Method for treating cancer by combined use

TECHNICAL FIELD

The present invention relates to a medicament and a method for treating cancer comprising a compound having murine double minute 2 (MDM2) inhibiting activity and a compound having Fms-like tyrosine kinase 3 (FLT3) inhibiting activity in combination.

BACKGROUND ART

p53 is known as an important factor for inhibiting canceration of cells. p53 is a transcription factor that induces the expression of genes involved in the cell cycle and cellular apoptosis in response to various stresses. p53 is thought to inhibit canceration of cells by a transcription regulating function thereof. In fact, deletion or mutation of the p53 gene is observed in about half of human cancer cases.

Meanwhile, overexpression of murine double minute 2 (MDM2), a type of E3 ubiquitin ligase, is known as a factor for canceration of cells that are cancerated in spite of the presence of normal p53. MDM2 is a protein whose expression is induced by p53. MDM2 negatively regulates p53 by binding to the transcription activity domain of p53 to decrease the transcription activity of p53, exporting p53 out of the nucleus, and mediating degradation of p53 by acting as an ubiquitination ligase against p53. Therefore, it is thought that inactivation of functions of and degradation of p53 are promoted in cells in which MDM2 is overexpressed, resulting in canceration (Non Patent Document 1).

Paying attention to such functions of MDM2, many approaches have been attempted using substances that inhibit the suppression of p53 functions by MDM2 as candidate anti-tumor agents. Examples of MDM2 inhibitors targeting the MDM2-p53 binding site have been reported, which include spirooxindole derivatives (Patent Documents 1 to 15 and Non Patent Documents 1 to 3), indole derivatives (Patent Document 16), pyrrolidine-2-carboxamide derivatives (Patent Document 17), pyrrolidinone derivatives (Patent Document 18), isoindolinone derivatives (Patent Document 19 and Non Patent Document 4) and dispiropyrrolidine compounds (Patent Document 20).

FLT3 is a protein belonging to receptor tyrosine kinase class III together with KIT, FMS and PDGFR, etc., and is thought to be involved in the hematopoietic system (Non Patent Documents 5 to 8). Its structure has an extracellular region composed of five immunoglobulin-like domains, one juxtamembrane region (JM domain), two tyrosine kinase domains (TK1 and TK2) divided by a kinase insert domain (KI domain), and a C-terminal domain. FLT3 is highly expressed in brain, placenta, liver and hematopoietic stem cells (Non Patent Documents 6 to 9).

A ligand of FLT3 (FL) is expressed in stromal cells of bone marrow and stimulates stem cells, either alone or in collaboration with other cytokines (Non Patent Documents 10 to 13). The ligand-receptor interaction between FL and FLT3 is considered to have important functions in the hematopoietic system.

Meanwhile, high expression of FLT3 is observed in most cases in samples of acute myeloid leukemia (AML) or acute lymphatic leukemia (ALL) patients, and high expression of FLT3 is also seen in chronic myeloid leukemia (CML). It is also known that growth of AML cells is more remarkably enhanced than that of ALL cells by stimulation of FL (Non Patent Documents 14 to 18). The FLT3 gene is the gene that is most frequently mutated in acute myeloid leukemia (AML) cases, and either of internal tandem duplications (ITDs) in the juxtamembrane region (Non Patent Document 19) or a mutation in the FLT3 activation loop region (Non Patent Document 20) is confirmed in approximately 30% to 35% of patients. The mutation of FLT3-ITD or the activation loop region is associated with constitutive activation of tyrosine kinase activity.

N-(5-tert-Butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea (quizartinib) having FLT3 inhibiting activity is known to have anti-tumor activity. Treatment of various cancers using quizartinib has been proposed in documents. Various dosing regimens have been reported. See, for example, Patent Documents 21 to 23 (which are incorporated herein by reference in their entirety). Also, the effects of combined use of quizartinib and anthracycline, a topoisomerase inhibitor or a tumor cell metabolic antagonist have been reported (Patent Document 24).

As for the relation between an MDM2 inhibitor and a FLT3 inhibitor, it has been reported that administration of an MDM2 inhibitor is preferred for patients whose cells contain FLT3 having an activating mutation (Patent Document 25). This document also states that combined administration of a FLT3 inhibitor and an MDM2 inhibitor is preferred for patients whose cells contain FLT3 having an activating mutation, but it does not disclose specific effects of the combined use of specific drugs.

There are various reports on the effects of combined use of various MDM2 inhibitors and various anti-tumor agents (Patent Documents 26 to 29).

CITATION LIST

Patent Documents

Patent Document 1: WO2006/091646 Patent Document 2: WO2006/136606 Patent Document 3: WO2007/104664 Patent Document 4: WO2007/104714 Patent Document 5: WO2008/034736 Patent Document 6: WO2008/036168 Patent Document 7: WO2008/055812 Patent Document 8: WO2008/141917 Patent Document 9: WO2008/141975 Patent Document 10: WO2009/077357 Patent Document 11: WO2009/080488 Patent Document 12: WO2010/084097 Patent Document 13: WO2010/091979 Patent Document 14: WO2010/094622 Patent Document 15: WO2010/121995 Patent Document 16: WO2008/119741 Patent Document 17: WO2010/031713 Patent Document 18: WO2010/028862 Patent Document 19: WO2006/024837 Patent Document 20: WO2012/121361 Patent Document 21: U.S. Patent Application Publication US 2007/0232604 Patent Document 22: U.S. Patent Application Publication US 2009/0123418 Patent Document 23: U.S. Patent Application Publication US 2009/0131426 Patent Document 24: WO2010/111172 Patent Document 25: WO2011/127058 Patent Document 26: EP1712235 Patent Document 27: WO2007/115289 Patent Document 28: WO2013/139724 Patent Document 29: WO2014/107713

Non Patent Documents

Non Patent Document 1: J. Am. Chem. Soc., 2005, 127, 10130-10131 Non Patent Document 2: J. Med. Chem., 2006, 49, 3432-3435 Non Patent Document 3: J. Med. Chem., 2009, 52, 7970-7973 Non Patent Document 4: J. Med. Chem., 2006, 49, 6209-6221 Non Patent Document 5: Genomics, 1991, 19, 380-385 Non Patent Document 6: Oncogene, 1991, 6, 1641-1650 Non Patent Document 7: Cell, 1991, 65, 1143-1152 Non Patent Document 8: Blood, 1993, 82, 1110-1119 Non Patent Document 9: Blood, 1996, 87, 1317-1325 Non Patent Document 10: Nature, 1994, 368, 643-648 Non Patent Document 11: Blood, 1995, 86, 3413-3420 Non Patent Document 12: Blood, 1995, 85, 1762-1768 Non Patent Document 13: Leukemia, 1996, 10, 1012-1018 Non Patent Document 14: Blood, 1995, 86, 4105-4114 Non Patent Document 15: Leukemia, 1996, 10, 1584-1591 Non Patent Document 16: Blood, 1996, 88, 3987-3997 Non Patent Document 17: Blood, 1992, 80, 2584-2593 Non Patent Document 18: Leukemia, 1996, 10, 261-270 Non Patent Document 19: Leukemia, 1996, 10, 1911-1918 Non Patent Document 20: Blood, 2001, 97 2434-2439

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to provide a medicament and a method for treating cancer comprising a compound having MDM2 inhibiting activity and a compound having FLT3 inhibiting activity in combination.

Solution to the Problem

As a result of extensive studies, the present inventors have found that use of (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''- -chloro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-di- hydrodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide, which is a compound having MDM2 inhibiting activity, or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea, which is a compound having FLT3 inhibiting activity, or a pharmaceutically acceptable salt thereof in combination particularly produces an excellent anti-tumor effect while keeping adverse reaction (e.g. weight loss) low, and accomplished the present invention.

Specifically, the present invention relates to the following [1] to [21]:

[1] A medicament for cancer treatment comprising (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof which are administered in combination.

[2] A medicament according to claim 1, wherein the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof are separately contained as active ingredients in different formulations and administered at the same time or different times.

[3] A medicament according to claim 1, wherein the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof are contained in a single formulation.

[4] A medicament according to claim 1, wherein the medicament is a kit formulation comprising the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof.

[5] A method for treating cancer comprising administering (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof in combination.

[6] A medicament according to any one of claims 1 to 4, wherein the respective salts of the compounds are (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide p-toluenesulfonate and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride.

[7] A treatment method according to claim 5, wherein the respective salts of the compounds are (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide p-toluenesulfonate and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride.

[8] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is blood cancer (leukemia, lymphoma or multiple myeloma), brain tumor, head and neck cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, anus cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, gastrointestinal stromal tumor, lung cancer, liver cancer, mesothelioma, thyroid cancer, renal cancer, prostate cancer, neuroendocrine tumor, melanoma, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, osteosarcoma, soft tissue sarcoma, Kaposi's sarcoma, myosarcoma, renal cancer, bladder cancer or testicular cancer.

[9] A treatment method according to claim 5 or 7, wherein the cancer is blood cancer (leukemia, lymphoma or multiple myeloma), brain tumor, head and neck cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, anus cancer, gallbladder cancer, cholangiocarcinoma cancer, pancreatic cancer, gastrointestinal stromal tumor, lung cancer, liver cancer, mesothelioma, thyroid cancer, renal cancer, prostate cancer, neuroendocrine tumor, melanoma, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, osteosarcoma, soft tissue sarcoma, Kaposi's sarcoma, myosarcoma, renal cancer, bladder cancer or testicular cancer.

[10] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is leukemia.

[11] A treatment method according to claim 5 or 7, wherein the cancer is leukemia.

[12] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is leukemia having an activating mutation of FLT3.

[13] A treatment method according to claim 5 or 7, wherein the cancer is leukemia having an activating mutation of FLT3.

[14] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is acute myeloid leukemia (AML).

[15] A treatment method according to claim 5 or 7, wherein the cancer is acute myeloid leukemia (AML).

[16] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is acute myeloid leukemia (AML) having a FLT3-ITD mutation.

[17] A treatment method according to claim 5 or 7, wherein the cancer is acute myeloid leukemia (AML) having a FLT3-ITD mutation.

[18] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer has wild-type TP53.

[19] A treatment method according to claim 5 or 7, wherein the cancer is cancer having wild-type TP53.

[20] A medicament according to any one of claims 1 to 4 and 6, wherein the cancer is cancer confirmed to be MDM2 inhibitor-sensitive using a gene signature.

[21] A treatment method according to claim 5 or 7, wherein the cancer is confirmed to be MDM2 inhibitor-sensitive using a gene signature.

Advantageous Effects of Invention

The present invention is useful as a method for treating cancer and/or an anti-cancer agent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 is a diagram showing in vivo effects of combined use of (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide tosylate (Compound A) and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea (quizartinib) on subcutaneously transplanted tumors of human acute myeloid leukemia cell line MOLM-13 having a FLT3-ITD mutation and wild-type TP53 in mice, and body weight change caused by combined administration thereof. The symbol x depicts an untreated control group, the symbol open circle depicts 0.5 mg/kg quizartinib, the symbol filled circle depicts 1 mg/kg quizartinib, the symbol open triangle depicts 25 mg/kg Compound A, the symbol open square depicts 25 mg/kg Compound A+0.5 mg/kg quizartinib, and the symbol filled square depicts 25 mg/kg Compound A+1 mg/kg quizartinib. The horizontal axis shows the number of days after tumor inoculation. The vertical axis of the upper panel shows estimated tumor volume calculated from tumor size. The vertical axis of the lower panel shows body weight change % relative to body weight on the first day of administration. The symbol filled triangle on the horizontal axis depicts the administration day of each compound. The error bar represents SE for the upper panel and SD for the lower panel.

FIG. 1-2 is a diagram showing in vivo effects of combined use of (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide tosylate (Compound A) and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea (quizartinib) on subcutaneously transplanted tumors of human acute myeloid leukemia cell line MOLM-13 having a FLT3-ITD mutation and wild-type TP53 in mice, and body weight change caused by combined administration thereof. The symbol x depicts an untreated control group, the symbol open circle depicts 0.5 mg/kg quizartinib, the symbol filled circle depicts 1 mg/kg quizartinib, the symbol filled triangle depicts 50 mg/kg Compound A, the symbol open square depicts 50 mg/kg Compound A+0.5 mg/kg quizartinib, and the symbol filled square depicts 50 mg/kg Compound A+1 mg/kg quizartinib. The abscissa shows the number of days after tumor inoculation. The ordinate of the upper panel shows estimated tumor volume calculated from tumor size. The ordinate of the lower panel shows body weight change % relative to body weight on the first day of administration. The symbol filled triangle on the abscissa depicts the administration day of each compound. The error bar represents SE for the upper panel and SD for the lower panel.

DESCRIPTION OF EMBODIMENTS

In the present invention, the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide is a compound of Example 70 in WO2012/121361. This compound can be produced by a method described in WO2012/121361 (WO2012/121361 is incorporated herein by reference in its entirety).

In the present invention, the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea is also referred to as 1-(5-tert-butyl-1,2-oxazol-3-yl)-3-(4-{7-[2-(morpholin-4-yl)ethoxy]imidaz- o[2,1-b][1,3]benzothiazol-2-yl}phenyl)urea or also referred to as quizartinib or AC220. This compound is represented by the following formula:

##STR00001## This compound can be produced by a method described in WO2007/109120 (WO2007/109120 is incorporated herein by reference in its entirety).

In the present invention, the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea may be various pharmaceutically acceptable salts.

Examples of the salts can include: hydrohalides such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formic acid, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate and maleate; amino acid salts such as ornithine salt, glutamate and aspartate; alkali metal salts such as sodium salt, potassium salt and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt and tris(hydroxymethyl)aminomethane salt.

The salt of the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide is preferably p-toluenesulfonate. The salt of the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea is preferably hydrochloride, particularly, dihydrochloride.

In the present invention, the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or their pharmaceutically acceptable salts may each be present in a free or solvate form. The compound represented by general formula (1) of the present invention or the salt thereof may be present in a hydrate form, for example, by absorbing moisture in the air. The solvate is not particularly limited so long as it is pharmaceutically acceptable. Specifically, the solvate is preferably a hydrate, an ethanol solvate or the like. Moreover, the compound represented by general formula (1) may be in an N-oxide form when containing a nitrogen atom. These solvate and N-oxide forms are also included in the present invention.

The (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-ch- loro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihyd- rodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imid- azo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or their pharmaceutically acceptable salts may have stereoisomers depending on their structures. The compounds or the salts also encompass all these stereoisomers and mixtures of these stereoisomers in any ratio. The stereoisomers are as defined in 1996 IUPC, Pure and Applied Chemistry 68, 2193-2222. When the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or their pharmaceutically acceptable salts are each present as tautomers, these tautomers may be present in equilibrium or a certain form may be dominantly present. All these cases are included in the scope of the present invention. The tautomers refer to isomers resulting from the shift of a proton of one atom of the molecule to another atom.

The (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-ch- loro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihyd- rodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imid- azo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or their pharmaceutically acceptable salts may each be a "pharmaceutically acceptable prodrug compound" that is converted to the desired compound through enzymatic oxidation, reduction, hydrolysis or the like or through hydrolysis or the like induced by gastric acid or the like, due to a reaction induced by an enzyme, gastric acid or the like under physiological conditions in vivo.

Examples of the prodrug include compounds obtained by acylation, alkylation or phosphorylation.

Prodrugs of the compounds can be produced from the compound (1) according to a method known in the art. Moreover, prodrugs of the compounds also include those converted to the desired compounds under physiological conditions as described in "Development of Pharmaceutical Products", vol. 7, Molecule Design, p. 163-198, Hirokawa-Shoten Ltd. (1990).

In the present invention, the terms "tumor" and "cancer" are used interchangeably. Furthermore, in the present invention, tumor, malignant tumor, cancer, malignant neoplasm, carcinoma, sarcoma, and the like may be collectively referred to as "tumor" or "cancer".

In the present invention, "FLT3" means Fms-like tyrosine kinase 3 (FLT3) and has the same meaning as FLK2, STK1, CD135 and FLK-2. FLT3 also includes homologs derived from various animal species. Human FLT3 is a molecule registered in NCBI under RefSEQ: accession NM_004119.2 (protein: RefSeq NP_004110.2).

FLT3 mRNA has a sequence as given below. However, it should be understood that even FLT3 having no mutation may differ in sequence among individuals due to polymorphism, etc.

TABLE-US-00001 acctgcagcgcgaggcgcgccgctccaggcggcatcgcagggctgggcc ggcgcggcctggggaccccgggctccggaggccatgccggcgttggcgc gcgacggcggccagctgccgctgctcgttgttttttctgcaatgatatt tgggactattacaaatcaagatctgcctgtgatcaagtgtgttttaatc aatcataagaacaatgattcatcagtggggaagtcatcatcatatccca tggtatcagaatccccggaagacctcgggtgtgcgttgagaccccagag ctcagggacagtgtacgaagctgccgctgtggaagtggatgtatctgct tccatcacactgcaagtgctggtcgacgccccagggaacatttcctgtc tctgggtctttaagcacagctccctgaattgccagccacattttgattt acaaaacagaggagttgtttccatggtcattttgaaaatgacagaaacc caagctggagaatacctactttttattcagagtgaagctaccaattaca caatattgtttacagtgagtataagaaataccctgctttacacattaag aagaccttactttagaaaaatggaaaaccaggacgccctggtctgcata tctgagagcgttccagagccgatcgtggaatgggtgctttgcgattcac agggggaaagctgtaaagaagaaagtccagctgttgttaaaaaggagga aaaagtgcttcatgaattatttgggacggacataaggtgctgtgccaga aatgaactgggcagggaatgcaccaggctgttcacaatagatctaaatc aaactcctcagaccacattgccacaattatttcttaaagtaggggaacc cttatggataaggtgcaaagctgttcatgtgaaccatggattcgggctc acctgggaattagaaaacaaagcactcgaggagggcaactactttgaga tgagtacctattcaacaaacagaactatgatacggattctgtttgcttt tgtatcatcagtggcaagaaacgacaccggatactacacttgttcctct tcaaagcatcccagtcaatcagctttggttaccatcgtagaaaagggat ttataaatgctaccaattcaagtgaagattatgaaattgaccaatatga agagttttgtttttctgtcaggtttaaagcctacccacaaatcagatgt acgtggaccttctctcgaaaatcatttccttgtgagcaaaagggtcttg ataacggatacagcatatccaagttttgcaatcataagcaccagccagg agaatatatattccatgcagaaaatgatgatgcccaatttaccaaaatg ttcacgctgaatataagaaggaaacctcaagtgctcgcagaagcatcgg caagtcaggcgtcctgtttctcggatggatacccattaccatcttggac ctggaagaagtgttcagacaagtctcccaactgcacagaagagatcaca gaaggagtctggaatagaaaggctaacagaaaagtgtttggacagtggg tgtcgagcagtactctaaacatgagtgaagccataaaagggttcctggt caagtgctgtgcatacaattcccttggcacatcttgtgagacgatcctt ttaaactctccaggccccttccctttcatccaagacaacatctcattct atgcaacaattggtgtttgtctcctcttcattgtcgttttaaccctgct aatttgtcacaagtacaaaaagcaatttaggtatgaaagccagctacag atggtacaggtgaccggctcctcagataatgagtacttctacgttgatt tcagagaatatgaatatgatctcaaatgggagtttccaagagaaaattt agagtttgggaaggtactaggatcaggtgcttttggaaaagtgatgaac gcaacagcttatggaattagcaaaacaggagtctcaatccaggttgccg tcaaaatgctgaaagaaaaagcagacagctctgaaagagaggcactcat gtcagaactcaagatgatgacccagctgggaagccacgagaatattgtg aacctgctgggggcgtgcacactgtcaggaccaatttacttgatttttg aatactgttgctatggtgatcttctcaactatctaagaagtaaaagaga aaaatttcacaggacttggacagagattttcaaggaacacaatttcagt ttttaccccactttccaatcacatccaaattccagcatgcctggttcaa gagaagttcagatacacccggactcggatcaaatctcagggcttcatgg gaattcatttcactctgaagatgaaattgaatatgaaaaccaaaaaagg ctggaagaagaggaggacttgaatgtgcttacatttgaagatcttcttt gctttgcatatcaagttgccaaaggaatggaatttctggaatttaagtc gtgtgttcacagagacctggccgccaggaacgtgcttgtcacccacggg aaagtggtgaagatatgtgactttggattggctcgagatatcatgagtg attccaactatgttgtcaggggcaatgcccgtctgcctgtaaaatggat ggcccccgaaagcctgtttgaaggcatctacaccattaagagtgatgtc tggtcatatggaatattactgtgggaaatcttctcacttggtgtgaatc cttaccctggcattccggttgatgctaacttctacaaactgattcaaaa tggatttaaaatggatcagccattttatgctacagaagaaatatacatt ataatgcaatcctgctgggcttttgactcaaggaaacggccatccttcc ctaatttgacttcgtttttaggatgtcagctggcagatgcagaagaagc gatgtatcagaatgtggatggccgtgtttcggaatgtcctcacacctac caaaacaggcgacctttcagcagagagatggatttggggctactctctc cgcaggctcaggtcgaagattcgtagaggaacaatttagttttaaggac ttcatccctccacctatccctaacaggctgtagattaccaaaacaagat taatttcatcactaaaagaaaatctattatcaactgctgcttcaccaga cttttctctagaagctgtctgcgtttactcttgttttcaaagggacttt tgtaaaatcaaatcatcctgtcacaaggcaggaggagctgataatgaac tttattggagcattgatctgcatccaaggccttctcaggctggcttgag tgaattgtgtacctgaagtacagtatattcttgtaaatacataaaacaa aagcattttgctaaggagaagctaatatgattttttaagtctatgtttt aaaataatatgtaaatttttcagctatttagtgatatattttatgggtg ggaataaaatttctactacagaattgcccattattgaattatttacatg gtataattagggcaagtcttaactggagttcacgaaccccctgaaattg tgcacccatagccacctacacattccttccagagcacgtgtgcttttac cccaagatacaaggaatgtgtaggcagctatggttgtcacagcctaaga tttctgcaacaacaggggttgtattgggggaagtttataatgaataggt gttctaccataaagagtaatacatcacctagacactttggcggccttcc cagactcagggccagtcagaagtaacatggaggattagtattttcaata aagttactcttgtccccacaaaaaaa.

FLT3 protein has an amino acid sequence as described below. However, it should be understood that even FLT3 having no mutation may differ in sequence among individuals due to polymorphism, etc.

TABLE-US-00002 mpalardggqlpllvvfsamifgtitnqdlpvikcvlinhknndssvgk sssypmvsespedlgcalrpgssgtvyeaaavevdvsasitlqvlvdap gnisclwvfkhsslncqphfdlqnrgvvsmvilkmtetgageyllfigs eatnytilftvsirntllytlrrpyfrkmengdalvcisesvpepivew vlcdsggesckeespavvkkeekvlhelfgtdirccarnelgrectrlf tidlnqtpqttlpqlflkvgeplwirckavhvnhgfgltwelenkalee gnyfemstystnrtmirilfafvssvarndtgyytcssskhpsqsalvt ivekgfinatnssedyeidgyeefcfsvrfkaypgirctwtfsrksfpc eqkgldngysiskfcnhkhqpgeyifhaenddagftkmftlnirrkpqv laeasasgascfsdgyplpswtwkkcsdkspncteeitegvwnrkanrk vfgqwvssstlnmseaikgflvkccaynslgtscetillnspgpfpfiq dnisfyatigvcllfivvltllichkykkgfryesqlqmvqvtgssdne yfyvdfreyeydlkwefprenlefgkvlgsgafgkvmnataygisktgv siqvavkmlkekadsserealmselkmmtqlgshenivnllgactlsgp iylifeyccygdllnylrskrekfhrtwteifkehnfsfyptfqshpns smpgsrevgihpdsdgisglhgnsfhsedeieyengkrleeeedlnvlt fedllcfayqvakgmeflefkscvhrdlaarnvlvthgkvvkicdfgla rdimsdsnyvvrgnarlpvkwmapeslfegiytiksdvwsygillweif slgvnpypgipvdanfykligngfkmdgpfyateeiyiimqscwafdsr krpsfpnitsflgcgladaeeamyqnvdgrvsecphtygnrrpfsremd lgllspqaqvedS.

In the present invention, "activating mutation of FLT3" means a mutation that causes ligand-independent activation of FLT3. Examples thereof include, but are not particularly limited to, internal tandem duplications (ITDs) in the juxtamembrane region (JM region), and point mutations D835V, D835E, D835N, D835Y and D835H that occur in the activation loop region of FLT3. The FLT3-ITD mutation occurs mainly in exon 14 of the JM region and is also found in exon 15.

In the present invention, "wild-type TP53" means that a gene TP53 encoding p53 protein is a gene having a sequence registered in NCBI under RefSEQ: accession NM_000546 (protein: RefSeq NP_000537).

Wild-type TP53 mRNA has a sequence given below. However, it should be understood that even TP53 having no mutation may differ in sequence among individuals due to polymorphism, etc.

TABLE-US-00003 gatgggattggggttttcccctcccatgtgctcaagactggcgctaaaag ttttgagcttctcaaaagtctagagccaccgtccagggagcaggtagctg ctgggctccggggacactttgcgttcgggctgggagcgtgctttccacga cggtgacacgcttccctggattggcagccagactgccttccgggtcactg ccatggaggagccgcagtcagatcctagcgtcgagccccctctgagtcag gaaacattttcagacctatggaaactacttcctgaaaacaacgttctgtc ccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgata ttgaacaatggttcactgaagacccaggtccagatgaagctcccagaatg ccagaggctgctccccccgtggcccctgcaccagcagctcctacaccggc ggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttccc agaaaacctaccagggcagctacggtttccgtctgggcttcttgcattct gggacagccaagtctgtgacttgcacgtactcccctgccctcaacaagat gttttgccaactggccaagacctgccctgtgcagctgtgggttgattcca cacccccgcccggcacccgcgtccgcgccatggccatctacaagcagtca cagcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctc agatagcgatggtctggcccctcctcagcatcttatccgagtggaaggaa atttgcgtgtggagtatttggatgacagaaacacttttcgacatagtgtg gtggtgccctatgagccgcctgaggttggctctgactgtaccaccatcca ctacaactacatgtgtaacagttcctgcatgggcggcatgaaccggaggc ccatcctcaccatcatcacactggaagactccagtggtaatctactggga cggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcg cacagaggaagagaatctccgcaagaaaggggagcctcaccacgagctgc ccccagggagcactaagcgagcactgcccaacaacaccagctcctctccc cagccaaagaagaaaccactggatggagaatatttcacccttcagatccg tgggcgtgagcgcttcgagatgttccgagagctgaatgaggccttggaac tcaaggatgcccaggctgggaaggagccaggggggagcgggctcactcca gccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactc atgttcaagacagaagggcctgactcagactgacattctccacttcttgt tccccactgacagcctcccacccccatctctccctcccctgccattttgg gttttgggtctttgaacccttgcttgcaataggtgtgcgtcagaagcacc caggacttccatttgctttgtcccggggctccactgaacaagttggcctg cactggtgttttgttgtggggaggaggatggggagtaggacataccagct tagattttaaggtttttactgtgagggatgtttgggagatgtaagaaatg ttcttgcagttaagggttagtttacaatcagccacattctaggtaggggc ccacttcaccgtactaaccagggaagctgtccctcactgttgaattttct ctaacttcaaggcccatatctgtgaaatgctggcatttgcacctacctca cagagtgcattgtgagggttaatgaaataatgtacatctggccttgaaac caccttttattacatggggtctagaacttgacccccttgagggtgcttgt tccctctccctgttggtcggtgggttggtagtttctacagttgggcagct ggttaggtagagggagttgtcaagtctctgctggcccagccaaaccctgt ctgacaacctcttggtgaaccttagtacctaaaaggaaatctcaccccat cccacaccctggaggatttcatctcttgtatatgatgatctggatccacc aagacttgttttatgctcagggtcaatttcttttttcttttttttttttt tttttctttttctttgagactgggtctcgctttgttgcccaggctggagt ggagtggcgtgatcttggcttactgcagcctttgcctccccggctcgagc agtcctgcctcagcctccggagtagctgggaccacaggttcatgccacca tggccagccaacttttgcatgttttgtagagatggggtctcacagtgttg cccaggctggtctcaaactcctgggctcaggcgatccacctgtctcagcc tcccagagtgctgggattacaattgtgagccaccacgtccagctggaagg gtcaacatcttttacattctgcaagcacatctgcattttcaccccaccct tcccctccttctccctttttatatcccatttttatatcgatctcttattt tacaataaaactttgctgccacctgtgtgtctgaggggtg.

TP53 protein has an amino acid sequence as described below. However, it should be understood that even TP53 having no mutation may differ in sequence among individuals due to polymorphism, etc.

TABLE-US-00004 meepgsdpsvepplsgetfsdlwkllpennvlsplpsgamddlmlspddi egwftedpgpdeaprmpeaappvapapaaptpaapapapswplsssvpsg ktyggsygfrlgflhsgtaksvtctyspalnkmfcglaktcpvglwvdst pppgtrvramaiykgsghmtevvrrophhercsdsdglappghlirvegn lrveylddrntfrhsvvvpyeppevgsdcttihynymcnsscmggmnrrp iltiitledssgnllgrnsfevrvcacpgrdrrteeenlrkkgephhelp pgstkralpnntssspgpkkkpldgeyftlgirgrerfemfrelnealel kdagagkepggsrahsshlkskkggstsrhkklmfktegpdsd.

In the present invention, "gene signature" means a single gene or a gene group consisting of a plurality of genes, a plurality of genes whose expression pattern is characteristic of a biological phenotype or a medical condition, such as morbidity of a certain disease, response to a certain medicament, or prognosis of a certain disease.

In the present invention, "biological sample" refers to tissues, liquids or cells isolated from an individual, or a mixture thereof. Examples thereof can include, but are not limited to, tumor biopsy, spinal fluid, pleural fluid, intra-abdominal fluid, lymph, skin sections, blood, urine, feces, sputum, respiratory organ, intestinal tract, genitourinary tract, saliva, milk, digestive organ, and cells collected therefrom. Preferred examples of a "biological sample" can include a portion of test subject-derived resected tissues obtained during surgery performed for the purpose of treating a cancer disease, a portion of tissues collected by biopsy or the like from a test subject suspected of having a cancer disease, and cells derived from pleural fluid or intra-abdominal fluid.

The biological sample may be protein extracts or nucleic acid extracts prepared from tissues, liquids or cells isolated from an individual, or a mixture thereof, etc. The protein extracts or the nucleic acid extracts can be prepared by use of a protein preparation method or a nucleic acid preparation method known per se in the art.

One aspect of the present invention relates to a medicament for cancer treatment comprising (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof which are administered in combination.

In the present invention, a "medicament" comprising (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof "which are administered in combination" is a medicament based on the assumption that both the drugs are administered in combination.

In the present invention, the "administration in combination" of (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof means that both the drugs are incorporated into the body of a recipient in a given period. A formulation containing both the drugs in a single formulation may be administered, or the drugs may be prepared into separate formulations and separately administered. In the case of preparing separate formulations, the timing of their administration is not particularly limited. The separate formulations may be administered at the same time or may be administered at different times or on different days in a staggered manner. In the case of administering the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof at different times or on different days, the order of their administration is not particularly limited. Usually, these formulations are administered according to their respective administration methods. Therefore, these formulations may be administered in the same number of doses or may be administered in a different number of doses. Also, in the case of preparing separate formulations, the respective administration methods (administration routes) of the formulations may be the same as each other, or these formulations may be administered by different administration methods (administration routes). Both the drugs do not have to exist at the same time in the body and may be incorporated into the body over a given period (e.g., 1 month, preferably 1 week, more preferably a few days, even more preferably 1 day). One of the active ingredients may have disappeared from the body at the time of administration of the other active ingredient.

Examples of a dosage form of the medicament of the present invention include 1) administration of a single formulation comprising (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof, 2) concurrent administration through the same administration route of two formulations separately prepared from (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof, 3) administration in a staggered manner through the same administration route of two formulations separately prepared from (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof, 4) concurrent administration through different administration routes of two formulations separately prepared from (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof, and 5) administration in a staggered manner through different administration routes of two formulations separately prepared from (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-c- hloro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihy- drodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof.

In the present invention, the two different formulations may be in the form of a kit comprising these formulations.

A medicament according to the present invention can contain (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and/or N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and can be administered as various injections such as intravenous injection, intramuscular injection, and subcutaneous injection or by various methods such as oral administration or percutaneous administration. A pharmaceutically acceptable carrier means a pharmaceutically acceptable material that is involved in transport of the compound of the present invention or a composition containing the compound of the present invention (e.g., an excipient, a diluent, an additive and a solvent) from a given organ to another organ.

A formulation can be prepared by selecting a suitable formulation form (e.g., oral formulation or injection) depending on the administration method and using various methods conventionally used for preparing a formulation. Examples of oral formulations can include tablets, powders, granules, capsules, pills, lozenges, solutions, syrups, elixirs, emulsions and oily or aqueous suspensions. In oral administration, the free compound or a salt form may be used. An aqueous formulation can be prepared by forming an acid adduct with a pharmaceutically acceptable acid or by forming an alkali metal salt such as sodium. As an injection, a stabilizer, a preservative, a dissolving aid, and the like can be used in the formulation. After filling a solution that may contain these aids and the like in a vessel, a formulation for use may be prepared as a solid formulation by lyophilization or the like. Furthermore, one dose may be filled in one vessel, or two or more doses may be filled in a vessel.

Examples of solid formulations include tablets, powders, granules, capsules, pills and lozenges. These solid formulations may contain pharmaceutically acceptable additives together with a compound of the present invention. Examples of additives include fillers, extenders, binders, disintegrating agents, dissolution promoting agents, skin wetting agents and lubricants. These additives can be selected and mixed as required to prepare a formulation.

Examples of liquid formulations include solutions, syrups, elixirs, emulsions and suspensions. Examples of additives include suspending agents and emulsifiers. These additives can be selected and mixed as required to prepare a formulation.

Examples of pharmaceutical materials can include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, arginine and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate and sodium bisulfite; buffers such as phosphate, citrate or borate buffers, sodium bicarbonate and Tris-HCl solutions; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA); complexing agents such as caffeine, polyvinylpyrrolidine, .beta.-cyclodextrin and hydroxypropyl-.beta.-cyclodextrin; bulking agents such as glucose, mannose and dextrin; other carbohydrates such as monosaccharides and disaccharides; coloring agents; corrigents; diluents; emulsifiers; hydrophilic polymers such as polyvinylpyrrolidine; low-molecular-weight polypeptides; salt-forming counterions; antiseptics such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide; solvents such as glycerin, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; surfactants such as sorbitan ester, polysorbates such as polysorbate 20 and polysorbate 80, triton, tromethamine, lecithin and cholesterol; stability enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol and sorbitol; transport agents; excipients; and/or pharmaceutical additives. The amount of these pharmaceutical materials added is preferably 0.01 to 100 times, particularly, 0.1 to 10 times the weight of the drug. The recipe of a preferred pharmaceutical composition in a formulation can be appropriately determined by those skilled in the art according to an applicable disease, an applicable administration route, etc.

An excipient or a carrier in a pharmaceutical composition may be liquid or solid. Appropriate excipients or carriers may be other materials usually used in injectable water, physiological saline, artificial cerebrospinal fluid, and parenteral administration. Neutral physiological saline or physiological saline containing serum albumin may be used as a carrier. The pharmaceutical composition can contain a Tris buffer of pH 7.0 to 8.5, an acetate buffer of pH 4.0 to 5.5, or a citrate buffer of pH 3.0 to 6.2. These buffers can also contain sorbitol or other compounds.

Preferred examples of the formulation of the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof include formulations described in WO2014/055397 (WO2014/055397 is incorporated herein by reference in its entirety).

The medicament of the present invention can be used in cancer treatment of mammals, particularly, humans. The dose and the administration interval of the medicament of the present invention can be suitably selected depending on the site of the disease, the patient's height, body weight, sex, or medical history, according to a physician's discretion. When the medicament of the present invention is administered to a human, the dose range is approximately 0.01 to 500 mg/kg body weight, preferably, approximately 0.1 to 100 mg/kg body weight, per day with respect to one type of active ingredient. Preferably, the active ingredient of the present invention is administered to a human once a day, or the dose is divided two to four times, and administration is repeated at an appropriate interval. Furthermore, the daily dose may exceed the above-mentioned dose at a physician's discretion, if necessary.

For examples of the administration method of the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof, see a method described in WO2009/061446, a method described in WO2010/132787 and a method described in U.S. Pat. No. 8,357,690, all of which are incorporated herein by reference in their entirety. This active ingredient may be administered once a day for 1 week, 2 weeks, 3 weeks, 4 weeks or 5 weeks. Preferred examples thereof include a method of continuously administering 12 to 450 mg, for example, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 90 mg, 135 mg, 200 mg, 300 mg or 450 mg, of this agent for 28 days, a method of continuously administering the above-mentioned dose for 8 to 21 days together with an additional anticancer agent, and a method of continuously administering the above-mentioned dose for 4 to 17 days together with an additional anticancer agent.

The type of cancer to be treated is not particularly limited as long as the cancer is confirmed to be sensitive to treatment by combined use of the present invention. Examples thereof include blood cancer (leukemia, lymphoma or multiple myeloma), brain tumor, head and neck cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, anus cancer, gallbladder cancer, cholangiocarcinoma cancer, pancreatic cancer, gastrointestinal stromal tumor, lung cancer, liver cancer, mesothelioma, thyroid cancer, renal cancer, prostate cancer, neuroendocrine tumor, melanoma, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, osteosarcoma, soft tissue sarcoma, Kaposi's sarcoma, myosarcoma, renal cancer, bladder cancer and testicular cancer. Among them, leukemia, particularly, acute myeloid leukemia (AML) is preferred. Leukemia having an activating mutation of FLT3 is more preferred, and acute myeloid leukemia having a FLT3-ITD mutation is particularly preferred.

In the present invention, a method for detecting the "activating mutation of FLT3" includes a method of detecting a mutation on genomic DNA as well as, when the mutation on the genomic DNA is reflected to base change in a transcription product or amino acid change in a translation product, a method of detecting this change in the transcription product or translation product (i.e., indirect detection), and a method based on detection of phosphorylated FLT3 because activation of FLT3 involves a rise in phosphorylation level.

In a preferred embodiment, a method for detecting a mutation includes a method of directly determining the nucleotide sequence of a gene region in a test subject-derived biological sample to thereby detect a mutation. In the present invention, the "FLT3 gene region" means a given region on genomic DNA containing the FLT3 gene. The region also contains the expression control regions (e.g., a promoter region and an enhancer region) of the FLT3 gene, the 3'-terminal untranslated region of the FLT3 gene, and the like. A mutation in these regions can influence, for example, the transcription activity of the FLT3 gene.

In this method, first, a DNA sample is prepared from a test subject-derived biological sample. Examples of the DNA sample include a genomic DNA sample and a cDNA sample prepared from RNA by reverse transcription.

The method for extracting genomic DNA or RNA from the biological sample is not particularly limited, and an approach known in the art can be appropriately selected for use. Examples of the method for extracting genomic DNA include a SDS phenol method (method which involves denaturing proteins in tissues preserved in a urea-containing solution or ethanol, using a proteolytic enzyme (proteinase K), a surfactant (SDS) and phenol, and extracting DNA by precipitation from the tissues using ethanol), and DNA extraction methods using Clean Columns.RTM. (manufactured by Nexttec Biotechnologie GmbH), AquaPure.RTM. (manufactured by Bio-Rad Laboratories, Inc.), ZR Plant/Seed DNA Kit (manufactured by Zymo Research Corp.), Aqua Genomic Solution.RTM. (manufactured by MoBiTec GmbH), prepGEM.RTM. (manufactured by ZyGEM NZ Ltd.) or BuccalQuick.RTM. (manufactured by TrimGen Corp.). Examples of the method for extracting RNA include extraction methods using phenol and a chaotropic salt (more specifically, extraction methods using a commercially available kit such as TRIzol (manufactured by Invitrogen Corp.) or ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.)), and methods using other commercially available kits (RNAPrep Total RNA Extraction Kit (manufactured by Beckman Coulter, Inc.), RNeasy Mini (manufactured by Qiagen N.V.), RNA Extraction Kit (manufactured by Pharmacia Biotech Inc.), etc.). Examples of reverse transcriptase for use in the preparation of cDNA from the extracted RNA include, but are not particularly limited to, reverse transcriptase derived from retrovirus such as RAV (Rous associated virus) or AMV (avian myeloblastosis virus), and reverse transcriptase derived from mouse retrovirus such as MMLV (Moloney murine leukemia virus).

In this embodiment, DNA containing a mutation site in the FLT3 gene region is subsequently isolated, and the nucleotide sequence of the isolated DNA is determined. The isolation of the DNA can be performed by, for example, PCR using a pair of oligonucleotide primers designed to flank the mutation in the FLT3 gene region, and the genomic DNA or the RNA as a template. The determination of the nucleotide sequence of the isolated DNA can be performed by a method generally known to those skilled in the art, such as the Maxam-Gilbert method or Sanger method.

The determined nucleotide sequence of the DNA or the cDNA can be compared with a control (e.g., the nucleotide sequence of DNA or cDNA derived from non-cancer tissues of the same test subject) to thereby determine the presence or absence of the mutation in the FLT3 gene region in the cancer cells of the test subject.

The method for detecting a mutation in the FLT3 gene region can be performed by various methods capable of detecting a mutation, in addition to the method of directly determining the nucleotide sequence of DNA or cDNA.

For example, in one of the methods, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, a reporter fluorescent dye- and quencher fluorescent dye-labeled oligonucleotide probe having a nucleotide sequence complementary to a nucleotide sequence containing the mutation in the FLT3 gene region is prepared. Then, the oligonucleotide probe is hybridized to the DNA sample. Then, the nucleotide sequence containing the mutation in the FLT3 gene region is amplified using the DNA sample hybridized with the oligonucleotide probe as a template. Then, fluorescence emitted by the reporter fluorescent dye through the decomposition of the oligonucleotide probe associated with the amplification is detected. Subsequently, the detected fluorescence is compared with a control. Examples of such a method include a double die probe method, so-called TaqMan.RTM. probe method.

In an alternative method, a DNA or cDNA sample is prepared from the biological sample. Subsequently, the nucleotide sequence containing the mutation in the FLT3 gene region is amplified using the DNA sample as a template in a reaction system containing an intercalator that emits fluorescence upon insertion between two strands of DNA. Then, the temperature of the reaction system is changed, and variation in the intensity of the fluorescence emitted by the intercalator is detected. The detected variation in the intensity of the fluorescence caused by the change in the temperature is compared with a control. Examples of such a method include a high resolution melting (HRM) method.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Then, the amplified DNA is cleaved with restriction enzymes. Subsequently, the DNA fragments are separated according to their sizes. Subsequently, the detected sizes of the DNA fragments are compared with a control. Examples of such a method include a method using restriction fragment length polymorphism (RFLP) and PCR-RFLP.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Then, the amplified DNA is dissociated into single-stranded DNA. Subsequently, the single-stranded DNA thus obtained by dissociation is separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with a control. Examples of such a method include PCR-SSCP (single-strand conformation polymorphism).

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Then, the amplified DNA is separated on a gel in which the concentration of a DNA denaturant is gradually elevated. Subsequently, the mobility of the separated DNA on the gel is compared with a control. Examples of such a method include denaturant gradient gel electrophoresis (DGGE).

A further alternative method is a method using DNA containing a mutation site in the FLT3 gene region prepared from the biological sample, and a substrate with immobilized oligonucleotide probes hybridizing to the DNA. Examples of such a method include a DNA array method.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Also, an "oligonucleotide primer having a nucleotide sequence complementary to the base downstream by one base from the base at the mutation site in the FLT3 gene region, and a downstream nucleotide sequence thereof" is prepared. Subsequently, a ddNTP primer extension reaction is performed using the DNA as a template and the primer. Subsequently, the primer extension reaction product is applied to a mass spectrometer to conduct mass spectrometry. Subsequently, the genotype is determined from the mass spectrometry results. Then, the determined genotype is compared with a control. Examples of such a method include MALDI-TOF/MS.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, an oligonucleotide probe consisting of 5'--"a nucleotide sequence complementary to the base at the mutation site in the FLT3 gene region, and an upstream nucleotide sequence thereof"--"a nucleotide sequence hybridizing neither to the base downstream by one base from the mutation site in the FLT3 gene region, nor to a downstream nucleotide sequence thereof"--3' (flap) is prepared. Also, an "oligonucleotide probe having a nucleotide sequence complementary to the base at the mutation site in the FLT3 gene region, and a downstream nucleotide sequence thereof" is prepared. Subsequently, the prepared DNA is hybridized to these two types of oligonucleotide probes. Subsequently, the hybridized DNA is cleaved with a single-stranded DNA-cleaving enzyme to release the flap. Examples of the single-stranded DNA-cleaving enzyme include, but are not particularly limited to, cleavase. In this method, a fluorescent reporter- and fluorescent quencher-labeled oligonucleotide probe having a sequence complementary to the flap is then hybridized to the flap. Subsequently, the intensity of the generated fluorescence is measured. Then, the measured intensity of the fluorescence is compared with a control. Examples of such a method include an invader method.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Then, the amplified DNA is dissociated into single strands, and only one of the single strands of the dissociated DNA is separated. Subsequently, an extension reaction is performed one by one from a base close to the base at the mutation site in the FLT3 gene region. Pyrophosphoric acid generated during this reaction is enzymatically allowed to develop light. The intensity of the light is measured. The measured intensity of the fluorescence is compared with a control. Examples of such a method include pyrosequencing.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Subsequently, an "oligonucleotide primer having a nucleotide sequence complementary to the base downstream by one base from the base at the mutation site in the FLT3 gene region, and a downstream nucleotide sequence thereof" is prepared. Subsequently, a single-base extension reaction is performed using the amplified DNA as a template and the prepared primer in the presence of fluorescently labeled nucleotides. Then, the degree of polarization of fluorescence is measured. Subsequently, the measured degree of polarization of fluorescence is compared with a control. Examples of such a method include the AcycloPrime method.

In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the FLT3 gene region is amplified. Subsequently, an "oligonucleotide primer having a nucleotide sequence complementary to the base downstream by one base from the base at the mutation site in the FLT3 gene region, and a downstream nucleotide sequence thereof" is prepared. Subsequently, a single-base extension reaction is performed using the amplified DNA as a template and the prepared primer in the presence of fluorescently labeled nucleotides. Subsequently, the base species used in the single-base extension reaction are determined. Then, the determined base species are compared with a control. Examples of such a method include the SNuPE method.

Provided that the mutation results in an amino acid change (e.g., substitution, deletion or insertion) in the FLT3 protein, a sample prepared from the biological sample may be a protein. In such a case, a method using a molecule (e.g., an antibody) specifically binding to a site having the amino acid change ascribable to the mutation can be used for detecting the mutation.

Since the activating mutation of FLT3 elevates the phosphorylation level of FLT3, the activating mutation of FLT3 can also be detected by the quantification of phosphorylated FLT3. A phosphorylated protein measurement method known in the art can be used as a method for quantitatively measuring the phosphorylated FLT3 protein. For example, various methods using antibodies against the phosphorylated FLT3 protein can be utilized. Specific examples thereof can include Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

A humanized antibody, a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody or the like can be appropriately used as an antibody against the mutated FLT3 protein or the phosphorylated FLT3 protein as long as the antibody is directed to the mutated FLT3 protein or the phosphorylated FLT3 protein as an antigen and specifically binds to the antigen. The antibody may be a polyclonal antibody or may be a monoclonal antibody. A monoclonal antibody is preferred from the viewpoint that homogeneous antibodies can be stably produced. The polyclonal antibody and the monoclonal antibody can be prepared by methods well known to those skilled in the art. A desired antibody can also be selected, for use, from commercially available antibodies.

A hybridoma producing the monoclonal antibody can basically be prepared by use of a technique known in the art as follows: the antigen of interest or cells expressing the antigen of interest is used as a sensitizing antigen, and a desired animal is immunized with this sensitizing antigen according to a conventional immunization method. The obtained immunocytes are fused with known parent cells by a conventional cell fusion method. Then, cells producing the desired monoclonal antibody (hybridoma cells) can be selected by a conventional screening method. The preparation of the hybridoma can be carried out according to, for example, the method of Millstein ("Methods of Enzymology", 1981, Vol. 73, p. 3-46).

In this context, the phosphorylated FLT3 protein or a fragment thereof can be used as the antigen for preparing the monoclonal antibody. Those skilled in the art can easily obtain the phosphorylated FLT3 protein or the fragment thereof according to a method described in a book, for example, Sambrook ed., "Molecular Cloning: A Laboratory Manual", the 2nd edition, Vol. 1-3, Cold Spring Harbor Laboratory Press, NY, 1989.

The protein or the fragment thereof and the antibody may be immobilized on a support and used for quantifying the phosphorylated FLT3 protein. The support is not limited as long as the support permits immobilization of proteins. General examples thereof can include: inorganic materials such as glass plates, silicon wafers and resins; natural polymer materials including nitrocellulose; and synthetic polymer materials including nylon and polystyrene.

More specific examples of the method for detecting the activating mutation of FLT3 include a method for detecting a FLT3-ITD mutation described in WO9817808 and its corresponding U.S. Pat. No. 6,846,630 (WO9817808 and U.S. Pat. No. 6,846,630 are incorporated herein by reference in their entirety). This method can be performed by using a detection kit commercially available from Takara Bio Inc., etc.

A method of performing RT-PCR using mRNA obtained from the test subject-derived biological sample, followed by capillary electrophoresis can also be used as another similar method (Leukemia, 2005, 19, 1479-1482, which is incorporated herein by reference in its entirety).

Specific examples of the method for detecting the phosphorylated FLT3 protein include a method described in WO2010/054185 (which is incorporated herein by reference in its entirety).

From another viewpoint, cancer sensitive to an MDM2 inhibitor and that having wild-type TP53 are preferable as the types of the cancer to be treated.

Various approaches mentioned above as methods for confirming a mutation in FLT3 can be similarly utilized as methods for confirming TP53 to be wild-type. More specific examples thereof include a microarray method using a probe specific for a mutated DNA sequence (AmpliChip p53, Roche Molecular Systems, Inc., etc., http://www.ncbi.nlm.nih.gov/pubmed/21319261), PCR using a probe specific for a mutated DNA sequence (qBiomarker Somatic Mutation PCR Arrays, Qiagen N.V., etc.), a method of reading the p53 gene sequence using a Sanger sequencer (http://p53.iarc.fr/Download/TP53_DirectSequencing_IARC.pdf), and a method of reading the p53 gene sequence using a next-generation sequencer (TruSeq Amplicon--Cancer Panel, Illumina http://www.illuminakk.co.jp/products/truseq_amplicon_cancer_panel.ilmn, Oncomine.RTM. Cancer Research Panel, Life Technologies Corp., http://www.lifetechnologies.com/jp/ja/home/clinical/preclinical-companion- -diagnostic-development/oncomine-cancer-research-panel-workflow.html, etc.).

A method using a gene signature can also be preferably used as a method for predicting sensitivity to an MDM2 inhibitor. Examples of the gene signature for predicting sensitivity to an MDM2 inhibitor include, but are not particularly limited to, a gene group described in WO2014/020502 (WO2014/020502 is incorporated herein by reference in its entirety). More specifically, a gene group comprising at least one gene selected from the group consisting of MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RPM2B, SESN1, CCNG1, XPC, TNFSF10B and AEN (the gene group may comprise all of these genes) can be preferably used. Other examples thereof include a gene group described in WO2015/000945 (WO2014/000945 is incorporated herein by reference in its entirety). More specifically, a gene group comprising at least one gene selected from the group consisting of BAX, RPS27L, EDA2R, XPC, DDB2, FDXR, MDM2, CDKN1A, TRIAP1, BBC3, CCNG1, TNFRSF10B and CDKN2A (the gene group may comprise all of these genes) can be preferably used. The number of genes contained in the gene group is not limited. A sensitive signature that allows the cancer to be confirmed as sensitive to an MDM2 inhibitor when the gene contained in the gene signature is highly expressed can be preferably used.

The medicament according to the present invention may be used in combination with an additional anti-tumor agent. Examples thereof include anti-tumor antibiotics, anti-tumor plant constituents, BRMs (biological response modifiers), hormones, vitamins, anti-tumor antibodies, molecular target drugs, alkylating agents, metabolic antagonists and other anti-tumor agents.

More specifically, examples of alkylating agents include: alkylating agents such as nitrogen mustard, nitrogen mustard N-oxide, bendamustine and chlorambucil; aziridine alkylating agents such as carboquone and thiotepa; epoxide alkylating agents such as dibromomannitol and dibromodulcitol; nitrosourea alkylating agents such as carmustine, lomustine, semustine, nimustine hydrochloride, streptozocin, chlorozotocin and ranimustine; and busulfan, improsulfan tosylate, temozolomide and dacarbazine.

Various examples of metabolic antagonists include: purine metabolic antagonists such as 6-mercaptopurine, 6-thioguanine and thioinosine; pyrimidine metabolic antagonists such as fluorouracil, tegafur, tegafur-uracil, carmofur, doxifluridine, broxuridine, cytarabine and enocitabine; and folic acid metabolic antagonists such as methotrexate and trimetrexate.

Examples of anti-tumor antibiotics include: mitomycin C, bleomycin, peplomycin, daunorubicin, aclarubicin, doxorubicin, idarubicin, pirarubicin, THP-adriamycin, 4'-epidoxorubicin and epirubicin; and chromomycin A3 and actinomycin D.

Examples of anti-tumor plant constituents and their derivatives include: vinca alkaloids such as vindesine, vincristine and vinblastine; taxanes such as paclitaxel, docetaxel and cabazitaxel; and epipodophyllotoxins such as etoposide and teniposide.

Examples of BRMs include tumor necrosis factors and indomethacin.

Examples of hormones include hydrocortisone, dexamethasone, methylprednisolone, prednisolone, prasterone, betamethasone, triamcinolone, oxymetholone, nandrolone, metenolone, fosfestrol, ethinylestradiol, chlormadinone, mepitiostane and medroxyprogesterone.

Examples of vitamins include vitamin C and vitamin A.

Examples of anti-tumor antibodies and molecular target drugs include trastuzumab, rituximab, cetuximab, nimotuzumab, denosumab, bevacizumab, infliximab, ipilimumab, nivolumab, pembrolizumab, avelumab, pidilizumab, atezolizumab, ramucirumab, imatinib mesylate, dasatinib, gefitinib, erlotinib, osimertinib, sunitinib, lapatinib, dabrafenib, trametinib, cobimetinib, pazopanib, palbociclib, panobinostat, sorafenib, crizotinib, vemurafenib, ibrutinib, bortezomib, carfilzomib, ixazomib and gilteritinib.

Examples of other anti-tumor agents include cisplatin, carboplatin, oxaliplatin, tamoxifen, letrozole, anastrozole, exemestane, toremifene citrate, fulvestrant, bicalutamide, flutamide, mitotane, leuprorelin, goserelin acetate, camptothecin, ifosfamide, cyclophosphamide, melphalan, L-asparaginase, aceglatone, sizofiran, picibanil, procarbazine, pipobroman, neocarzinostatin, hydroxyurea, ubenimex, azacitidine, decitabine, thalidomide, lenalidomide, pomalidomide, eribulin, tretinoin and krestin.

Another aspect of the present invention relates to a method for predicting responsiveness to treatment of cancer with (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof in combination, comprising using a test subject-derived biological sample, detecting the presence or absence of an activating mutation of FLT3 contained in the biological sample, and confirming the test subject having the detected activating mutation of FLT3 to be responsive to the treatment of cancer with the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof in combination.

An alternative aspect of the present invention relates to a method for selecting a subject for treatment of cancer with (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or a pharmaceutically acceptable salt thereof and N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or a pharmaceutically acceptable salt thereof in combination, comprising using a test subject-derived biological sample, detecting the presence or absence of an activating mutation of FLT3 in the biological sample, and selecting the test subject having the detected activating mutation of FLT3 as the subject for the treatment of cancer with the (3'R,4'S,5'R)--N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6''-chloro- -4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodi- spiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide or the pharmaceutically acceptable salt thereof and the N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[- 2,1-b][1,3]benzothiazol-2-yl]phenyl}urea or the pharmaceutically acceptable salt thereof in combination.

The present invention further relates to a method for predicting responsiveness to treatment of cancer or a method for selecting a subject for treatment of cancer, wherein the activating mutation of FLT3 is a FLT3-ITD mutation.

A method for collecting the test subject-derived biological sample and a method for detecting the activating mutation of FLT3 or the FLT3-ITD mutation in the biological sample are as mentioned above.

EXAMPLES

Hereinafter, the present invention will be specifically explained with reference to the Examples given below. However, the present invention is not limited to these examples, and they should not be construed in any limitative way.

Test Example 1 Study on In Vivo Effect of Combined Use of Compound A and Quizartinib

Human acute myeloid leukemia cell line MOLM-13 cells having a FLT3-ITD mutation and wild-type TP53 were suspended to 5.times.10.sup.7 cells/mL using phosphate-buffered saline. 0.1 mL of the prepared cell suspension was subcutaneously transplanted to each NOD-SCID mouse (male, 5 to 7 weeks old). On 6 days after the tumor inoculation, after confirmation that the average tumor volume exceeded 100 mm.sup.3, the mice were randomized (N=6/group) on the basis of their tumor volume values. 25 mg/kg or 50 mg/kg Compound A or 0.5 mg/kg or 1 mg/kg quizartinib (LC Laboratories) was administered by oral gavage to the mice. For a combined use group, 25 mg/kg or 50 mg/kg Compound A and 0.5 mg/kg or 1 mg/kg quizartinib were orally administered sequentially by forced administration. The administration was performed once a day for 5 consecutive days (6 to 10 days after the tumor inoculation) from the date of randomization (6 days after the tumor inoculation), and after a 2-day drug holiday, performed once a day for 4 consecutive days (13 to 16 days after the tumor inoculation). The length (mm) and width (mm) of tumor were measured over time using an electronic digital caliper. Tumor growth inhibition % (TGI %) on the date of assessment (17 days after the tumor inoculation) calculated according to calculation formula (4) shown below was used in evaluation. Also, the body weights were measured over time using an automatic balance for small animals, and body weight change % was calculated according to calculation formula (5) shown below to assess the influence of drug administration on the body weights. In addition, the results of the last body weight measurement were used in dose calculation. TGI (%)=(1-A/B).times.100 (4)

A: Average tumor volume of the compound-administered group on the date of assessment (*)

B: Average tumor volume of the untreated control group on the date of assessment (*)

*: The tumor volume was calculated according to 1/2.times.[Major axis of tumor].times.[Minor axis of tumor].times.[Minor axis of tumor]. Body weight change (%)=Average body weight change % of the individuals Body weight change % of each individual=(1-BWn/BWs).times.100 (5)

BWn: Body weight on day n

BWs: Body weight on the start day of administration

The results are shown in FIG. 1 and Tables 1 to 3.

TABLE-US-00005 TABLE 1 Group TGI (%) Compound A 25 mg/kg 38 Compound A 50 mg/kg 82 Compound A 25 mg/kg + Quizartinib 0.5 mg/kg 57 Compound A 50 mg/kg + Quizartinib 0.5 mg/kg 85 Compound A 25 mg/kg + Quizartinib 1 mg/kg 85 Compound A 50 mg/kg + Quizartinib 1 mg/kg 97 Quizartinib 0.5 mg/kg 29 Quizartinib 1 mg/kg 61

TABLE-US-00006 TABLE 2 Estimated tumor volume (mm.sup.3) Days after tumor inoculation Group (N = 6) 6 8 10 13 15 17 1. Untreated average 163 302 625 1227 1667 2667 SE 12 39 64 136 152 195 2. DS-3032b 25 mg/kg average 169 266 425 812 983 1661 SE 14 24 41 78 63 104 3. DS-3032b 50 mg/kg average 162 195 210 325 375 467 SE 12 15 23 37 49 64 4. DS-3032b 25 mg/kg + Quizartinib 0.5 mg/kg average 169 168 220 673 753 1149 SE 14 10 21 50 66 83 5. DS-3032b 50 mg/kg + Quizartinib 0.5 mg/kg average 164 134 106 290 297 387 SE 13 12 11 37 47 81 6. DS-3032b 25 mg/kg + Quizartinib 1 mg/kg average 165 132 98 314 318 387 SE 12 12 11 45 40 43 7. DS-3032b 50 mg/kg + Quizartinib 1 mg/kg average 171 111 62 116 96 67 SE 15 8 7 18 17 14 8. Quizartinib 0.5 mg/kg average 167 239 477 897 1212 1893 SE 13 28 36 61 96 154 9. Quizartinib 1 mg/kg average 163 168 203 593 663 1039 SE 13 11 18 39 57 67

TABLE-US-00007 TABLE 3 Body weight change (%) Days after tumor inoculation Group (N = 6) 6 8 10 13 15 17 1. Untreated average 0.0 2.6 6.0 10.8 15.2 19.2 SD 0.0 1.2 1.3 1.0 2.0 2.8 2. DS-3032b 25 mg/kg average 0.0 2.1 3.1 6.4 8.0 10.6 SD 0.0 1.1 1.9 1.9 2.5 2.7 3. DS-3032b 50 mg/kg average 0.0 1.5 2.7 5.5 6.8 5.8 SD 0.0 1.4 2.3 2.5 3.5 3.4 4. DS-3032b 25 mg/kg + Quizartinib 0.5 mg/kg average 0.0 0.3 1.4 5.1 5.5 6.1 SD 0.0 2.1 2.9 3.1 4.1 4.2 5. DS-3032b 50 mg/kg + Quizartinib 0.5 mg/kg average 0.0 1.0 0.3 2.9 4.2 3.6 SD 0.0 2.0 3.1 3.8 4.6 4.2 6. DS-3032b 25 mg/kg + Quizartinib 1 mg/kg average 0.0 1.1 0.7 4.7 2.8 3.3 SD 0.0 2.4 3.7 2.8 4.1 4.5 7. DS-3032b 50 mg/kg + Quizartinib 1 mg/kg average 0.0 -0.6 -0.3 2.5 2.6 3.2 SD 0.0 2.6 4.8 3.3 5.3 5.1 8. Quizartinib 0.5 mg/kg average 0.0 4.2 4.2 8.5 9.6 10.7 SD 0.0 2.9 2.6 2.1 1.6 2.8 9. Quizartinib 1 mg/kg average 0.0 0.2 1.8 4.6 5.1 5.8 SD 0.0 1.7 1.9 3.2 3.4 3.0

Free Text of Sequence Listing SEQ ID NO: 1: FLT3 mRNA encoding FLT3 protein (SEQ ID NO: 2). SEQ ID NO: 2: Amino acid sequence of the FLT3 protein. SEQ ID NO: 3: TP53 mRNA encoding TP53 protein (SEQ ID NO: 4). SEQ ID NO: 4: Amino acid sequence of the TP53 protein.

SEQUENCE LISTINGS

1

413848DNAHomo sapiensmisc_featureFLT3 mRNA encoding FLT3 protein (SEQ ID NO.2)CDS(83)..(3064) 1acctgcagcg cgaggcgcgc cgctccaggc ggcatcgcag ggctgggccg gcgcggcctg 60gggaccccgg gctccggagg cc atg ccg gcg ttg gcg cgc gac ggc ggc cag 112 Met Pro Ala Leu Ala Arg Asp Gly Gly Gln 1 5 10ctg ccg ctg ctc gtt gtt ttt tct gca atg ata ttt ggg act att aca 160Leu Pro Leu Leu Val Val Phe Ser Ala Met Ile Phe Gly Thr Ile Thr 15 20 25aat caa gat ctg cct gtg atc aag tgt gtt tta atc aat cat aag aac 208Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn His Lys Asn 30 35 40aat gat tca tca gtg ggg aag tca tca tca tat ccc atg gta tca gaa 256Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser Glu 45 50 55tcc ccg gaa gac ctc ggg tgt gcg ttg aga ccc cag agc tca ggg aca 304Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr 60 65 70gtg tac gaa gct gcc gct gtg gaa gtg gat gta tct gct tcc atc aca 352Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile Thr75 80 85 90ctg caa gtg ctg gtc gac gcc cca ggg aac att tcc tgt ctc tgg gtc 400Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp Val 95 100 105ttt aag cac agc tcc ctg aat tgc cag cca cat ttt gat tta caa aac 448Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln Asn 110 115 120aga gga gtt gtt tcc atg gtc att ttg aaa atg aca gaa acc caa gct 496Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln Ala 125 130 135gga gaa tac cta ctt ttt att cag agt gaa gct acc aat tac aca ata 544Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile 140 145 150ttg ttt aca gtg agt ata aga aat acc ctg ctt tac aca tta aga aga 592Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg155 160 165 170cct tac ttt aga aaa atg gaa aac cag gac gcc ctg gtc tgc ata tct 640Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val Cys Ile Ser 175 180 185gag agc gtt cca gag ccg atc gtg gaa tgg gtg ctt tgc gat tca cag 688Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys Asp Ser Gln 190 195 200ggg gaa agc tgt aaa gaa gaa agt cca gct gtt gtt aaa aag gag gaa 736Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys Lys Glu Glu 205 210 215aaa gtg ctt cat gaa tta ttt ggg acg gac ata agg tgc tgt gcc aga 784Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg 220 225 230aat gaa ctg ggc agg gaa tgc acc agg ctg ttc aca ata gat cta aat 832Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn235 240 245 250caa act cct cag acc aca ttg cca caa tta ttt ctt aaa gta ggg gaa 880Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys Val Gly Glu 255 260 265ccc tta tgg ata agg tgc aaa gct gtt cat gtg aac cat gga ttc ggg 928Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His Gly Phe Gly 270 275 280ctc acc tgg gaa tta gaa aac aaa gca ctc gag gag ggc aac tac ttt 976Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe 285 290 295gag atg agt acc tat tca aca aac aga act atg ata cgg att ctg ttt 1024Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg Ile Leu Phe 300 305 310gct ttt gta tca tca gtg gca aga aac gac acc gga tac tac act tgt 1072Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys315 320 325 330tcc tct tca aag cat ccc agt caa tca gct ttg gtt acc atc gta gaa 1120Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Val Glu 335 340 345aag gga ttt ata aat gct acc aat tca agt gaa gat tat gaa att gac 1168Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp 350 355 360caa tat gaa gag ttt tgt ttt tct gtc agg ttt aaa gcc tac cca caa 1216Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln 365 370 375atc aga tgt acg tgg acc ttc tct cga aaa tca ttt cct tgt gag caa 1264Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln 380 385 390aag ggt ctt gat aac gga tac agc ata tcc aag ttt tgc aat cat aag 1312Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys Asn His Lys395 400 405 410cac cag cca gga gaa tat ata ttc cat gca gaa aat gat gat gcc caa 1360His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp Asp Ala Gln 415 420 425ttt acc aaa atg ttc acg ctg aat ata aga agg aaa cct caa gtg ctc 1408Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro Gln Val Leu 430 435 440gca gaa gca tcg gca agt cag gcg tcc tgt ttc tcg gat gga tac cca 1456Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro 445 450 455tta cca tct tgg acc tgg aag aag tgt tca gac aag tct ccc aac tgc 1504Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys 460 465 470aca gaa gag atc aca gaa gga gtc tgg aat aga aag gct aac aga aaa 1552Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala Asn Arg Lys475 480 485 490gtg ttt gga cag tgg gtg tcg agc agt act cta aac atg agt gaa gcc 1600Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu Ala 495 500 505ata aaa ggg ttc ctg gtc aag tgc tgt gca tac aat tcc ctt ggc aca 1648Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr 510 515 520tct tgt gag acg atc ctt tta aac tct cca ggc ccc ttc cct ttc atc 1696Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile 525 530 535caa gac aac atc tca ttc tat gca aca att ggt gtt tgt ctc ctc ttc 1744Gln Asp Asn Ile Ser Phe Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe 540 545 550att gtc gtt tta acc ctg cta att tgt cac aag tac aaa aag caa ttt 1792Ile Val Val Leu Thr Leu Leu Ile Cys His Lys Tyr Lys Lys Gln Phe555 560 565 570agg tat gaa agc cag cta cag atg gta cag gtg acc ggc tcc tca gat 1840Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser Ser Asp 575 580 585aat gag tac ttc tac gtt gat ttc aga gaa tat gaa tat gat ctc aaa 1888Asn Glu Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys 590 595 600tgg gag ttt cca aga gaa aat tta gag ttt ggg aag gta cta gga tca 1936Trp Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser 605 610 615ggt gct ttt gga aaa gtg atg aac gca aca gct tat gga att agc aaa 1984Gly Ala Phe Gly Lys Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys 620 625 630aca gga gtc tca atc cag gtt gcc gtc aaa atg ctg aaa gaa aaa gca 2032Thr Gly Val Ser Ile Gln Val Ala Val Lys Met Leu Lys Glu Lys Ala635 640 645 650gac agc tct gaa aga gag gca ctc atg tca gaa ctc aag atg atg acc 2080Asp Ser Ser Glu Arg Glu Ala Leu Met Ser Glu Leu Lys Met Met Thr 655 660 665cag ctg gga agc cac gag aat att gtg aac ctg ctg ggg gcg tgc aca 2128Gln Leu Gly Ser His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr 670 675 680ctg tca gga cca att tac ttg att ttt gaa tac tgt tgc tat ggt gat 2176Leu Ser Gly Pro Ile Tyr Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp 685 690 695ctt ctc aac tat cta aga agt aaa aga gaa aaa ttt cac agg act tgg 2224Leu Leu Asn Tyr Leu Arg Ser Lys Arg Glu Lys Phe His Arg Thr Trp 700 705 710aca gag att ttc aag gaa cac aat ttc agt ttt tac ccc act ttc caa 2272Thr Glu Ile Phe Lys Glu His Asn Phe Ser Phe Tyr Pro Thr Phe Gln715 720 725 730tca cat cca aat tcc agc atg cct ggt tca aga gaa gtt cag ata cac 2320Ser His Pro Asn Ser Ser Met Pro Gly Ser Arg Glu Val Gln Ile His 735 740 745ccg gac tcg gat caa atc tca ggg ctt cat ggg aat tca ttt cac tct 2368Pro Asp Ser Asp Gln Ile Ser Gly Leu His Gly Asn Ser Phe His Ser 750 755 760gaa gat gaa att gaa tat gaa aac caa aaa agg ctg gaa gaa gag gag 2416Glu Asp Glu Ile Glu Tyr Glu Asn Gln Lys Arg Leu Glu Glu Glu Glu 765 770 775gac ttg aat gtg ctt aca ttt gaa gat ctt ctt tgc ttt gca tat caa 2464Asp Leu Asn Val Leu Thr Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gln 780 785 790gtt gcc aaa gga atg gaa ttt ctg gaa ttt aag tcg tgt gtt cac aga 2512Val Ala Lys Gly Met Glu Phe Leu Glu Phe Lys Ser Cys Val His Arg795 800 805 810gac ctg gcc gcc agg aac gtg ctt gtc acc cac ggg aaa gtg gtg aag 2560Asp Leu Ala Ala Arg Asn Val Leu Val Thr His Gly Lys Val Val Lys 815 820 825ata tgt gac ttt gga ttg gct cga gat atc atg agt gat tcc aac tat 2608Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met Ser Asp Ser Asn Tyr 830 835 840gtt gtc agg ggc aat gcc cgt ctg cct gta aaa tgg atg gcc ccc gaa 2656Val Val Arg Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu 845 850 855agc ctg ttt gaa ggc atc tac acc att aag agt gat gtc tgg tca tat 2704Ser Leu Phe Glu Gly Ile Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr 860 865 870gga ata tta ctg tgg gaa atc ttc tca ctt ggt gtg aat cct tac cct 2752Gly Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro875 880 885 890ggc att ccg gtt gat gct aac ttc tac aaa ctg att caa aat gga ttt 2800Gly Ile Pro Val Asp Ala Asn Phe Tyr Lys Leu Ile Gln Asn Gly Phe 895 900 905aaa atg gat cag cca ttt tat gct aca gaa gaa ata tac att ata atg 2848Lys Met Asp Gln Pro Phe Tyr Ala Thr Glu Glu Ile Tyr Ile Ile Met 910 915 920caa tcc tgc tgg gct ttt gac tca agg aaa cgg cca tcc ttc cct aat 2896Gln Ser Cys Trp Ala Phe Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn 925 930 935ttg act tcg ttt tta gga tgt cag ctg gca gat gca gaa gaa gcg atg 2944Leu Thr Ser Phe Leu Gly Cys Gln Leu Ala Asp Ala Glu Glu Ala Met 940 945 950tat cag aat gtg gat ggc cgt gtt tcg gaa tgt cct cac acc tac caa 2992Tyr Gln Asn Val Asp Gly Arg Val Ser Glu Cys Pro His Thr Tyr Gln955 960 965 970aac agg cga cct ttc agc aga gag atg gat ttg ggg cta ctc tct ccg 3040Asn Arg Arg Pro Phe Ser Arg Glu Met Asp Leu Gly Leu Leu Ser Pro 975 980 985cag gct cag gtc gaa gat tcg tag aggaacaatt tagttttaag gacttcatcc 3094Gln Ala Gln Val Glu Asp Ser 990ctccacctat ccctaacagg ctgtagatta ccaaaacaag attaatttca tcactaaaag 3154aaaatctatt atcaactgct gcttcaccag acttttctct agaagctgtc tgcgtttact 3214cttgttttca aagggacttt tgtaaaatca aatcatcctg tcacaaggca ggaggagctg 3274ataatgaact ttattggagc attgatctgc atccaaggcc ttctcaggct ggcttgagtg 3334aattgtgtac ctgaagtaca gtatattctt gtaaatacat aaaacaaaag cattttgcta 3394aggagaagct aatatgattt tttaagtcta tgttttaaaa taatatgtaa atttttcagc 3454tatttagtga tatattttat gggtgggaat aaaatttcta ctacagaatt gcccattatt 3514gaattattta catggtataa ttagggcaag tcttaactgg agttcacgaa ccccctgaaa 3574ttgtgcaccc atagccacct acacattcct tccagagcac gtgtgctttt accccaagat 3634acaaggaatg tgtaggcagc tatggttgtc acagcctaag atttctgcaa caacaggggt 3694tgtattgggg gaagtttata atgaataggt gttctaccat aaagagtaat acatcaccta 3754gacactttgg cggccttccc agactcaggg ccagtcagaa gtaacatgga ggattagtat 3814tttcaataaa gttactcttg tccccacaaa aaaa 38482993PRTHomo sapiens 2Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195 200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu 595 600 605Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val 610 615 620Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln625 630 635 640Val Ala Val Lys Met Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu 645 650 655Ala Leu Met Ser Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu 660 665 670Asn Ile Val Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr 675 680 685Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg 690 695 700Ser Lys Arg Glu Lys Phe His Arg Thr Trp Thr Glu Ile Phe Lys Glu705

710 715 720His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Ser His Pro Asn Ser Ser 725 730 735Met Pro Gly Ser Arg Glu Val Gln Ile His Pro Asp Ser Asp Gln Ile 740 745 750Ser Gly Leu His Gly Asn Ser Phe His Ser Glu Asp Glu Ile Glu Tyr 755 760 765Glu Asn Gln Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr 770 775 780Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys Gly Met Glu785 790 795 800Phe Leu Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn 805 810 815Val Leu Val Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu 820 825 830Ala Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Asn Ala 835 840 845Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile 850 855 860Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu865 870 875 880Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala 885 890 895Asn Phe Tyr Lys Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pro Phe 900 905 910Tyr Ala Thr Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe 915 920 925Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly 930 935 940Cys Gln Leu Ala Asp Ala Glu Glu Ala Met Tyr Gln Asn Val Asp Gly945 950 955 960Arg Val Ser Glu Cys Pro His Thr Tyr Gln Asn Arg Arg Pro Phe Ser 965 970 975Arg Glu Met Asp Leu Gly Leu Leu Ser Pro Gln Ala Gln Val Glu Asp 980 985 990Ser32591DNAHomo sapiensmisc_featureTP53 mRNA encoding TP53 protein (SEQ ID NO.4)CDS(203)..(1384) 3gatgggattg gggttttccc ctcccatgtg ctcaagactg gcgctaaaag ttttgagctt 60ctcaaaagtc tagagccacc gtccagggag caggtagctg ctgggctccg gggacacttt 120gcgttcgggc tgggagcgtg ctttccacga cggtgacacg cttccctgga ttggcagcca 180gactgccttc cgggtcactg cc atg gag gag ccg cag tca gat cct agc gtc 232 Met Glu Glu Pro Gln Ser Asp Pro Ser Val 1 5 10gag ccc cct ctg agt cag gaa aca ttt tca gac cta tgg aaa cta ctt 280Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu 15 20 25cct gaa aac aac gtt ctg tcc ccc ttg ccg tcc caa gca atg gat gat 328Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp 30 35 40ttg atg ctg tcc ccg gac gat att gaa caa tgg ttc act gaa gac cca 376Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro 45 50 55ggt cca gat gaa gct ccc aga atg cca gag gct gct ccc ccc gtg gcc 424Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala 60 65 70cct gca cca gca gct cct aca ccg gcg gcc cct gca cca gcc ccc tcc 472Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser75 80 85 90tgg ccc ctg tca tct tct gtc cct tcc cag aaa acc tac cag ggc agc 520Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser 95 100 105tac ggt ttc cgt ctg ggc ttc ttg cat tct ggg aca gcc aag tct gtg 568Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val 110 115 120act tgc acg tac tcc cct gcc ctc aac aag atg ttt tgc caa ctg gcc 616Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala 125 130 135aag acc tgc cct gtg cag ctg tgg gtt gat tcc aca ccc ccg ccc ggc 664Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly 140 145 150acc cgc gtc cgc gcc atg gcc atc tac aag cag tca cag cac atg acg 712Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr155 160 165 170gag gtt gtg agg cgc tgc ccc cac cat gag cgc tgc tca gat agc gat 760Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp 175 180 185ggt ctg gcc cct cct cag cat ctt atc cga gtg gaa gga aat ttg cgt 808Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg 190 195 200gtg gag tat ttg gat gac aga aac act ttt cga cat agt gtg gtg gtg 856Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val 205 210 215ccc tat gag ccg cct gag gtt ggc tct gac tgt acc acc atc cac tac 904Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr 220 225 230aac tac atg tgt aac agt tcc tgc atg ggc ggc atg aac cgg agg ccc 952Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro235 240 245 250atc ctc acc atc atc aca ctg gaa gac tcc agt ggt aat cta ctg gga 1000Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly 255 260 265cgg aac agc ttt gag gtg cgt gtt tgt gcc tgt cct ggg aga gac cgg 1048Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg 270 275 280cgc aca gag gaa gag aat ctc cgc aag aaa ggg gag cct cac cac gag 1096Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu 285 290 295ctg ccc cca ggg agc act aag cga gca ctg ccc aac aac acc agc tcc 1144Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser 300 305 310tct ccc cag cca aag aag aaa cca ctg gat gga gaa tat ttc acc ctt 1192Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu315 320 325 330cag atc cgt ggg cgt gag cgc ttc gag atg ttc cga gag ctg aat gag 1240Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu 335 340 345gcc ttg gaa ctc aag gat gcc cag gct ggg aag gag cca ggg ggg agc 1288Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser 350 355 360agg gct cac tcc agc cac ctg aag tcc aaa aag ggt cag tct acc tcc 1336Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser 365 370 375cgc cat aaa aaa ctc atg ttc aag aca gaa ggg cct gac tca gac tga 1384Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp 380 385 390cattctccac ttcttgttcc ccactgacag cctcccaccc ccatctctcc ctcccctgcc 1444attttgggtt ttgggtcttt gaacccttgc ttgcaatagg tgtgcgtcag aagcacccag 1504gacttccatt tgctttgtcc cggggctcca ctgaacaagt tggcctgcac tggtgttttg 1564ttgtggggag gaggatgggg agtaggacat accagcttag attttaaggt ttttactgtg 1624agggatgttt gggagatgta agaaatgttc ttgcagttaa gggttagttt acaatcagcc 1684acattctagg taggggccca cttcaccgta ctaaccaggg aagctgtccc tcactgttga 1744attttctcta acttcaaggc ccatatctgt gaaatgctgg catttgcacc tacctcacag 1804agtgcattgt gagggttaat gaaataatgt acatctggcc ttgaaaccac cttttattac 1864atggggtcta gaacttgacc cccttgaggg tgcttgttcc ctctccctgt tggtcggtgg 1924gttggtagtt tctacagttg ggcagctggt taggtagagg gagttgtcaa gtctctgctg 1984gcccagccaa accctgtctg acaacctctt ggtgaacctt agtacctaaa aggaaatctc 2044accccatccc acaccctgga ggatttcatc tcttgtatat gatgatctgg atccaccaag 2104acttgtttta tgctcagggt caatttcttt tttctttttt tttttttttt ttctttttct 2164ttgagactgg gtctcgcttt gttgcccagg ctggagtgga gtggcgtgat cttggcttac 2224tgcagccttt gcctccccgg ctcgagcagt cctgcctcag cctccggagt agctgggacc 2284acaggttcat gccaccatgg ccagccaact tttgcatgtt ttgtagagat ggggtctcac 2344agtgttgccc aggctggtct caaactcctg ggctcaggcg atccacctgt ctcagcctcc 2404cagagtgctg ggattacaat tgtgagccac cacgtccagc tggaagggtc aacatctttt 2464acattctgca agcacatctg cattttcacc ccacccttcc cctccttctc cctttttata 2524tcccattttt atatcgatct cttattttac aataaaactt tgctgccacc tgtgtgtctg 2584aggggtg 25914393PRTHomo sapiens 4Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln1 5 10 15Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro65 70 75 80Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met145 150 155 160Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser225 230 235 240Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys305 310 315 320Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380Phe Lys Thr Glu Gly Pro Asp Ser Asp385 390