JP2008261764A - Measuring method of kinase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring a kinase inhibitor contained in a biological sample using the biological sample containing the kinase inhibitor and a cell for developing target kinase, an immunological measuring method for measuring the kinase inhibitor, and a reagent, kit, or the like for immunological measurement. <P>SOLUTION: This measuring method of the kinase inhibitor contained in the biological sample includes a process (a) of bringing the biological sample into contact with the cell for developing the kinase working as the target of the kinase inhibitor, and a process (b) of measuring the substrate of the kinase as the target existing inside the cell and/or the phosphorylation level of kinase on the downstream side of the kinase as the target. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring a kinase inhibitor contained in a biological sample using a biological sample and a cell expressing a kinase that is a target of the kinase inhibitor. The present invention also relates to a reagent or kit for measuring the kinase inhibitor, including cells that express the kinase.

Kinases are involved in various information transmissions in the living body, and have been attracting attention as a target for drug discovery, and kinase inhibitors are expected as therapeutic agents for various diseases.
Fms like tyrosine kinase 3 (hereinafter referred to as FLT3) is a receptor-type protein tyrosine kinase (PTK) belonging to the platelet-induced growth factor receptor (PDGFR) family, and is bound by the binding of its ligand FLT3 ligand. It is activated by being quantified and is a kinase that phosphorylates various proteins that are intracellular substrates, and is involved in cell proliferation and differentiation. In particular, FLT3 or FLK2 (Fetai liver kinase-2) is expressed in hematopoietic stem cells and is known to play an important role in their proliferation [Cell, 65, 1143 (1991)]. Recently, as a result of studies on leukemia patients, FLT3 activity without ligand binding due to a mutation in which a tyrosine residue repeat (Internal Tandem Duplication, ITD) is inserted in the FLT3 cell membrane region (Juxter membrane) [Leukemia, 11, 1447 (1997)]. In addition, it has been shown that FLT3 is activated by a mutation in which the amino acid sequence in the region near the cell membrane of FLT3 becomes longer or shorter [Blood, 96, 3907 (2000) ]]. In addition, it has been shown that FLT3 is constitutively activated by amino acid point mutations in the kinase region of FLT3 [Blood, 97, 2434 (2001)]. Furthermore, it has been shown that overexpression of FLT3 results in constitutive activation of FLT3 [Blood, 103, 1901 (2004)].

  As mentioned above, FLT3 mutations currently include repetitive tyrosine residues in the region near the cell membrane, changes in the length of the region near the cell membrane, amino acid point mutations in the kinase region of FLT3, FLT3 kinase Overexpression is known, and it is thought that FLT3 is constantly activated in about half of patients with acute myeloid leukemia. It is known that by introducing these mutant genes into a cell line dependent on cytokines, for example, 32D cells, proliferative ability independent of cytokines is obtained. Therefore, FLT3 inhibitory substances are considered useful as therapeutic agents for various cancers including leukemia. These constitutive activations based on FLT3 mutations are thought to cause infinite proliferation of cells by transmitting cell proliferation signals and to be an important cause of leukemia. On the other hand, phosphorylation of STAT5 has been reported as a signal transduction molecule common to the activation of FLT3. That is, it has been reported that STAT5 is constantly phosphorylated in the mutant FLT3 cell line [Blood, 105, 4792 (2005)].

It has been reported that addition of a FLT3 inhibitor to leukemia cells in which FLT3 is constitutively activated decreases intracellular FLT3 phosphorylation and STAT5 phosphorylation.
Bcr-Abelson kinase (Bcr-Abl) is a fusion protein produced by fusion of Abl kinase and BCR gene by Philadelphia chromosome (Ph chromosome) translocation. It is a chronic myeloid leukemia (CML) and acute lymphoblast. It is a molecule identified as a causative gene product in acute lymphoblastic leukemia (ALL) [Nature, 315, 550 (1985); 325, 631 (1987)]. Bcr-Abl is a tyrosine kinase that controls the canceration and infinite growth of Ph-positive CML and ALL cells. Imatinib, which selectively inhibits Abl kinase, has low toxicity and high clinical efficacy for CML and ALL patients. It has been clinically applied as a drug shown [New England Journal of Medicine, 345, 645 (2002)].

  In addition, in recent years, drug resistance to imatinib has occurred in CML and ALL treatment with imatinib. One of the causes of the resistance mechanism is a point mutation in the kinase domain of Bcr-Abl, which reduces the sensitivity to imatinib. [Nature Review Drug Discovery, Vol. 3, page 1001 (2004)]. Thus, inhibitors of Bcr-Abl that have point mutations that reduce the sensitivity of imatinib are useful against leukemia that has acquired resistance to imatinib. One of the most frequently detected Bcr-Abl point mutations in imatinib-resistant CML and ALL patients has been reported to be a gene mutation (T315I mutation) in which the threonine residue at position 315 is replaced with isoleucine [ Science, 293, 876 (2001)], an inhibitor against leukemia cells having the Abl T315I mutation is useful against leukemia that has acquired resistance to imatinib.

  Kinase insert domain-containing receptor (KDR) is one of the subtypes of Vascular endothelial growth factor receptor (VEGFR), a receptor-type protein tyrosine kinase. It plays an important role in differentiation [Nature Medicine, 7, 199 (2001); Blood, 99, 2179 (2002)]. Sunitinib, a drug that inhibits KDR and the like, has been shown to be effective in kidney cancer and has been approved as an anticancer drug.

  Fibroblast growth factor receptor (FGFR), a receptor-type protein tyrosine kinase, is activated by dimerization by the binding of its ligand, FGF, and has various intracellular substrates. It is an enzyme that phosphorylates proteins. Four subtypes of FGFR1 to FGFR4 are known for FGFR. These FGFRs play an important role in fibroblast cell proliferation and differentiation [Expert Opinion on Therapeutic Targets, Vol. 6, p. 469 (2002)] . In tumor tissue, fibroblasts surround tumor cells and are thought to play an important role in tumor growth [Cell, 100, 57 (2000)]. On the other hand, as a result of examination of specimens from multiple myeloma patients, it was found that FGFR3 gene overexpression was caused by chromosomal translocation in about 25% of multiple myeloma [Blood, Vol. 92 3025 (1998)]. In addition, FGF expression is high in bone marrow of patients with multiple myeloma, and it is considered that activation of FGFR signal occurs in multiple myeloma cells expressing FGFR3 [Blood, 101 Volume 2, p. 2775 (2003)]. Furthermore, active mutations of FGFR3 are known in multiple myeloma cell lines and patient specimens, and this constant activation causes cell proliferation signals, thereby causing infinite proliferation of cells and multiple It is thought to be an important cause of myeloma [Blood, 97, 729 (2001)]. In addition, FGF or FGFR overexpression and active mutations may cause many cancers other than multiple myeloma (eg, pituitary tumors, myeloproliferative diseases, kidney cancer, bladder cancer, colon cancer, head and neck cancer, skin cancer, Gastric cancer, non-Hodgkin lymphoma, brain tumor, breast cancer, ovarian cancer, etc.) [Expert Opinion on Therapeutic Targets, 6, 469 (2002); Nature, 411, 355 (2001)]. Therefore, FGFR inhibitors are thought to be useful as therapeutic agents for various cancers.

  Aurora is a serine / threonine kinase that is activated during cell division (G2 / M phase) and has been reported to be involved in centrosome duplication, chromosome segregation, cytokinesis, and the like. Aurora has three subtypes, A, B, and C. Among them, Aurora A is present on chromosome 20q13, which has been reported to be amplified in various cancers, breast cancer, colon cancer, and bladder Cancer, pancreatic cancer, gastric cancer, etc. are not only frequently overexpressed, but also have been reported to correlate with malignancy and prognosis [Trends in Cell Biology, 9, 454 (1999); British Journal of Cancer, 84, 824 (2001); Journal of National Cancer Institute 94, 1320 (2002)]. Aurora B has also been reported to be overexpressed in clinical specimens of breast and colon cancer with Aurora A [Oncogene, 14, 2195 (1997); EMBO Journal 17, p. 3052 (1998)]. Regarding Aurora C, the function has not been clarified yet, but its expression has been confirmed in cancer cell lines such as breast cancer [The Journal of Biological Chemistry] 274, 7334 (1999)]. Such abnormal mitotic kinases are considered to be one of the causes of chromosomal instability, a characteristic of many cancer cells. Aurora inhibitors are used to treat various cancers including colorectal cancer. It is considered useful as an agent.

  When kinase inhibitors are administered to patients, the protein binding rate of each drug and the plasma concentration in cancer patients differ greatly, and various metabolites are generated in vivo. It is a difficult situation to analyze the drug concentration in plasma sufficient to reduce phosphorylation of the kinase or downstream of its target kinase using the post-drug patient.

  An object of the present invention is to provide a method for measuring an active substance of a kinase inhibitor contained in a patient's living body using a biological sample derived from a patient administered with the kinase inhibitor and a cell expressing a target kinase. There is to do.

The present invention relates to the following (1) to (15).
(1) A method for measuring a kinase inhibitor contained in a biological sample, comprising the following steps a and b;
(A) contacting the biological sample with a cell expressing a kinase that is a target of a kinase inhibitor;
(B) A step of measuring a phosphorylation level of a target kinase present in the cell and / or a kinase downstream of the target kinase.

(2) The measuring method according to the above (1), wherein the kinase is tyrosine kinase or serine / threonine kinase.
(3) The measuring method according to (2), wherein the tyrosine kinase is a tyrosine kinase selected from the group consisting of hummus-like tyrosine kinase 3, mutant Abelson kinase, vascular endothelial growth factor receptor 2 and fibroblast growth factor receptor. .
(4) The measuring method according to the above (2), wherein the serine / threonine kinase is Aurora.
(5) The measurement method according to any one of (1) to (4), wherein the biological sample is blood, plasma, or serum.
(6) The measurement method according to any one of (1) to (5), wherein the biological sample is a biological sample of a patient administered with a kinase inhibitor.

(7) The measurement method according to any one of (1) to (6), wherein the cell that expresses the target kinase is a cancer cell.
(8) The measurement method according to (7), wherein the cancer cells are solid cancer cells or hematopoietic tumor cells.
(9) The measurement method according to (8), wherein the hematopoietic tumor cell is a leukemia cell.
(10) The measurement method according to any one of (1) to (6), wherein the cell that expresses the target kinase is a cancer cell derived from a patient administered with a kinase inhibitor.
(11) The target kinase substrate is a signal transduction molecule selected from the group consisting of signal transducer and activator of transcription 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), histone H3 and CRKL (1 ) To (10).

(12) The steps (1) to (1), wherein the step of measuring the phosphorylation level of a substrate of a target kinase existing inside the cell and / or a kinase downstream of the target kinase comprises an immunological measurement method. 11) The measuring method described in any one of the above.
(13) The measurement method according to (12), wherein the immunological measurement method is Western blotting or immune cell staining.
(14) The measurement reagent according to any one of (1) to (11) above, which comprises a cell expressing a target kinase.
(15) The measurement kit according to any one of (1) to (11), comprising a cell expressing a target kinase.

  According to the present invention, there is provided a method for measuring a kinase inhibitor contained in a patient biological sample using a biological sample derived from a patient administered with the kinase inhibitor and a cell expressing a kinase that is a target of the kinase inhibitor. Provided.

The method for measuring a kinase inhibitor in a biological sample of the present invention includes the following steps a, b and c.
Step a: Contacting the biological sample with a cell expressing the target kinase Step b: The target kinase substrate present in the cell and / or the phosphorylation level of the kinase downstream of the target kinase Measuring step Kinase is an enzyme that transfers a phosphate group from a molecule having a high-energy phosphate bond, such as ATP, to a substrate or target molecule. Protein kinase is an enzyme that transfers a phosphate group to a protein molecule. That's it.

  Although it does not restrict | limit especially as a kinase in this invention, Protein kinase is preferable. Examples of protein kinases include serine / threonine kinases and tyrosine kinases. Examples of tyrosine kinases include receptor-type tyrosine kinases and non-receptor-type tyrosine kinases, and any of them may be used. Specifically, epidermal growth factor receptor (EGFR) kinase, platelet derived growth factor receptor (PDGFR) kinase, insulin receptor and insulin-like growth factor (IGF) receptor kinase, stem cell factor (SCF) receptor kinase ( c-Kit), Janus kinase (JAK), ERBB / HER protein kinase, SRC kinase, FLT3 kinase, Abelson kinase (Abl), vascular endothelial growth factor receptor 2 (KDR), fibroblast growth factor receptor (FGFR) Etc. Examples of serine / threonine kinases include Aurora.

In the present invention, the kinase inhibitor may be any substance as long as it has kinase inhibitory activity. In addition to the kinase inhibitor administered to the living body, the kinase inhibitor is derived from the administered kinase inhibitor such as an active metabolite in vivo. Including substances having kinase inhibitory activity.
The kinase inhibitor used in the present invention is not particularly limited as long as it is a compound having kinase inhibitory activity, and examples thereof include compounds described in WO2005 / 012257, WO2005 / 012258, WO2005 / 095382, WO2005 / 095341, and the like. . More specifically, for example, the formula (I)

（式中、R¹は置換もしくは非置換のアリールまたは置換もしくは非置換の複素環基を表す）で表されるインダゾール誘導体またはその薬理学的に許容される塩、式（Ia）

Wherein R ¹ represents a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group, or a pharmacologically acceptable salt thereof, formula (Ia)

［式中、R²はCONR^4aR^4b（式中、R^4a及びR^4bは、同一または異なって水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のアリール、置換もしくは非置換のアラルキルまたは置換もしくは非置換の複素環基を表すか、またはR^4a及びR^4bが隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する）またはNR^5aR^5b（式中、R^5aは置換もしくは非置換の低級アルキルスルホニルまたは置換もしくは非置換のアリールスルホニルを表し、R^5bは水素原子または置換もしくは非置換の低級アルキルを表す）を表し、
R³は水素原子、ハロゲン、シアノ、ニトロ、ヒドロキシ、カルボキシ、低級アルコキシカルボニル、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルコキシ、置換もしくは非置換の低級アルカノイル、CONR^6aR^6b（式中、R^6a及びR^6bは、同一または異なって水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のアリール、置換もしくは非置換のアラルキルまたは置換もしくは非置換の複素環基を表すか、またはR^6a及びR^6bが隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する）またはNR^7aR^7b（式中、R^7a及びR^7bは、同一または異なって水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルカノイル、置換もしくは非置換のアロイル、置換もしくは非置換のヘテロアロイル、置換もしくは非置換のアラルキル、置換もしくは非置換の低級アルキルスルホニルまたは置換もしくは非置換のアリールスルホニルを表す）を表す］で表されるインダゾール誘導体またはその薬理学的に許容される塩、式（Ib）

[Wherein R ² is CONR ^4a R ^4b (wherein R ^4a and R ^4b are the same or different, a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl; Or a substituted or unsubstituted heterocyclic group, or R ^4a and R ^4b together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group) or NR ^5a R ^5b (wherein R ^5a represents substituted or unsubstituted lower alkylsulfonyl or substituted or unsubstituted arylsulfonyl, and R ^5b represents a hydrogen atom or substituted or unsubstituted lower alkyl).
R ³ is a hydrogen atom, halogen, cyano, nitro, hydroxy, carboxy, lower alkoxycarbonyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkanoyl, CONR ^6a R ^6b (formula R ^6a and R ^6b are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heterocyclic group, Or R ^6a and R ^6b together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group) or NR ^7a R ^7b (wherein R ^7a and R ^7b are the same or different and are hydrogen atoms; Substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted Teroaroyl, substituted or unsubstituted aralkyl, substituted or unsubstituted lower alkylsulfonyl or substituted or unsubstituted arylsulfonyl)] or an pharmacologically acceptable salt thereof, Ib)

［式中、R^8a、R^8b及びR^8cは同一または異なって、水素原子、ハロゲン、ニトロ、ニトロソ、カルボキシ、シアノ、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルカノイル、置換もしくは非置換の低級アルコキシカルボニル、置換もしくは非置換のアリール、NR^9aR^9b（式中、R^9a及びR^9bは同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換の低級アルコキシ、置換もしくは非置換の低級アルカノイル、置換もしくは非置換のアリール、置換もしくは非置換のアロイル、置換もしくは非置換の複素環基または置換もしくは非置換のヘテロアロイルを表すか、またはR^9a及びR^9bが隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する）またはOR¹⁰（式中、R¹⁰は水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のアリール、置換もしくは非置換のアロイルまたは置換もしくは非置換の複素環基を表す）を表す］で表されるインダゾール誘導体またはその薬理学的に許容される塩、式（Ic）

[In the formula, R ^8a , R ^8b and R ^8c are the same or different and each represents a hydrogen atom, halogen, nitro, nitroso, carboxy, cyano, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or non-substituted. Substituted lower alkoxycarbonyl, substituted or unsubstituted aryl, NR ^9a R ^9b (wherein R ^9a and R ^9b are the same or different and represent a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl Substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, substituted or unsubstituted aroyl, substituted or unsubstituted heterocyclic group or substituted or Represents unsubstituted heteroaroyl or R ^9a and R ^9b together with the adjacent nitrogen atom To form a substituted or unsubstituted heterocyclic group) or OR ¹⁰ (wherein R ¹⁰ is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aroyl or substituted) Or represents an unsubstituted heterocyclic group)] or an pharmacologically acceptable salt thereof, represented by formula (Ic)

｛式中、R¹¹は置換もしくは非置換の複素環基［該置換複素環基における置換基は、同一または異なって置換数1〜3の、オキソ、ホルミル、カルボキシ、低級アルコキシカルボニル、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルコキシ、CONR^12aR^12b（式中、R^12a及びR^12bは、同一または異なって水素原子または置換もしくは非置換の低級アルキルを表す）、NR^13aR^13b（式中、R^13a及びR^13bは、同一または異なって水素原子、低級アルカノイル、低級アルコキシカルボニル、アラルキル、置換もしくは非置換の低級アルキル、置換もしくは非置換のアリール、置換もしくは非置換のアロイルまたは置換もしくは非置換の複素環基を表す）または-O(CR^14aR^14b)_nO-（式中、R^14a及びR^14bは、同一または異なって水素原子または低級アルキルを表し、nは2または3を表し、末端の2つの酸素原子は置換複素環基における複素環基上の同一炭素原子に結合する）である］を表す｝で表されるインダゾール誘導体またはその薬理学的に許容される塩、式（II）

{In the formula, R ¹¹ represents a substituted or unsubstituted heterocyclic group [the substituents in the substituted heterocyclic group are the same or different and have 1 to 3 substituents such as oxo, formyl, carboxy, lower alkoxycarbonyl, substituted or non-substituted. Substituted lower alkyl, substituted or unsubstituted lower alkoxy, CONR ^12a R ^12b (wherein R ^12a and R ^12b are the same or different and each represents a hydrogen atom or substituted or unsubstituted lower alkyl), NR ^13a R ^13b Wherein R ^13a and R ^13b are the same or different and each represents a hydrogen atom, lower alkanoyl, lower alkoxycarbonyl, aralkyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aroyl or substituted or an unsubstituted heterocyclic group) or _{^{-O (CR 14a R 14b) n}} O- ( wherein, R ^14a and R ^14b is a hydrogen atom or lower same or different Indazole derivatives represented by the formula: wherein n represents 2 or 3, and the two oxygen atoms at the ends are bonded to the same carbon atom on the heterocyclic group in the substituted heterocyclic group) Pharmacologically acceptable salt, formula (II)

〔式中、-X-Y-Z-は-O-CR¹⁷=N-｛式中、R¹⁷は水素原子、ヒドロキシ、カルボキシ、低級アルキル、以下の置換基群Ａより選ばれる同一のもしくは異なる1つ〜4つの置換基で置換された低級アルキル［置換基群Ａ：ハロゲン、アミノ、アミノスルホニル、ニトロ、ヒドロキシ、メルカプト、シアノ、ホルミル、カルボキシ、カルバモイル、低級アルカノイルオキシ、低級アルカノイルアミノ、モノまたはジ(低級アルキル)アミノカルボニル、低級アルコキシカルボニル、モノまたはジ低級アルキルアミノ、N-アリール-N-低級アルキルアミノ、低級アルキルスルホニル、低級アルキルスルフィニル、モノまたはジ(低級アルキルスルホニル)アミノ、モノまたはジ(アリールスルホニル)アミノ、トリ低級アルキルシリル、低級アルキルチオ、芳香族複素環アルキルチオ、低級アルカノイル、以下の置換基群ａより選ばれる同一のまたは異なる1つ〜3つの置換基で置換された低級アルカノイル（置換基群ａ：ハロゲン及びヒドロキシ）、低級アルコキシ、前記置換基群ａより選ばれる同一のまたは異なる1つ〜3つの置換基で置換された低級アルコキシ、アリールオキシ、前記置換基群ａより選ばれる同一のまたは異なる1つ〜3つの置換基で置換されたアリールオキシ、アラルキルオキシ及び前記置換基群ａより選ばれる同一のまたは異なる1つ〜3つの置換基で置換されたアラルキルオキシ；なお該置換された低級アルキルが置換メチル、置換エチルまたは置換プロピルであるときは、その置換基は-NR^18aR^18b（式中、R^18a及びR^18bは同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級シクロアルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換のアラルキル、置換もしくは非置換の芳香族複素環アルキル、置換もしくは非置換の脂環式複素環アルキル、置換もしくは非置換のアリール、置換もしくは非置換の芳香族複素環基または置換もしくは非置換の脂環式複素環基を表す）であってもよい］、低級シクロアルキル、前記置換基群Ａより選ばれる同一のもしくは異なる1つ〜4つの置換基で置換された低級シクロアルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換のアラルキル、置換もしくは非置換の芳香族複素環アルキル、置換もしくは非置換の脂環式複素環アルキル、置換もしくは非置換のアリール、置換もしくは非置換の芳香族複素環基、置換もしくは非置換の脂環式複素環基、置換もしくは非置換の低級アルコキシ、置換もしくは非置換の低級アルコキシカルボニル、置換もしくは非置換の低級アルキルチオ、置換もしくは非置換の低級アルカノイルまたは-C(=O)NR^19aR^19b（式中、R^19a及びR^19bは同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級シクロアルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換のアラルキル、置換もしくは非置換の芳香族複素環アルキル、置換もしくは非置換の脂環式複素環アルキル、置換もしくは非置換のアリール、置換もしくは非置換の芳香族複素環基または置換もしくは非置換の脂環式複素環基を表すか、またはR^19a及びR^19bが隣接する窒素原子と一緒になって置換もしくは非置換の脂環式複素環基を形成する）を表す｝、-N=CR^17a-O-（式中、R^17aは前記R¹⁷と同義である）、-O-N=CR^17b-（式中、R^17bは前記R¹⁷と同義である）、-O-C(=O)-NR²⁰-（式中、R²⁰は水素原子、低級アルキル、前記置換基群Ａより選ばれる同一のもしくは異なる1つ〜4つの置換基で置換された低級アルキル、低級シクロアルキル、前記置換基群Ａより選ばれる同一のもしくは異なる1つ〜4つの置換基で置換された低級シクロアルキルまたは置換もしくは非置換の脂環式複素環アルキルを表す）、-N=N-NR²¹-（式中、R²¹は置換もしくは非置換の低級アルキル、置換もしくは非置換の低級シクロアルキルまたは置換もしくは非置換の脂環式複素環アルキルを表す）または-NR^21a-N=N-（式中、R^21aは前記R²¹と同義である）を表し、

[Wherein, -XYZ- represents —O—CR ¹⁷ ═N— {wherein R ¹⁷ represents a hydrogen atom, hydroxy, carboxy, lower alkyl, the same or different one selected from the following substituent group A to 4 Lower alkyl substituted with one substituent [Substituent group A: halogen, amino, aminosulfonyl, nitro, hydroxy, mercapto, cyano, formyl, carboxy, carbamoyl, lower alkanoyloxy, lower alkanoylamino, mono or di (lower alkyl ) Aminocarbonyl, lower alkoxycarbonyl, mono- or di-lower alkylamino, N-aryl-N-lower alkylamino, lower alkylsulfonyl, lower alkylsulfinyl, mono or di (lower alkylsulfonyl) amino, mono or di (arylsulfonyl) Amino, tri-lower alkylsilyl, lower alkylthio, aromatic heterocyclic al Kirthio, lower alkanoyl, lower alkanoyl substituted with the same or different 1 to 3 substituents selected from the following substituent group a (substituent group a: halogen and hydroxy), lower alkoxy, the aforementioned substituent group a Lower alkoxy substituted with the same or different 1 to 3 substituents selected from aryloxy, aryloxy substituted with the same or different 1 to 3 substituents selected from the substituent group a, Aralkyloxy substituted with the same or different 1 to 3 substituents selected from aralkyloxy and the substituent group a; when the substituted lower alkyl is substituted methyl, substituted ethyl or substituted propyl, during the substituent -NR ^18a R ^18b (wherein, R ^18a and R ^18b may be the same or different, a hydrogen atom, a substituted or unsubstituted lower alkyl, location Or unsubstituted lower cycloalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic heterocyclic alkyl, substituted or unsubstituted alicyclic A heterocyclic alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aromatic heterocyclic group or a substituted or unsubstituted alicyclic heterocyclic group)], a lower cycloalkyl, and the above substituent Lower cycloalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted, substituted with the same or different 1 to 4 substituents selected from group A Substituted aromatic heterocyclic alkyl, substituted or unsubstituted alicyclic heterocyclic alkyl, substituted or Unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted alicyclic heterocyclic group, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted Lower alkylthio, substituted or unsubstituted lower alkanoyl or —C (═O) NR ^19a R ^19b (wherein R ^19a and R ^19b are the same or different and represent a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted Substituted lower cycloalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic heterocyclic alkyl, substituted or unsubstituted alicyclic heterocyclic ring Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic group or substituted or unsubstituted Or R ^19a and R ^19b together with the adjacent nitrogen atom form a substituted or unsubstituted alicyclic heterocyclic group}, —N═CR ^17a -O- (wherein R ^17a has the same meaning as R ¹⁷ above), -ON = CR ^17b- (wherein R ^17b has the same meaning as R ¹⁷ above), -OC (= O) -NR ^20- (wherein R ²⁰ is a hydrogen atom, lower alkyl, lower alkyl substituted with the same or different 1 to 4 substituents selected from substituent group A, lower cycloalkyl, substituent group A represent a more same or different one to four of the lower cycloalkyl substituted with a substituent or a substituted or unsubstituted aliphatic heterocycle-alkyl is selected), - N = N-NR 21 - ( wherein, R ²¹ represents a substituted or unsubstituted lower alkyl, substituted or unsubstituted lower cycloalkyl or substituted or unsubstituted aliphatic heterocycle-alkyl Or -NR ^21a -N = N- (wherein, R ^21a is a is same meaning as the R ²¹⁾ represent,

R ¹⁵ represents —NR ^22a R ^22b (wherein R ^22a and R ^22b are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower cycloalkyl, substituted or unsubstituted lower alkenyl; Substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic heterocyclic alkyl, substituted or unsubstituted alicyclic heterocyclic alkyl, substituted or unsubstituted monocyclic aryl, substituted Or an unsubstituted monocyclic aromatic heterocyclic group or a substituted or unsubstituted alicyclic heterocyclic group, or R ^22a and R ^22b together with the adjacent nitrogen atom are substituted or unsubstituted A cyclic heterocyclic group, provided that when one of R ^22a or R ^22b is a hydrogen atom, the other of R ^22a or R ^22b is substituted or unsubstituted pyrazol-3-yl and substituted or unsubstituted; Is not unsubstituted 1,2,4-triazol-3-yl) or -OR ²³ (wherein R ²³ is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower cycloalkyl, substituted Or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic heterocyclic alkyl, substituted or unsubstituted alicyclic heterocyclic alkyl, substituted or unsubstituted Represents an aryl, a substituted or unsubstituted aromatic heterocyclic group or a substituted or unsubstituted alicyclic heterocyclic group),

R ¹⁶ is —NR ^24a R ^24b {wherein R ^24a and R ^24b are the same or different and each represents a hydrogen atom, lower alkyl, or the same or different one to four substituents selected from the following substituent group B: Substituted lower alkyl [substituent group B: halogen, amino, aminosulfonyl, nitro, hydroxy, mercapto, cyano, formyl, carboxy, carbamoyl, lower alkanoyloxy, lower alkanoylamino, mono or di (lower alkyl) aminocarbonyl, Lower alkoxycarbonyl, mono- or di-lower alkylamino, N-aryl-N-lower alkylamino, lower alkylsulfonyl, lower alkylsulfinyl, mono or di (lower alkylsulfonyl) amino, mono or di (arylsulfonyl) amino, tri-lower Alkylsilyl, lower alkylthio, aromatic heterocyclic alkyl Thio, lower alkanoyl, lower alkanoyl substituted with the same or different 1 to 3 substituents selected from the following substituent group a (substituent group a: halogen and hydroxy), lower alkoxy, the aforementioned substituent group a Lower alkoxy substituted with the same or different 1 to 3 substituents selected from aryloxy, aryloxy substituted with the same or different 1 to 3 substituents selected from the substituent group a, Aralkyloxy substituted with the same or different 1 to 3 substituents selected from aralkyloxy and the substituent group a], lower cycloalkyl, and the same or different 1 to 4 selected from the substituent group B Lower cycloalkyl substituted with one substituent, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted Together with unsubstituted aliphatic heterocycle-alkyl, substituted or unsubstituted monocyclic aryl or a substituted or unsubstituted alicyclic heterocyclic group, or R ^24a and the nitrogen atom to which R ^24b is adjacent Thus, a substituted or unsubstituted alicyclic heterocyclic group or a substituted or unsubstituted aromatic heterocyclic group is formed. However, R ^24a and R ^24b cannot simultaneously be hydrogen atoms}, —NR ²⁵ CR ^26a R ^26b —Ar {wherein R ²⁵ represents a hydrogen atom, lower alkyl or lower cycloalkyl, and R ^26a and R ^26b represent The same or different hydrogen atom, lower alkyl, lower alkyl substituted with the same or different 1 to 3 substituents selected from the following substituent group b (substituent group b: halogen, hydroxy and hydroxymethyl), Represents a lower cycloalkyl or a lower cycloalkyl substituted with the same or different 1 to 3 substituents selected from the above-mentioned substituent group b, and Ar represents an aryl, the same or different selected from the following substituent group C Aryl substituted with 1 to 3 substituents [Substituent group C: halogen, amino, nitro, hydroxy, mercapto, cyano, carboxy, aminosulfonyl, lower alkyl A lower alkyl substituted with the same or different 1 to 3 substituents selected from the substituent group b, and a same or different 1 to 3 substituents selected from the substituent group b. Lower cycloalkyl, lower alkoxy, lower alkylthio, mono or dilower alkylamino, lower alkanoylamino, mono or di (lower alkylsulfonyl) amino, lower alkoxycarbonylamino, alicyclic heterocyclic alkyloxy and alkylenedi substituted with Oxy], an aromatic heterocyclic group, or an aromatic heterocyclic group substituted with the same or different one to three substituents selected from the substituent group C} or —NR ²⁵ CR ^26a R ^26b CR ^27a R ^27b -Ar (wherein R ²⁵ , R ^26a , R ^26b and Ar are as defined above, and R ^27a and R ^27b are as defined above as R ^26a and R ^26b , respectively) Or a pharmacologically acceptable salt thereof, represented by formula (III)

〔式中、Wは-C(=O)-または-CHR³¹-（式中、R³¹は水素原子、ヒドロキシ、置換もしくは非置換の低級アルキルまたは置換もしくは非置換の低級アルコキシを表す）を表し、
R²⁸は

[Wherein W represents —C (═O) — or —CHR ³¹ — (wherein R ³¹ represents a hydrogen atom, hydroxy, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower alkoxy). ,
R ²⁸

｛式中、Ar³はアリール、以下の置換基群Aから選ばれる同一のもしくは異なる1つまたは2つの置換基で置換されたアリール、単環性芳香族複素環基または以下の置換基群Aから選ばれる同一のもしくは異なる1つまたは2つの置換基で置換された単環性芳香族複素環基を表す；置換基群C［ハロゲン、ニトロ、ヒドロキシ、シアノ、カルボキシ、低級アルコキシカルボニル、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルコキシ、置換もしくは非置換の低級アルカノイル、-CONR^32aR^32b（式中、R^32a及びR^32bは同一または異なって水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のアリールまたは置換もしくは非置換のアラルキルを表すか、またはR^32a及びR^32bが隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する）及び-NR^33aR^33b（式中、R^33a及びR^33bは同一または異なって水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルカノイル、置換もしくは非置換の低級アルキルスルホニル、置換もしくは非置換のアリール、置換もしくは非置換のアラルキル、置換もしくは非置換のアロイル、置換もしくは非置換のアリールスルホニルまたは置換もしくは非置換のヘテロアロイルを表す）］｝を表し、
R²⁹は水素原子または

{Wherein Ar ³ is aryl, aryl substituted with the same or different one or two substituents selected from the following substituent group A, monocyclic aromatic heterocyclic group or the following substituent group A Represents a monocyclic aromatic heterocyclic group substituted with the same or different one or two substituents selected from: Substituent group C [halogen, nitro, hydroxy, cyano, carboxy, lower alkoxycarbonyl, substituted or Unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkanoyl, -CONR ^32a R ^32b (wherein R ^32a and R ^32b are the same or different and represent a hydrogen atom, substituted or unsubstituted lower alkyl, substituted together with the nitrogen atom to which, or R ^32a and R ^{32 b} are adjacent a substituted or unsubstituted aryl or substituted or unsubstituted aralkyl or During substitution to form a heterocyclic group) and -NR ^33a R ^33b (wherein, R ^33a and R ^33b are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or Represents an unsubstituted lower alkylsulfonyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aroyl, substituted or unsubstituted arylsulfonyl or substituted or unsubstituted heteroaroyl))}
R ²⁹ is a hydrogen atom or

（式中、Ar⁴は前記Ar³と同義である）を表し、
R³⁰は水素原子、ハロゲン、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルカノイル、置換もしくは非置換のアリール、-NR^34aR^34b［式中、R^34a及びR^34bは同一または異なって水素原子、置換もしくは非置換の低級アルキルまたは-C(=O)-R³⁵（式中、R³⁵は水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルコキシまたは置換もしくは非置換のアラルキルを表す）を表す］または

(Wherein Ar ⁴ has the same meaning as Ar ³ ),
R ³⁰ is a hydrogen atom, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted aryl, —NR ^34a R ^34b [wherein R ^34a and R ^34b are the same or different; A hydrogen atom, substituted or unsubstituted lower alkyl or —C (═O) —R ³⁵ (wherein R ³⁵ is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted) Represents an aralkyl of)] or

（式中、Ar⁵は前記Ar³と同義である）を表す。但しR²⁹が水素原子であり、かつAr¹がアリール、2つの低級アルコキシで置換されたアリール、または1つの低級アルキルもしくは低級アルコキシのみで置換されたアリールである場合、R³⁰は水素原子ではない〕で表される含窒素複素環化合物またはその薬理学的に許容される塩等が挙げられる。
本発明のキナーゼ阻害物質の測定方法の対象となるキナーゼまたはその基質のリン酸化レベルは前記疾患のバイオマーカーとして用いることもできる。

(Wherein Ar ⁵ has the same meaning as Ar ³ ). However, when R ²⁹ is a hydrogen atom and Ar ¹ is aryl, aryl substituted with two lower alkoxys, or aryl substituted only with one lower alkyl or lower alkoxy, R ³⁰ is not a hydrogen atom ] The nitrogen-containing heterocyclic compound represented by this, or its pharmacologically acceptable salt etc. are mentioned.
The phosphorylation level of the kinase or its substrate, which is the target of the method for measuring a kinase inhibitor of the present invention, can also be used as a biomarker for the disease.

  The biological sample of the present invention is not particularly limited as long as it is a sample in a living body in which a kinase inhibitor is present or suspected to exist, or a processed product thereof, such as blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, etc. Is mentioned. In particular, a biological sample of a patient administered with a kinase inhibitor is preferable. A patient who has been administered a kinase inhibitor refers to a patient who has been administered a kinase inhibitor to treat various diseases. Specifically, cancer (for example, chronic myelogenous leukemia, acute myeloid leukemia, leukemia such as plasma cell leukemia, myeloma such as multiple myeloma, lymphoma, breast cancer, endometrial cancer, cervical cancer, prostate cancer , Bladder cancer, kidney cancer, stomach cancer, esophageal cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, oral and neck cancer, osteosarcoma, melanoma, cancer caused by brain tumor, etc.), allergic diseases (eg, asthma, Rhinitis, allergic conjunctivitis, atopic dermatitis, etc.), various inflammatory diseases, osteoarthritis, arthritis, osteoporosis, autoimmune disease, infection, sepsis, cachexia, cerebral infarction, Alzheimer's disease, chronic lung inflammation Disease, chronic obstructive pulmonary disease (COPD), reperfusion injury, thrombosis, glomerulonephritis, diabetes, anti-host graft rejection, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, Castleman disease, intraatrial myxoma, Examples include psoriasis, dermatitis, gout, adult respiratory distress syndrome (ARDS), arteriosclerosis, restenosis after percutaneous transcoronary dilatation (PTCA), pancreatitis, pain, rheumatism and the like.

  A cell that expresses a kinase that is a target of a kinase inhibitor (hereinafter referred to as a target kinase) may be any cell that expresses a target kinase. It may be a cell. For example, MOLM-13 (Deutsche Sammlung von Mikroorganismen und Zellkulturen), MV-4-11 (American Type Culture Collection), K562 (manufactured by American Type culture Collection), BV173 (manufactured by Deutsche Sammlung von Mikroorganismen und Zellkulturen), TCC-Y And leukemia cell lines such as TCC-Ysr and TCC-Ywr (WO2004 / 094628). Moreover, the cell derived from the patient who administered the kinase inhibitor can also be used. In addition, cells expressing mutant FLT3, cells expressing Abl, cells expressing Aurora, and the like can also be used.

The target kinase substrate present in the cell in the step b represents a substrate that exists in a cell that expresses the target kinase and phosphorylates when the target kinase is activated, for example, signal transducer and Examples include activator of transcription 1 (STAT1), STAT2, STAT3, STAT4, STAT5, STAT6, Histone H3, and CRKL.
In step b, the kinase downstream of the target kinase is a kinase present in the cell expressing the target kinase (hereinafter referred to as intracellular kinase), which is located downstream of the signal transduction pathway of the target kinase, This refers to a kinase in which phosphorylation occurs when activated by. The method for measuring the phosphorylation level of the target kinase substrate and / or the kinase downstream of the target kinase is not particularly limited as long as it can measure the phosphorylation level of the target kinase and / or the kinase downstream of the kinase. However, an immunoassay method using an antibody or an antibody fragment that specifically recognizes a phosphorylated target kinase substrate and / or a phosphorylated intracellular kinase. Examples of the antibody include a polyclonal antibody, a monoclonal antibody, or an antibody fragment thereof.

Immunological measurement methods in the present invention include immunohistochemical staining methods such as fluorescent antibody method, immunoenzyme antibody method (ELISA), radioactive substance-labeled immunoantibody method (RIA), immunohistochemical staining method, immunocytochemical staining method ( ABC method, CSA method, etc.), dot blotting method, western blotting method, immunoprecipitation method, enzyme immunoassay method, sandwich ELISA method [Monoclonal antibody experiment manual (Kodansha Scientific, 1987), secondary biochemistry experiment course 5 Immunobiochemical research method (Tokyo Kagaku Dojin, 1986)].
The fluorescent antibody method can be performed using a method described in the literature [Monoclonal Antibodies: Principles and practice, Third edition (Academic Press, 1996), monoclonal antibody experiment manual (Kodansha Scientific, 1987)], etc. it can. Specifically, the biological sample is contacted with a cell expressing the target kinase, and after lysis, the antibody that specifically recognizes the phosphorylated target kinase substrate or intracellular kinase is reacted, This is a method in which an anti-immunoglobulin antibody or binding fragment labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC) or phycoerythrin is reacted, and then the fluorescent dye is measured with a flow cytometer.

In the immunoenzyme antibody method (ELISA), an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase after contacting a biological sample with a cell expressing the target kinase and lysing the cell. In this method, after the reaction, an anti-immunoglobulin antibody or a binding fragment to which an enzyme label such as peroxidase or biotin is applied is further reacted, and then the coloring dye is measured with an absorptiometer.
Radioactive substance-labeled immunoantibody method (RIA) specifically recognizes a phosphorylated target kinase substrate or intracellular kinase after contacting a biological sample with a cell expressing a target kinase and lysing the cell. In this method, after the antibody is reacted, an anti-immunoglobulin antibody or binding fragment to which radiation labeling has been applied is further reacted, and then measured with a scintillation counter or the like.

Immunohistochemical staining methods (ABC method, CSA method, etc.) such as immunocytostaining and immunohistochemical staining can be found in literature [Monoclonal Antibodies: Principles and practice, Third edition (Academic Press, 1996), Monoclonal Antibody Experiment Manual ( Kodansha Scientific, 1987)] and the like.
In the immune cell staining method, a biological sample is contacted with a cell expressing a target kinase, and the cell is lysed, followed by reacting an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase. This is a method of measuring using a flow cytometer after further reacting an anti-immunoglobulin antibody or binding fragment labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC), an enzyme such as peroxidase or biotin.
In the immunohistochemical staining method, a biological sample and a cell expressing a target kinase are contacted, and the cell is lysed, and then a phosphorylated target kinase substrate or an antibody that specifically recognizes an intracellular kinase is reacted. This is a method in which an anti-immunoglobulin antibody or binding fragment labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC), an enzyme such as peroxidase or biotin is further reacted, and then observed using a microscope.

In the dot blotting method, a biological sample is contacted with cells expressing a target kinase, the cells are lysed, the lysate is blotted onto a PVDF membrane or a nitrocellulose membrane, and the target kinase phosphorylated on the membrane After reacting with an antibody that specifically recognizes a substrate or intracellular kinase, react with an anti-mouse IgG antibody or binding fragment labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase or biotin, and then confirm the label. Is the method.
In Western blotting, a biological sample and cells expressing a target kinase are contacted, and the cells are lysed, and then the lysate is subjected to SDS-polyacrylamide gel electrophoresis (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). The gel is blotted on a PVDF membrane or a nitrocellulose membrane, and the membrane is reacted with an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase, and FITC and the like. This is a method for confirming the label after reacting with an anti-mouse IgG antibody or binding fragment labeled with an enzyme such as a fluorescent substance, peroxidase, biotin or the like.

In the immunoprecipitation method, a biological sample is contacted with a cell expressing a target kinase, and the cell is lysed and then reacted with an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase. Thereafter, a carrier having specific binding ability to immunoglobulin such as protein G-Sepharose is added to precipitate the antigen-antibody complex.
The sandwich ELISA method involves reacting the target antibody in the test solution bound by the antigen-antibody reaction and the first antibody against the target substance with the second antibody against the target substance simultaneously with or separately from the first antibody. And measuring the target substance in the test liquid. The two antibodies used recognize different epitopes. Specifically, an antibody that specifically recognizes a target kinase substrate or intracellular kinase is adsorbed on a plate in advance, a biological sample is brought into contact with cells expressing the target kinase, and the lysate obtained by dissolving the cells is added to the plate. After that, an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase is reacted. Then, after reacting an anti-immunoglobulin antibody or binding fragment labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC) or phycoerythrin, the fluorescent dye is measured with a flow cytometer. Using the antibody labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC) or phycoerythrin, an antibody that specifically recognizes a phosphorylated target kinase substrate or intracellular kinase that is a secondary antibody, and then Fluorescent dyes may be measured.

A specific measurement method using STAT5 as an example of the target kinase substrate is shown below.
Cells expressing the target kinase are centrifuged and washed. This is suspended in phosphate buffer, then dispensed into an Eppendorf tube at 3 × 10 ⁶ / tube, and centrifuged. After centrifugation, the supernatant is removed, and 500 μL of human plasma containing a kinase inhibitor is added and contacted in a carbon dioxide incubator set at 37 ° C. for 1 hour. The cells after contact are washed with a phosphate buffer, and then 2% formaldehyde aqueous solution is added, and the mixture is reacted at 37 ° C. for 10 minutes to be fixed. Then 100% methanol is added and allowed to react on ice for 30 minutes to penetrate.
Dissolve the fixed and permeated cells into a test tube, add Phamingen Stain Buffer (BSA), centrifuge and wash the cells. Then 1.0 mg / mL Alexa488-labeled anti-phospho-STAT5 antibody [anti-phospho-STAT5 Antibody KM3461 labeled with Alexa Flour488 (Molecular PROBES) labeled with Alexa488] or Alexa488 labeled Rat IgG [serum was collected from SD rat, and the IgG fraction obtained from the serum was collected with Alexa Flour488 labeled reagent (Molecular PROBES The product is labeled with Alexa488] (produced by the company) for 30 minutes on ice. After the reaction, the cells were washed by centrifuging with a phosphate buffer containing 0.2 W / V% bovine serum albumin and 0.09 W / V% sodium azide, suspended in phosphate buffer, and then flow cytometer (EPICS Measure red fluorescence of 10,000 cells at 488 nm excitation wavelength, 575 nm fluorescence wavelength using ELITE or Cytomics FC500, Beckman Coulter).

The measurement method of the present invention includes a method for detecting and quantifying a kinase inhibitor in a biological sample. Examples of the quantification method of the present invention include the following methods using the immunological measurement method described above.
Centrifuge the cells expressing the target kinase.
A body fluid is collected from a patient to which the kinase inhibitor is administered, and a cell expressing the target kinase is brought into contact with the body fluid. In order to create a standard curve, each concentration of kinase inhibitor is added to a cell line isolated from a human and cultured, and the cell line is contacted with a cell expressing the target kinase. After administration of each concentration of kinase inhibitor to a healthy person, a body fluid is collected, and cells expressing the target kinase are brought into contact with the body fluid.
After the cells after contact are lysed, an antibody reactive to the labeled target kinase substrate is reacted. After the reaction, the red fluorescence per 1 × 10 ⁴ cells is measured using a flow cytometer at an excitation wavelength of 488 nm and a fluorescence wavelength of 575 nm.
The standard curve can be represented by the value of red fluorescence and the concentration of kinase inhibitor.
Therefore, the concentration of the kinase inhibitor contained in the biological sample can be determined from the red fluorescence value obtained by the above measurement using a standard curve.

The kit of the present invention is composed of a combination of reagents. However, the composition or form is different as long as it contains a substance that is essentially the same as each component described below or a substance that is essentially the same as a part thereof. Is included in the kit of the present invention.
The reagent includes a cell for expressing the target kinase, a reagent for measuring the target kinase substrate and / or the phosphorylation level of the intracellular kinase, and a biological sample diluent, if necessary.
Examples of the diluted solution of the biological sample include an aqueous solution containing a stabilizer in a surfactant or a buffer. When whole blood is used as a sample, an aqueous solution is prepared in an isotonic solution with salts, sugars, buffers, etc. for the purpose of preventing changes in serum component concentration due to expansion and contraction of blood cells such as red blood cells. It is preferred that Although there is no restriction | limiting in particular as salts, For example, halogenated alkali metal salts, such as sodium chloride and potassium chloride, etc. are mentioned. The saccharide is not particularly limited, and examples thereof include sugar alcohols such as mannitol and sorbitol.

Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples.
In Example 1 below, the test compound was 4- {5- (piperazin-1-ylmethyl) [1,3,4] oxazol-2-yl} -4- (n-propylamino) -2- [2 -(Pyridin-4-yl) ethyl] pyrimidine (hereinafter referred to as Compound 1) was used. Compound 1 can be synthesized by the method described in WO2005 / 095382.

After compound 1 was administered to male Balb / c mice (Claire Japan), blood was collected from the animals. Blood samples were collected before administration of Compound 1, 4 hours, 8 hours and 24 hours after administration. The obtained blood was centrifuged at 1,500 g for 5 minutes to isolate plasma. The insoluble fraction of the obtained plasma was removed by centrifugation at 15,000 g for 5 minutes. Control plasma was prepared from mice not administered Compound 1 by the same procedure.
MOLM-13 cells are washed with phosphate buffer and dispensed into tubes (1.5 mL polypropylene tubes, Treff AG, catalog number 96.7246.9.01) at 5 × 10 ^{5 to} 5 × 10 ⁶ cells / tube. And pelleted by centrifugation at 750 g for 5 minutes. The plasma or control plasma was added to the pelleted cells at 500 μL / tube, followed by reaction at 37 ° C. under 5% CO ₂ for 60 minutes. The mixture after the reaction was centrifuged at 750 g for 5 minutes to remove plasma. The obtained cells were washed with a phosphate buffer.

To solubilize intracellular proteins, lysis buffer [50 mmol / L HEPES, 150 mmol / L NaCl, 1 vol% Nonidet-P40, 50 mmol / L NaF, 2 mmol / L Na3VO4, 1 vol% Protease inhibitor cocktail for General Use (Nacalai Tesque)] was added to the cells and treated on ice for 10 minutes. This was centrifuged at 15000 g for 5 minutes, and the resulting supernatant was used as a cell extract. The protein concentration contained in each sample was measured using a protein assay kit (manufactured by Bio Rad), and diluted with lysis buffer so that the protein mass between samples was constant. Further, 2 × sample buffer (manufactured by Nacalai Tesque) was added and heat-treated at 95 ° C. for 5 minutes was used as a sample for SDS-PAGE.
The SDS-PAGE sample prepared above was separated by SDS-PAGE, and then transferred to a PVDF membrane (Polyvinylidene Fluoride membrane, MILLIPORE, catalog number: IPVH00010). Blocking buffer after transfer (50 mmol / L Tris-buffered saline (pH 8.0) containing 0.05 vol% Tween 20 (Bethyl Laboratories, Montgomery, TX, USA), 5 w / v% skim milk (Nacalai Tesque) The primary antibody and the secondary antibody were added and allowed to react. As the primary antibody, anti-phosphorylated STAT5 antibody KM3461 and anti-STAT5 antibody (manufactured by Santa Cruz) prepared by the method of Reference Example 1 were used. After reacting the antibody, the X-ray film reacted with ECL Western blotting detection reagent (Amersham Biosciences) was exposed to detect the target band. The results are shown in Figure 1. The band corresponding to phosphorylated STAT5 was classified into No reduction (N), Partial reduction (P), and Complete reduction (C), and a dot plot was made with the vertical axis representing the total plasma drug concentration. The definitions of N, P, and C are as follows.
No reduction (N): A group in which a decrease in STAT5 phosphorylation level cannot be confirmed visually compared to the control band.
Partial reduction (P): Group that is visually confirmed to have decreased to STAT5 phosphorylation level compared to the control band
Complete reduction (C): Group in which no band corresponding to STAT5 phosphorylation level can be confirmed visually

The concentration of the compound in plasma was measured using LC / MS / MS under the following analytical conditions. FIG. 2 shows the plotted results with respect to the concentration of Compound 1 and the level of STAT5 phosphorylation measured by LC / MS / MS.
Analysis conditions by LC / MS / MS
HPLC system: Agilent 1100 (Agilent Technologies)
Autosampler: HTC PAL (CTC Analytics)
Mass spectrometer: API2000 (Applied Biosystems / MDS Sciex)
Analysis software: Analyst 1.3.1 (Applied Biosystems / MDS Sciex)
Guard filter: Pre-filter (manufactured by Nomura Chemical Co., Ltd.)
Column: CAPCELL PAK C18 MG (3 μm, 3 mm id x 35 mm, manufactured by Shiseido)
Column temperature: Room temperature Mobile phase conditions: A) 10 mmol / L ammonium acetate aqueous solution, B) Acetonitrile

Flow rate: 0.8 mL / min
Injection volume: 60 μL
Ionization method: Atmospheric pressure chemical ionization, positive
Curtain gas: Nitrogen 207 kPa
Nebulizer gas: Air 552 kPa
Auxiliary gas: Air 138 kPa
Collision gas: Nitrogen 27.6 kPa
Ion source temperature: 450 ° C
Interface heater: On
Needle current: 3 μA
Orifice voltage: 16 V
Ring voltage: 370 V
Collision energy: 23 eV
Detection mass (m / z: Q1 / Q3): 424.08 / 319.15
Reference Example 1: Preparation of monoclonal antibody that can specifically detect phosphorylation of tyrosine 694 of STAT5 (1) Preparation of antigenic peptide (abbreviation)
Abbreviations for amino acids and their protecting groups used in the present invention are the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature on Biochemical Nomenclature [European Journal of Biochemistry] 138, 9 (1984)].

The following abbreviations represent the corresponding amino acids below unless otherwise specified.
Ala: L-alanine
Asp: L-aspartic acid
Asx: L-Aspartic acid or L-Asparagine
Arg: L-Arginine
Cys: L-cysteine
Gln: L-glutamine
Glx: L-glutamic acid
Gly: Glycine
Ile: L-isoleucine
Leu: L-leucine
Met: L-methionine
PHe: L-Phenylalanine
Ser: L-serine
Thr: L-threonine
Tyr: L-tyrosine
pTyr: L-phosphotyrosine
Met: L-methionine
Val: L-valine The protein sequence of human STAT5 was analyzed, and the following sequence was selected as a partial sequence considered to be suitable as an antigen from the hydrophilicity and secondary structure in the portion containing tyrosine at position 694. Ac represents acetyl.

Compound PY694 (SEQ ID NO: 1)
Ac-Ala Lys Ala Val Asp Gly pTyr Val Lys Pro Gln Ile Lys Gln Val Val Pro Glu PHe Val Cys-OH
Moreover, the following sequences were used as negative control antigens used for hybridoma screening.

Compound Y694 (SEQ ID NO: 2)
Ac-Ala Lys Ala Val Asp Gly Tyr Val Lys Pro Gln Ile Lys Gln Val Val Pro Glu PHe Val Cys-OH
Compounds PY694 and PY694 were synthesized according to the synthesis program of Shimadzu Corporation using an automatic synthesizer, purified by HPLC, and used as antigens.
(2) Preparation of immunogen Compound PY694 obtained in (1) was used as an immunogen by preparing a conjugate with KLH (Calbiochem) by the following method for the purpose of enhancing immunogenicity. That is, KLH was dissolved in PBS to adjust to 10 mg / mL, and 1/10 volume of 25 mg / mL MBS [N- (m-Maleimidobenzoyloxy) succinimide; Nacalai Tesque] was added dropwise and stirred for 30 minutes to react. I let you. KLH-MBS 2.5 mg obtained by removing free MBS in a gel filtration column (Sephadex G-25 column) previously equilibrated with PBS was dissolved in 0.1 mol / L sodium phosphate buffer (PH7.0) 1 It was mixed with mg of compound PY694 and stirred at room temperature for 3 hours to be reacted. After the reaction, dialyzed with PBS was used as an immunogen.
(3) Immunization of animals and preparation of antibody-producing cells 50 µg of KLH conjugate of compound PY694 prepared in (2) was added to each with aluminum hydroxide adjuvant (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, page 99, 1988). 2 mg and pertussis vaccine (Chiba Prefectural Serum Research Laboratories) 1 × 10 ⁹ cells were administered to 3 4-week-old female SD rats (Shizuoka Prefectural Experimental Animal Agricultural Cooperative Association). From 2 weeks after the administration, 50 μg of KLH conjugate of compound PY694 was administered once a week for a total of 4 times. Partial blood was collected from the tail vein of the rat, the serum antibody titer thereof was examined by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the rat showing a sufficient antibody titer.

The spleen was shredded in MEM (Minimum Essential Medium) medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1200 rpm, 5 minutes). Tris-ammonium chloride buffer (PH7.6) was added to the obtained precipitate fraction, and erythrocytes were removed by treatment for 1-2 minutes. The resulting precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.
(4) Enzyme immunoassay [binding ELISA (Enzyme-Linked Immuno Sorbent Assay)]
As the antigen for assay, PY694 and Y694 obtained in (1) were conjugated with thyroglobulin (hereinafter abbreviated as THY). The preparation method was as described in (2), but SMCC [4- (N-Maleimidomethyl) -cyclohexane-1-carboxylic acid N-hydroxysuccinimido ester; Sigma] was used instead of MBS as the crosslinking agent. The conjugate prepared as described above was dispensed at 5 μg / mL and 50 μL / well onto a 96-well ELISA plate (Greiner), and allowed to stand overnight at 4 ° C. for adsorption. After the plate was washed, 100 μL / well of 1% bovine serum albumin (BSA) -Dulbeccoline buffer (PBS) was added and left at room temperature for 1 hour to block the remaining active groups.

After standing, discard 1% BSA-PBS, and dispense 50 μL / well of the immunodeficient rat antiserum partially collected in (2) as the primary antibody and the hybridoma culture supernatant obtained in (6) below. , Left for 2 hours. The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate [(ICI trademark Tween 20 equivalent: manufactured by Wako Pure Chemical Industries)] / PBS (hereinafter referred to as Tween-PBS) as a secondary antibody. Peroxidase-labeled rabbit anti-rat immunoglobulin (Dako) was added at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing the plate with Tween-PBS, ABTS substrate solution [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium, 1 mmoL / L ABTS / 0.1moL / L citrate buffer (PH4.2)] The color was developed and the absorbance at OD 415 nm was measured using a plate reader (Emax; MoLecuLar Devices).
(5) Preparation of mouse myeloma cells
8-Azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1: purchased from ATCC) is cultured in 10% fetal bovine serum-supplemented RPMI1640 (Invitrogen), and 2 × 10 ⁷ or more cells are secured during cell fusion And used as a parent strain for cell fusion.
(6) Production of hybridoma The rat spleen cells obtained in (3) and the myeloma cells obtained in (5) were mixed at 10: 1 and centrifuged (1200 rpm, 5 minutes). After thoroughly loosened and the resulting precipitate fraction group of cells, with stirring, at 37 ° C., polyethylene glycol -1000 (PEG-1000) 1 g , MEM medium 1 mL, and dimethyl sulfoxide 0.35 mL of a mixture of 10 ⁸ 0.5 mL per mouse spleen cell was added, and 1 mL of MEM medium was added several times to the suspension every 1 to 2 minutes, and then the MEM medium was added so that the total volume became 50 mL.

After centrifuging the suspension (900 rpm, 5 minutes) and gently loosening the cells of the obtained precipitate fraction, the cells were gently removed by sucking and sucking with a pipette into the HAT medium [10% fetal calf. Suspension in 100 mL of a medium supplemented with HAT Media Supplement (manufactured by Invitrogen) in RPMI1640 medium supplemented with serum]. The suspension was dispensed at 200 μL / well into a 96-well culture plate and cultured at 37 ° C. for 10 days in a 5% CO ₂ incubator.

After culture, the culture supernatant is examined by the enzyme immunoassay described in (4), a hole that reacts with compound PY694 and does not react with compound Y694 is selected, and cloning by limiting dilution is repeated twice from the cells contained therein. Anti-PY694 monoclonal antibody-producing hybridoma strains KM3461, KM3462, and KM3463 were established.
(7) Purification of monoclonal antibody Eight-week-old nude female mouse (BALB / c, Shizuoka Prefecture experiment) in which pristane (2,6,10,14-tetramethylpentadecane, Sigma) was administered intraperitoneally 0.5 mL / mouse 3 days ago The hybridoma strain obtained in (6) was injected intraperitoneally into the animal agriculture cooperative) at 5 to 20 × 10 ⁶ cells / animal. After 10 to 21 days, ascites was collected (1 to 8 mL / animal) from a mouse in which ascites accumulated due to the hybridoma becoming ascites tumor.

The ascites was centrifuged (3000 rpm, 5 minutes) to remove solids. Purified IgG monoclonal antibody was obtained by purification by the caprylic acid precipitation method (Antibodies-A Laboratory ManuaL, Cold Spring Harbor Laboratory, 1988). The subclass of the monoclonal antibody was determined by ELISA using a sub-clustering kit. The subclasses of KM3461, KM3462 and KM3463 were all IgG2a.
Reference Example 2: Examination of reactivity of anti-PY694 monoclonal antibody (1) Reactivity with antigen compound in antigen solid-layer system (binding ELISA)
This was carried out according to the method shown in (4) of Reference Example 1. However, as the primary antibody, the hybridoma culture supernatant obtained in (5) of Reference Example 1 was used after 5-fold dilution from the stock solution at a 5-fold dilution. The results are shown in Figure 3. KM3461, KM3462, and KM3463 reacted only with compound PY694, and did not react with compound Y694 at all.
(2) Reactivity with antigenic compounds in the antigen liquid phase system (Inhibition ELISA)
The reactivity of the KM3461, KM3462 and KM3463 to the antigen compound in the liquid phase system was examined by an inhibition ELISA.

  As shown in Reference Example 1 (4), a plate on which an antigen was immobilized was prepared, and the compound PY694 obtained in Reference Example 1 (1) was diluted stepwise at a 5-fold dilution from 20 μg / mL. After dispensing compound Y694 at 50 μL / well, dilute each culture supernatant of KM3461, KM3462 and KM3463 (dilution ratio: KM3461: x10, KM3462: x50, KM3463: x7) and dispense at 50 μL / well Then, they were mixed in the well and reacted at room temperature for 2 hours. The wells were washed with Tween-PBS, diluted peroxidase-labeled rabbit anti-rat immunoglobulin (Dako) was added at 50 μL / well, reacted at room temperature for 1 hour, washed with Tween-PBS, ABTS substrate solution [2.2-azinobis Color was developed using (3-ethylbenzothiazole-6-sulfonic acid) ammonium], and the absorbance at OD 415 nm was measured with a plate reader (Emax; Wako Pure Chemical Industries).

As shown in FIG. 4, all of KM3461, KM3462, and KM3463 showed specific reactivity to compound PY694 in the liquid phase system.
(3) Phosphorylated STAT5 detection by Western blot It has been reported that STAT5 is constantly phosphorylated [Leukemia Research, 27, 803-805 (2003)] cell line K562 (RIKEN Cell Bank) ) Solubilization buffer [50 mmol / L Tris-HCl (PH7.2), 150 mmol / L Sodium chloride, 1% TritonX, 2 mmol / L Magnesium chloride, 2 mmol / L Calcium cholride, 0.1% Sodium azide, 5 μmol / L Phenylmethylsulfonyl fluoride, 50 mmol / L N-etylmaleimide, 1 mg / mL Leupeptin, 100 μmol / L 1,4-dithiothreitol, 2 mmol / L Sodiumorthovanadate, 1 mmol / L Sodium fluoride, 10 mmol / L βglycerophosphate] After adding 1 mL of 5 × 10 ⁷ cells and reacting at 4 ° C. for 30 minutes, the centrifuged supernatant was used as a solubilized fraction. A solubilized fraction equivalent to 1.5 × 10 ⁵ cells per lane was fractionated by SDS-polyacrylamide electrophoresis, and the gel after migration was transferred to a PVDF membrane. After blocking the membrane with 1% BSA-PBS, each culture supernatant of KM3461, KM3642, and KM3463, anti-phosphorylated STAT5 monoclonal antibody Clone47 (Becton Dickinson) 5 μg / mL / 1% BSA- as a positive control antibody PBS and KM1762 (anti-avermectin antibody) 5 μg / mL / 1% BSA-PBS as a negative control antibody were reacted at room temperature for 2 hours. The membrane reacted with the antibody was thoroughly washed with Tween-PBS, and then diluted peroxidase-labeled rabbit anti-rat immunoglobulin (Dako) was reacted at room temperature for 1 hour. The membrane was thoroughly washed with Tween-PBS, and the band was detected using Western blotting detection reagent (ECL Western blotting detection Regents (registered trademark), manufactured by Amersham Pharmacia).

As shown in FIG. 5, KM3461, KM3462, and KM3463 detected a band around 95 kilodaltons similarly to the commercially available anti-phosphorylated STAT5 monoclonal antibody Clone47 (Becton Dickinson). Therefore, in combination with the results of (1) and (2), it was considered that KM3461, KM3462 and KM3463 are antibodies that specifically recognize phosphorylated STAT5. It was also shown that these antibodies can be used for detection of phosphorylated STAT5 by Western blot.
(4) Fluorescent antibody staining using cells (flow cytometry)
K562 cells were cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum for 2-3 days, and the cells collected by centrifugation were washed with PBS. In order to increase the membrane permeability of cells, 2% neutral buffered formalin solution (Nacalai Tesque) was reacted at 37 ° C for 10 minutes, washed with PBS and centrifuged at 100% methanol at ice temperature. For 30 minutes. After washing with PBS, in order to avoid non-specific adsorption of the antibody, blocking was performed for 20 minutes at ice temperature using human immunoglobulin IgG (Mitsubishi Pharma) / 1% BSA-PBS. Dispense it into a 96-well U-shaped plate so that 1 × 10 ⁶ cells / 100 μL / 1% BSA-PBS, centrifuge (1800 rpm, 2 minutes), remove the supernatant, and use KM3461 as the primary antibody. , KM3462 and KM3463 culture supernatant, anti-phosphorylated STAT5 monoclonal antibody Clone47 (Becton Dickinson) 10 μg / mL / 10% fetal calf serum RPMI1640 as a positive control antibody, and KM511 [anti-GCSF mutation as a negative control antibody Antibody, Agricultural and Biological Chemistry, 53, 1095 (1989)] or rat IgG2a control antibody (Beckman Coulter) 10 μg / mL / 10% fetal bovine serum Added RPMI1640 was dispensed at 50 μL / well and allowed to react for 30 minutes at ice temperature. Wash 3 times with PBS, add 20 μL / well of Alexa 488-labeled anti-rat immunoglobulin G (H + L) (manufactured by Molecular Probes) as a secondary antibody, and allow to react for 30 minutes under ice-cold conditions. It was. The sample was again washed with PBS three times, suspended in PBS, and measured with a flow cytometer (Beckman Coulter, Inc.) at a wavelength of 510 to 530 nm excited with argon-ion laser light (488 nm).

  As shown in FIG. 6, KM3461, KM3462, and KM3463 were shown to be antibodies capable of detecting phosphorylated STAT5 inside K562 cells.

2 is a diagram showing the results of SDS-PAGE in Example 1. FIG. FIG. 3 is a graph showing STAT5 phosphorylation levels in Example 1. Each symbol represents N (No reduction), P (Partial reduction), C (complete reduction), and the vertical axis represents plasma total drug concentration (ng / mL). ○ indicates a dose of Compound 1 of 50 mg / kg, ▲ indicates a dose of Compound 1 of 100 mg / kg, and ● indicates a dose of Compound 1 of 200 mg / kg. It is a figure which shows the reactivity of the monoclonal antibodies KM3461, KM3462, and KM3463 in binding ELISA. The horizontal axis shows the dilution of each culture supernatant of monoclonal antibodies KM3461, KM3462, and KM3463, and the vertical axis shows the binding activity of each culture supernatant of monoclonal antibodies KM3461, KM3462, and KM3463. ○ indicates a dose of Compound 1 of 50 mg / kg, ▲ indicates a dose of Compound 1 of 100 mg / kg, and ● indicates a dose of Compound 1 of 200 mg / kg. It is a figure which shows the reactivity of the monoclonal antibodies KM3461, KM3462, and KM3463 in inhibition ELISA. The horizontal axis represents the concentration of compound PY694 and compound Y694, and the vertical axis represents the binding activity of each culture supernatant of monoclonal antibodies KM3461, KM3462, and KM3463. Inhibition 0 shows the binding activity of each culture supernatant of monoclonal antibodies KM3461, KM3462, and KM3463 when compound PY694 or compound Y694 is not added. ○ indicates a dose of Compound 1 of 50 mg / kg, ▲ indicates a dose of Compound 1 of 100 mg / kg, and ● indicates a dose of Compound 1 of 200 mg / kg. It is a figure which shows the reactivity of the monoclonal antibodies KM3461, KM3462, and KM3463 in a Western blot. From the left of the lane, 1. molecular weight marker, 2.K562 solubilized fraction are shown. It is a figure which shows the reactivity of the monoclonal antibodies KM3461, KM3462, and KM3463 in flow cytometry. The vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity.

SEQ ID NO: 1-Description of artificial sequence: synthetic DNA
SEQ ID NO: 2-description of artificial sequence: synthetic DNA

Claims (15)

A method for measuring a kinase inhibitor contained in a biological sample, comprising the following steps a and b;
(A) contacting the biological sample with a cell expressing a kinase that is a target of a kinase inhibitor;
(B) A step of measuring a phosphorylation level of a target kinase present in the cell and / or a kinase downstream of the target kinase. The method according to claim 1, wherein the kinase is a tyrosine kinase or a serine / threonine kinase. The method according to claim 2, wherein the tyrosine kinase is a tyrosine kinase selected from the group consisting of hummus-like tyrosine kinase 3, mutant Abelson kinase, vascular endothelial growth factor receptor 2 and fibroblast growth factor receptor. The method according to claim 2, wherein the serine / threonine kinase is an aurora. The measurement method according to claim 1, wherein the biological sample is blood, plasma, or serum. The measurement method according to claim 1, wherein the biological sample is a biological sample of a patient administered with a kinase inhibitor. The measurement method according to claim 1, wherein the cell that expresses the target kinase is a cancer cell. The measurement method according to claim 7, wherein the cancer cells are solid cancer cells or hematopoietic tumor cells. The measurement method according to claim 8, wherein the hematopoietic tumor cell is a leukemia cell. The measurement method according to any one of claims 1 to 6, wherein the cell expressing the target kinase is a cancer cell derived from a patient administered with a kinase inhibitor. 11. The target kinase substrate is a signal transduction molecule selected from the group consisting of signal transducer and activator of transcription 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), histone H3, and CRKL. The measuring method in any one. The step of measuring a phosphorylation level of a substrate of a target kinase present in the cell and / or a kinase downstream of the target kinase is a step comprising an immunological measurement method. The measuring method described. The measuring method according to claim 12, wherein the immunological measuring method is Western blotting or immune cell staining. The reagent for a measurement in any one of Claims 1-11 containing the cell which expresses the target kinase. The measurement kit according to claim 1, comprising a cell expressing a target kinase.

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