically acceptable salt thereof.
The reducing agent according to claim 2, wherein R 2 is phenyl or phenyl substituted with 1 to 3 substituents.
The reducing agent according to claim 2, wherein R 2 is a substituted or unsubstituted aromatic heterocyclic group.
The reducing agent according to any one of claims 2 to 4, wherein R 3 , R 4 and R 5 are hydrogen atoms.
The reducing agent according to any one of claims 2 to 5, wherein R 1 is CONR 7 R 8 (wherein R 7 and R 8 are as defined above).
R 1 is CONR 7a R 8a (wherein R 7a and R 8a are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, or a substituted or unsubstituted heterocyclic alkyl). The reducing agent according to any one of 5.
(Wherein, R 7b and R 8b form a substituted or unsubstituted heterocyclic group together with the adjacent nitrogen atom) R 1 is CONR 7b R 8b according to any one of claims 2 to 5, which is a Reducing agent.
The reducing agent according to any one of claims 2 to 8, wherein R 6 is a hydrogen atom, lower alkyl, halogen or aryl.
The reducing agent according to any one of claims 2 to 8, wherein R 6 is lower alkyl.
The reducing agent according to any one of claims 2 to 8, wherein R 6 is ethyl.
Heat shock protein 90 (Hsp90) family protein inhibitor is 2- {2-ethyl-3,5-dihydroxy-6- [3-methoxy-4- (2-morpholinoethoxy) benzoyl] phenyl} -N, N-bis (2-methoxyethyl) acetamide or 2- [2-ethyl-3,5-dihydroxy-6- (4-methoxybenzoyl) phenyl] -N- (2-hydroxyethyl) -N- (2-methoxyethyl) acetamide Or a reducing agent according to claim 1, which is a pharmacologically acceptable salt thereof.
Heat shock protein 90 (Hsp90) family protein inhibitors are represented by formula (II)

{Where n1 represents an integer from 0 to 10,
R 11 is a hydrogen atom, hydroxy, cyano, carboxy, nitro, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or Unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aroyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted heterocyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted Arylsulfonyl, substituted or unsubstituted heterocyclic group, CONR 17 R 18 (wherein R 17 and R 18 are the same or different, a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, Substituted or unsubstituted lower alkanoyl, substituted or Represents unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclic alkyl or substituted or unsubstituted aroyl, or nitrogen in which R 17 and R 18 are adjacent NR 19 R 20 [wherein R 19 and R 20 are the same or different, and represent a hydrogen atom, a substituted or unsubstituted lower alkylsulfonyl, and a substituted or unsubstituted heterocyclic group together with an atom] Substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl, substituted or unsubstituted substituted heterocyclealkyl, during aroyl substituted or unsubstituted, or CONR 21 R 22 (wherein, the R 21 and R 22 each R 17 and R 18 Or represents a a) synonymous, or R 19 and R 20 form a substituted or unsubstituted heterocyclic group together with the adjacent nitrogen atom] or OR 23 (wherein, R 23 is a substituted or unsubstituted Lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl or substituted or unsubstituted heterocyclic ring Represents alkyl)
R 12 represents a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heterocyclic group;
R 13 and R 15 are the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Lower alkylsulfonyl, substituted or unsubstituted arylsulfonyl, carbamoyl, sulfamoyl, substituted or unsubstituted lower alkylaminocarbonyl, substituted or unsubstituted di-lower alkylaminocarbonyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted Represents substituted heterocyclic carbonyl, substituted or unsubstituted aralkyl or substituted or unsubstituted aroyl;
R 14 and R 16 are the same or different and are a hydrogen atom, hydroxy, halogen, cyano, nitro, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted Lower alkoxy, substituted or unsubstituted cycloalkyl, amino, lower alkylamino, di-lower alkylamino, carboxy, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aryloxy, substituted or unsubstituted aryl, substituted Or a substituted or unsubstituted lower alkanoyl, a substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclic alkyl}, or a pharmaceutically acceptable salt thereof The reducing agent according to claim 1.
R 11 is hydroxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted A heterocyclic group, substituted or unsubstituted aryl, CONR 17 R 18 (wherein R 17 and R 18 are as defined above) or NR 19 R 20 (wherein R 19 and R 20 are as defined above, respectively) 14. The reducing agent according to claim 13, wherein
The reducing agent according to claim 13 or 14, wherein R 12 is substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group.
The reducing agent according to claim 13 or 14, wherein R 12 is substituted or unsubstituted phenyl.
The reducing agent according to claim 13 or 14, wherein R 12 is substituted or unsubstituted furyl.
The reducing agent according to any one of claims 13 to 17, wherein R 13 , R 14 and R 15 are hydrogen atoms.
The reducing agent according to any one of claims 13 to 18, wherein R 16 is a hydrogen atom, halogen or substituted or unsubstituted lower alkyl.
Heat shock protein 90 (Hsp90) family protein inhibitor is 17-allylamino-17-demethoxygeldanamycin (17-AAG) or 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) or its The reducing agent according to claim 1, which is a pharmacologically acceptable salt.
The reducing agent according to any one of claims 1 to 20, wherein the cancer stem cells and / or cancer precursor cells are cells having tumorigenic activity.
Metastasis of cancer stem cells and / or cancer progenitor cells having tumorigenic activity, including a cell population present in a tumor primary lesion, a cell population present in a tumor recurrence lesion, and a micrometastasis composed of one or more cells Cell population present in tumor nest, cell population present in bone marrow, side population cell population, quiescent cell population, GO phase cell population, RNA content is relative to average content in all cancer cell populations Low cell population, cell population with relatively low uptake of pyronin Y than the average uptake in all cancer cell populations, CD34 positive cell population, CD38 negative cell population, CD138 negative cell population, CD44 positive cell population, CD24 Cell population whose expression level is relatively lower than the average expression level in all cancer cell populations, epithelial-specific antigen (ESA) positive cell population, sphere cell population, CD24 positive cell population It belongs to at least one cell population selected from a CD166 positive cell population, an α2β1 integrin positive cell population, a CD133 positive cell population, a CD90 positive cell population, a CD55 positive cell population, and a cell population having high aldehyde dehydrogenase (Aldehyde Dehydrogenase: ALDH) activity The reducing agent according to any one of claims 1 to 20, which is a cell.
The reducing agent according to any one of claims 1 to 20, wherein the cancer stem cells and / or cancer precursor cells are cells belonging to a side population cell population having tumorigenic activity.
The reducing agent according to any one of claims 1 to 23, wherein the cancer is a cancer caused by a hematopoietic tumor or a solid cancer.
The reducing agent according to any one of claims 1 to 23, wherein the cancer is lung cancer.
26. The reducing agent according to claim 25, wherein the lung cancer is small cell lung cancer.
26. The reducing agent according to claim 25, wherein the lung cancer is non-small cell lung cancer.
The reducing agent according to any one of claims 1 to 23, wherein the cancer is breast cancer.
The reducing agent according to any of claims 1 to 28, for simultaneous or sequential administration of at least one compound.
30. The reducing agent according to claim 29, wherein at least one compound is an antitumor agent.
A preventive agent for cancer recurrence and / or metastasis, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
Heat shock protein 90 (Hsp90) family protein inhibitors are represented by formula (I)

(Wherein, n, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are as defined above) or a pharmacologically acceptable salt thereof Item 31 is a prophylactic agent.
Heat shock protein 90 (Hsp90) family protein inhibitors are represented by formula (II)

(Wherein n1, R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are as defined above) or a pharmacologically acceptable salt thereof Item 31 is a prophylactic agent.
The preventive agent according to any one of claims 31 to 33, wherein the cancer stem cells and / or cancer precursor cells are cells having tumorigenic activity.
Metastasis of cancer stem cells and / or cancer progenitor cells having tumorigenic activity, including a cell population present in a tumor primary lesion, a cell population present in a tumor recurrence lesion, and a micrometastasis composed of one or more cells Cell population present in tumor nest, cell population present in bone marrow, side population cell population, quiescent cell population, GO phase cell population, RNA content relative to average content in all cancer cell populations Low cell population, cell population with relatively low uptake of pyronin Y than the average uptake in all cancer cell populations, CD34 positive cell population, CD38 negative cell population, CD138 negative cell population, CD44 positive cell population, CD24 Cell population whose expression level is relatively lower than the average expression level in all cancer cell populations, epithelial-specific antigen (ESA) positive cell population, sphere cell population, CD24 positive cell population It belongs to at least one cell population selected from a CD166 positive cell population, an α2β1 integrin positive cell population, a CD133 positive cell population, a CD90 positive cell population, a CD55 positive cell population, and a cell population having high aldehyde dehydrogenase (Aldehyde Dehydrogenase: ALDH) activity The preventive agent according to any one of claims 31 to 33, which is a cell.
The preventive agent according to any one of claims 31 to 33, wherein the cancer stem cells and / or cancer precursor cells are cells belonging to a side population cell population having tumorigenic activity.
The preventive agent according to any one of claims 31 to 36, wherein the cancer is a cancer caused by a hematopoietic tumor or a solid cancer.
The preventive agent according to any of claims 31 to 36, wherein the cancer is lung cancer.
39. The preventive agent according to claim 38, wherein the lung cancer is small cell lung cancer.
The preventive agent according to claim 38, wherein the lung cancer is non-small cell lung cancer.
The preventive agent according to any of claims 31 to 36, wherein the cancer is breast cancer.
The preventive agent according to any one of claims 31 to 41, for simultaneous or sequential administration of at least one compound.
43. The preventive agent according to claim 42, wherein at least one compound is an antitumor agent.
An agent for reducing cancer stem cells and / or cancer precursor cells in a patient having cancer stem cells and / or cancer precursor cells, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
A preventive agent for cancer recurrence and / or metastasis in a patient having cancer stem cells and / or cancer precursor cells, comprising a heat shock protein 90 (Hsp90) family protein inhibitor as an active ingredient.
A method for reducing cancer stem cells and / or cancer progenitor cells, comprising a step of administering an effective amount of a heat shock protein 90 (Hsp90) family protein inhibitor.
A method for preventing cancer recurrence and / or metastasis, comprising a step of administering an effective amount of a heat shock protein 90 (Hsp90) family protein inhibitor.
A heat shock protein 90 (Hsp90) family protein inhibitor for use in reducing cancer stem cells and / or cancer precursor cells.
A heat shock protein 90 (Hsp90) family protein inhibitor for use in preventing cancer recurrence and / or metastasis.
Use of a heat shock protein 90 (Hsp90) family protein inhibitor for the production of an agent for reducing cancer stem cells and / or cancer precursor cells.
Use of a heat shock protein 90 (Hsp90) family protein inhibitor for the manufacture of an agent for preventing cancer recurrence and / or metastasis.