WO2005061707A1 - METHOD OF JUDGING SENSITIVITY OF CANCER CELL TO Eg5 INHIBITOR - Google Patents

Abstract

A method which comprises measuring the sensitivities to an Eg5 inhibitor of two or more human cancer cell lines and the expression amounts of one or more human genes and identifying a gene showing a correlation between its expression amount and the sensitivity to the Eg5 inhibitor as a gene relating to the sensitivity to the Eg5 inhibitor. A method which comprises selecting one or more genes from the genes relating to the sensitivity to the Eg5 inhibitor thus identified, measuring the expression amounts of the selected genes in cancer cells, assigning scores depending on the expression amounts and thus judging the sensitivity of the cancer cells to the Eg5 inhibitor.

Description

 Specification

 Method for determining sensitivity of cancer cells to Eg5 inhibitors

 The present invention provides a method for identifying a gene involved in the sensitivity of a cancer cell to a 5-inhibitor, a method for determining the sensitivity of a cancer patient to an Eg5 inhibitor, and a method for screening a substance that enhances the sensitivity of a cancer cell to an Eg5 inhibitor About. Background art

 Cancer chemotherapy with various anticancer drugs is one of the most important methods in treating cancer. For effective treatment with anticancer drugs, it is important to predict the sensitivity of patients to anticancer drugs and to select appropriate drugs. Finding genes that determine susceptibility to anticancer drugs is one method for predicting susceptibility. To date, many genes have been reported as determinants of sensitivity to multiple anticancer drugs. For example, the drug transporters MDR1 (J, Biol. Chem., 265, 506-514, 1990) and MRP2 (Cancer Res., 57, .3537-3547, 1997; Cancer Res 56> 4124-4129, 1996 ), A drug-metabolizing enzyme, cytochrome P450 (hereinafter abbreviated as CYP), Family 1 (Curr. Pharm. Des., 8, .1335-1347, 2002), Guruya Thion S-transferrase (J. Biol) Chem., 261, 15544-15549, 1986), and acetylacetyltransferase (Pharraacogenomics, 3, 349-366, 2002). Further, for example, the following genes have been reported as genes regulating the sensitivity to each anticancer drug. Yellow milk hydrolase (Biochemistry, 12, 3923-3927, 1973,) and dihydrofolate reductase (Cancer, 57, 1912-1917, 1986) are resistance factors to methotrexate. In addition, thymidylate synthase (J. Clin. Oncol., 6, 1653-1664, 1988), meta-mouth thionine (Cancer Treat. Res., 57, 233-249, 1991) and cytidine deamine

 (Cancer, 40> 509-518, 1977), each of which is a resistance factor to 5-fluorouracil, cisbratin and cytosine arabinoside. Enhanced DT-diaphorase activity is a susceptibility factor for mitomycin C (Cancer Res., 52> 6936-6939, 1992). Thus, many genes are known as genes that determine susceptibility to anticancer drugs. However, these known genes alone are not sufficient to explain susceptibility to anticancer drugs, and further research is needed.

On the other hand, attempts are being made to predict the sensitivity to anticancer drugs or to predict genes involved in sensitivity to anticancer drugs using analyzes such as cDNA microarrays and SNPs. Human cancer cell line panel-Analysis of the correlation between gene expression in xenografts of human cancer and sensitivity to anticancer drugs has also been reported (Nat. Genet., 24, 236-244, 2000; Cancer Res., 62 518-527, 2002; Cancer Res., 62, 1139-1147, 2002; Proc. Natl. Acad. Sci. USA, 98, 10787-10792, 2001; JP 2003-61678). The genes found in these studies are used to predict the susceptibility to anticancer drugs. —Can be used as a force. In addition, some of these genes may actually determine sensitivity to anticancer drugs.

 Spindle function is essential for the centrosome localization and accurate chromosome segregation during cell division (cell cycle M phase). Inhibition of its function inhibits normal cell division and causes cell death in cancer cells It is known to induce

 M-phase kinesin is a protein involved in M-phase spindle control and plays an essential role in M-phase progression of the cell cycle. These proteins have a function of moving proteins along microtubules by utilizing energy generated by ATP hydrolysis, and are a group of functional proteins generally called “molecular motors”. In the M phase, it is deeply involved in the elongation and maintenance of the spindle and the formation of a structure called the spindle pole, and further controls the progression of correct cell division through the movement of chromosomes along spindle microtubules. ing.

 Eg5 is one of the M-phase kinesins that forms an evolutionarily conserved subfamily. It is known that the expression of に お け る 5 in normal human tissues is limited to the testis and thymus, etc.In addition, the results of analysis of cancer patient tissues show that the expression is higher in cancerous parts than in non-cancerous parts (US6414121; Proc. Natl. Acad. Sci. USA, 99> 4465-4470, 2002).

 Therefore, M-phase kinesin Eg5 is important as a target molecule of a novel M-phase agonist, and its inhibitor is considered to be promising as a therapeutic agent for diseases caused by abnormal cell growth control such as cancer. Monastrol is a compound exhibiting Eg5 inhibitory activity.

 (monastrol) (Science, 286, 971-974, 1999), quinazoline derivative

 (WO01 / 98278), phenathiazine derivatives (WO02 / 057244), triphenylmethane derivatives (WO02 / 056880), dihydropyrimidine derivatives (WO02 / 079149;

WO02 / 079169) has been reported.

 Factors involved in drug sensitivity of anticancer drugs that act on microtubules, which are constituents of the M-phase spindle, include MDR1 which is a drug transporter for paclitaxel, which belongs to taxanes. It has been reported (Curr. Cancer Drug Targets, 3, 1-19, 2003; Cancer Res., 63 »1515-1519, 2003), and comprehensive analysis using DNA microarrays has also been performed (Cancer Res 63> 2200-2205, 2003). Another transposon, vincristine, belonging to the bin alkaloids, such as MRP1, has been reported (Clin. Cancer Res., 5, 673-680, 1999). However, there have been no reports to date on the prediction of the sensitivity of Eg5 inhibitors or the genes involved in sensitivity to Eg5 inhibitors.

 On the other hand, thiadiazoline derivatives showing antitumor activity are known

 (WO03 / 051854). Also, cysteine derivatives effective for the treatment of leukemia are known (J. Med. Chem., 13, 414-418, 1970; J. Med. Chem., 15, 13-16, 1972). Disclosure of the invention

An object of the present invention is to provide a method for identifying genes involved in the sensitivity of cancer cells to an Eg5 inhibitor, and to use these genes to increase the sensitivity of cancer cells to an Eg5 inhibitor. The purpose is to provide a method for determining.

 The present inventors have conducted intensive studies to solve the above problems, and first measured the sensitivity to Eg5 inhibitors and the expression levels of 119 human genes in a panel of 38 human cancer cell lines. We selected 19 genes whose expression was correlated with sensitivity to Eg5 inhibitors, particularly in 26, lung cancer-derived strains. In addition, the present inventors constructed a statistical model for predicting the sensitivity of cancer cells to an Eg5 inhibitor from the expression levels of those genes, and further reduced the number of genes used for prediction to 11 Was also successfully constructed.

 By using this model and examining the expression of a small number of selected genes, it has become possible to provide a method for determining the susceptibility of cancer patients to anticancer drugs. Furthermore, by increasing or decreasing the expression of those genes, it has become possible to provide a method for enhancing sensitivity to an anticancer agent, and a method for screening a substance that enhances sensitivity to an anticancer agent. The present invention has been completed based on these findings.

 That is, the present invention relates to the following (1) to (54).

 (1) The following steps (a) to (c)

 (a) measuring the sensitivity to an Eg5 inhibitor and the expression level of one or more human genes for two or more human cancer cell lines,

 (b) analyzing the correlation between the expression level of the gene in each cell line and the sensitivity of the cell line to the Eg5 inhibitor for each gene whose expression level was measured in step (a);

(c) selecting a gene that was correlated in step (b),

 A method for identifying a gene involved in sensitivity of a cancer cell to an Eg5 inhibitor.

(2) The human cancer cell lines used for measurement in step (a) are A549, Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226, NC H345, N "7, NCI-H460 NCI-'H1299, SBC-3, PC-14, PC- / DTX ヽ PC-14 / CDDP, MRC-5, IMR-90, EBC-1, NCI-H292, SHP-77, NC Cell lines derived from lung cancer consisting of NCI-H838, A427, NCI-H522, Calu-6 and PC-9, colon cancer derived from Colo205s HT-29, HCT116, SW480, DLD-l and WiDr A group consisting of a cell line, a cell line derived from ovarian cancer consisting of PANC-U MIA PaCa-2, AsPC-1 and BxPC-3, and an ovarian cancer cell line consisting of SK-0V-3 and 0VCAR-3 The method according to (1), which is a cell line selected from all or some cell lines.

(3) Cell lines selected are A549 _s Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226 ヽ NCI-H345, N417, NCI-H460 NCI-H596, NCI-H1299, SBC -3, PC-14, PC-14, PC-14 / CDDP, MRC-5, IMR-90, EBC-1, NCI-H292, SHP-77, NC Awakening ヽ NCI-H838, A427, NCI- (2) The method according to (2), which is all or a part of a cell line selected from a lung cancer-derived cell line consisting of H522, Calu-6 and PC-9.

 (4) The method according to (1), wherein the human cancer cell line used for the measurement in step (a) is a cancer cell line derived from one specific organ.

 (5) The method according to (4), wherein one specific organ is a lung.

(6) The Eg5 inhibitor for measuring the sensitivity is represented by the general formula (I)

<Wherein

R ¹ and R are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted Represents an aryl or substituted or unsubstituted heterocyclic group;

R ² is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted A heterocyclic group of the formula: 1 C (= W) R ⁶ [wherein W represents an oxygen atom or a sulfur atom, and R ⁶ is a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl A substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocyclic group, one NR ⁷ R ⁸ (wherein R ⁷ and R ⁸ is the same or different and is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted Or an unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted heterocyclic group wherein and R ⁸ together with an adjacent nitrogen atom ), —OR ⁹ , wherein R ⁹ is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or Represents an unsubstituted aryl or a substituted or unsubstituted heterocyclic group) or -SR ¹⁰ (wherein, R ^1G is as defined above for R ⁹ )], one NR "R ¹² {wherein R "and R ¹² are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted Conversion cycloalkyl, substituted or unsubstituted Ariru or substitution or unsubstituted heterocyclic璟基or ^{- C (= 0) R 13} [ wherein, R ¹³ is a hydrogen atom, a substituted if Ku is unsubstituted lower alkyl, Substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, — NR ^7A R ^8A ( Wherein R ^7A and R ^8A are as defined above for R ⁷ and R ⁸ ), — 0R ^9A (where R ^9A is as defined for R ⁹ above) or — SR (formula among, R ^1QA is "(in the formula, R" above represents the R ⁹ as synonymous) representing a]} or a S0 ₂ R is or represents the same meaning) and said R ^9, or R ¹ and R ² together with the adjacent nitrogen atom forms a substituted or unsubstituted heterocyclic group,

R ⁶ is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, Represents a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group, or R ⁴ and R ⁵ are taken together 1 ( ^{15 A} R ^15B — Q— (CR ¹⁵ ¾ ^15D ) _m2 — {wherein Q represents a single bond, substituted or unsubstituted phenylene or cycloalkylene, and ml and Π12 are the same or different Where ml and m2 are not simultaneously 0 and R] ^5A , ^R15B and ^R15D are the same or different and each represents a hydrogen atom, halogen, substituted or unsubstituted lower alkyl. , —OR ¹⁶ [wherein R ¹⁶ is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted Substituted heterocycle , - CONR ^7B R ^8B (wherein and R ^8B are the same or different, a hydrogen atom, a substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substitution or unsubstituted lower alkynyl, substituted or unsubstituted Represents a substituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted R ™ and R ^8B together with an adjacent nitrogen atom; To form an unsubstituted heterocyclic group), -S0 ₂ NR 78 ^(I (wherein R ^7G and R ^8e have the same meanings as R ^7B and R ^8B , respectively)) or —C0R ¹⁷ (wherein R ¹⁷ is a hydrogen atom, a substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower § Rukiniru, substituted or unsubstituted cycloalkyl, substituted or unsubstituted § Li one le or location Or an unsubstituted heterocyclic璟基) representing a], in one NR ¹⁸ R ¹⁹ [wherein, R "and R ¹⁹ are the same or different, a hydrogen atom, a substituted or unsubstituted lower alkyl, substituted or unsubstituted Lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, — C0R ² ° (where R ² ° is Hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted complex Ring group, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy, amino, substituted or unsubstituted lower alkyl Ruamino, substituted or unsubstituted di-lower alkylamino or substituted or represents unsubstituted Ariruamino) or a S0 ₂ R "(wherein, R ²¹ is properly be substituted unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl represents a representative) substituted or unsubstituted Ariru or substituted or unsubstituted heterocyclic璟基] or - C0 ₂ R ²² (wherein. Wherein R ²² is as defined above for R ¹⁷ ) or R ^15A and R ^15B or R ^15G and R ^15D represent an oxygen atom, and ml or m2 is an integer of 2 or more Where each of ⁵⁴ , R ^15B , and R ^15D may be the same or different, and H ^15A , R ^15B s R ^15G and R ^{15D, which} are bonded to two adjacent carbon atoms, are taken together. Become a bond May be formed)

Represents a hydrogen atom or —C (= W ^A ) R ²³ ′ (wherein W ^A and R ²³ have the same meanings as W and R ⁶ above), and a thiadiazoline derivative represented by> or a pharmacology thereof. The method according to any one of (1) to (5), which is a salt that is specifically acceptable. (7) R ¹ is hydrogen atom, R ² is ^{- C (= 0) R 6A} ( wherein, R ^6A represents a lower alkyl), R ³ is - C (= 0) R (wherein , R represents lower alkyl), R ⁴ is substituted lower alkyl, and R ⁵ is phenyl.

(8) R ⁴ is _{- (C) na NHS0 2 R} a ( wherein, na is 1 or 2, the R ^a represents a substituted or unsubstituted lower alkyl) to a (6) or (7) The described method.

 (9) The thiadiazoline derivative represented by the general formula (I) is a compound selected from the group consisting of compounds represented by the following formulas (A) to (C) and (E) to (P)

感受性の強さを測定する 5阻害剤が、 一般式 （II)

The five inhibitors that measure the sensitivity are represented by the general formula (II)

 (Π)

(Wherein, R ²⁴ represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group, and ⁵ and! ^ Are the same or different and each represents a hydrogen atom, a halogen, a lower alkyl, a lower alkoxy, , Hydroxy, carboxy, or hydroxymethyl, or an oxygen atom, a sulfur atom or a bond with R ^M and ⁶ Represents a cysteine derivative or a pharmacologically acceptable salt thereof,

The method according to any one of (I) to (5).

 (II) The method according to (10), wherein the cysteine derivative is an L-cysteine derivative. .

(12) R ²⁴ is phenyl, 4-methylphenyl, 2-naphthyl, 4-pyridyl, or 1,3-benzodioxoyl-5-yl, and R ²⁵ and R ²⁶ are the same or different and are each a hydrogen atom , Halogen, lower alkyl, lower alkoxy, hydroxy, carboxy, or hydroxymethyl.

(13) The Eg5 inhibitor for which the sensitivity is to be measured is a compound represented by the following formula (Q) or a pharmacologically acceptable salt thereof, which is one of (1) to (5). The method described in section.

(14) 以下の （a) 〜 （e) の工程

(14) The following steps (a) to (e)

 (a) selecting one or more genes from the genes identified by the method according to any one of (1) to (13) and determining a score according to the expression level;

 (b) measuring the expression level of the gene selected in step (a) for the test cancer cell;

 (c) assigning, for each gene, the score determined in step (a) based on the expression level measured in step (b),

 (d) obtaining a numerical value indicative of the sensitivity of the test cancer cells to the Eg5 inhibitor from the assigned scores for all the selected genes,

 (e) comparing a predetermined threshold value with the value obtained in (d), a method for determining sensitivity of a cancer cell to an Eg5 inhibitor.

 (15) The method according to (14), wherein the gene selected in the step (a) is one or more genes selected from the following (i) to (xix) genes consisting of the respective genes: .

 (ί) Cytochrome Ρ450 · Family 24 · Subfamily A · Polypeptide 1 (CYP24A1)

 (ii) Lebutin receptor gene-related protein (0BRGRP)

 (iii) Factor B (BF)

(iv) Adenine phosphoribosyl transcriptase (APRT) (v) Bakilowis IAP repeat-containing protein (BIRC3)

 (vi) Sequence software 1 (SQSTM1)

 (vii) Tumor necrosis factor spike inducible protein 2 (TNFAIP2)

 (viii) integral membrane protein 2B (ITM2B)

 (ix) ATP binding cassette 'Subfamily 1 C · Member 1 2 (ABCC2)

 (x) Annexin A3 (ANXA3)

 (xi) Homologous sequence 3 containing family 1 'member 1 3 (FAM3C)

 (xii) ATP binding cassette / subfamily C'member 1 (ABCC3)

 (xiii) hypothetical protein BC007436 (BC007436)

 (xiv) Microtubule-associated protein IB (MAP1B)

 (XV) Cyclin Bl (CCNB1)

 (xvi) Heat shock 90 kDa protein 1 h (HSPCA)

 (xvii) Cyclin B2 (CCNB2)

 (xviii) Virtual protein FLJ23269 (FLJ23269)

 (xix) Solute Carrier 1 'Family 1 12 Member 1 2 (SLC12A2)

 (16) The method according to (15), wherein the genes selected in the step (a) are CYP24A1, 0BRGRP, APRT, TNFAIP2, ITM2B, ABCC2, BC007436 MAP1B ヽ HSPCA. CCNB2 and FU23269.

 (17) The Eg5 inhibitor is the thiadiazoline derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof (14) to (16). The method described in the section.

 (18) The cysteine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof, wherein the Eg5 inhibitor is any one of (14) to (16). The method described in the section.

 (19) The method according to any one of (14) to (16), wherein the Eg5 inhibitor is the compound according to (13) or a pharmacologically acceptable salt thereof.

 (20) The method according to any one of (14) to (19), wherein the cancer cells are lung cancer cells.

 (21) The method according to any one of (1) to (20), wherein the expression level of the gene is measured by quantifying mRNA, which is a transcription product of the gene, using a DNA microarray.

 (22) The method according to any one of (1) to (20), wherein the expression level of the gene is measured by quantifying mRNA, which is a transcription product of the gene, by quantitative PCR.

 (23) The expression method described in any one of (1) to (20), wherein the expression level of the gene is measured by quantifying mRNA, which is a transcript of the gene, by a Northern plot. .

 (24) A reagent for quantifying mRNA, comprising an oligonucleotide complementary to the mRNA, for use in the method according to (22).

(25) The expression level of a gene is measured by immunizing a protein which is a gene product of the gene. The method according to any one of (1) to (20), which is performed by quantification by a chemical method.

 (26) The method according to (25), wherein the expression level of the gene is measured by quantifying a protein which is a gene product of the gene by ELISA.

 (27) The method according to (25), wherein the expression level of the gene is measured by quantifying a protein which is a gene product of the gene by a stamp lot.

 (28) An immunoassay reagent containing an antibody against the protein for use in the method according to any one of (25) to (27).

 (29) BC007436, MAP1B, CC thigh, HSPCA, CCNB2, FLJ23269 and one or more genes selected from the group consisting of SLC12A2, which enhance the sensitivity to Eg5 inhibitors by forcibly expressing them in cancer cells An agent for enhancing sensitivity to an Eg5 inhibitor, comprising a gene exhibiting activity or a protein encoded by the gene. (30) The sensitivity enhancer according to (29), wherein the cell is a lung cancer cell.

 (3 1) The Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof, and the sensitivity described in (29) or (30). Enhancer.

 (32) 'The Eg5 inhibitor is a cystine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof, as described in (29) or (30). Sensitivity enhancer.

 (33) The sensitivity enhancer according to (29) or (30), wherein the Eg5 inhibitor is the compound according to (13) or a pharmacologically acceptable salt thereof.

 (34) A test substance is added to the cells, and the expression level of at least one or more genes selected from the group consisting of BC007436, MAP1B, CCNB1, HSPCA, CCNB2, FLJ23269 and SLC12A2 in the cells is measured, and the test substance is added. A method for screening a substance that enhances the sensitivity to an Eg5 inhibitor, wherein a substance that increases the expression of the gene as compared to the expression level of the gene in the absence of the gene is selected as a candidate substance that enhances the sensitivity to the 5 inhibitor.

 (35) The screening method according to (34), wherein the cell is a lung cancer cell.

 (36) The screening method according to (34) or (35), wherein the Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof. .

 (37) The Eg5 inhibitor is a cystine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof, and a screen described in (34) or (35). Method.

 (38) The screening method according to (34) or (35), wherein the Eg5 inhibitor is the compound according to (13) or a pharmacologically acceptable salt thereof.

(39) CYP 4A1 _S 0B GRP _s BF, APRT, BIRC3, SQSTM1 _S TNFAIP2, ITM2B, ABCC2, AMA3, FAM3C, and one or more genes selected from the group consisting of: SiRNA or a gene that suppresses the expression of a gene that exhibits activity to enhance sensitivity to an Eg5 inhibitor by suppressing gene expression Antisense polynucleotide.

 (40) The siRNA or antisense polynucleotide according to (39), wherein the cancer cell is a lung cancer cell.

 (41) The Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof, or the siRNA according to (39) or (40). Or an antisense polynucleotide.

 (42) The Eg5 inhibitor is the cystine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof, or the siRNA according to (39) or (40). Antisense polynucleotide.

 (43) The siRNA or antisense polynucleotide according to (39) or (40), wherein the Eg5 inhibitor is the compound according to (13) or a pharmaceutically acceptable salt thereof.

 (44) A polynucleotide expressing the siRNA or antisense polynucleotide according to any one of (39) to (43).

 (45) An agent for enhancing sensitivity to an Eg5 inhibitor, comprising the siRNA or antisense polynucleotide according to any one of (39) to (43) or the vector according to (44).

(46) CYP24A1, 0BRGRP ヽ BF ヽ APRT ヽ BIRC3, SQSTMU TNFAIP2, ITM2B, ABCC2, ANXA3 _S Any one selected from FAM3C and ABCC3, by suppressing the expression of the gene in cancer cells, A sensitivity enhancer for an Eg5 inhibitor, comprising an antibody against a protein encoded by a gene exhibiting an activity of enhancing sensitivity to an Eg5 inhibitor.

 (47) The Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof.

(48) The sensitivity enhancer according to (46), wherein the Eg5 inhibitor is the cystine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof.

(49) The sensitivity enhancer according to (46), wherein the Eg5 inhibitor is the compound according to (13) or a pharmacologically acceptable salt thereof.

(50) A test substance is added to the cells, and at least one gene selected from the group consisting of CYP24A1, 0BRGRP, BF, APRT, BIRC3, SQSTMU TNFAIP2 _S ITM2B, ABCC2, ANXA3, FAM3C and ABCC3 in the cells is added. The expression level is measured, and a substance that decreases the expression of the gene compared to the expression level of the gene when no test substance is added is selected as a candidate substance that enhances the sensitivity to the Eg5 inhibitor. A method for screening a substance that enhances sensitivity to an agent.

 (51) The screening method according to (50), wherein the cell is a lung cancer cell.

(52) The screening described in (50) or (51), wherein the Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof. Method. (53) The Eg5 inhibitor is a cystine derivative according to any one of (10) to (12) or a pharmacologically acceptable salt thereof according to (50) or (51). Screening method.

 (54) The screening method according to (50) or (51), wherein the Eg5 inhibitor is the compound according to (13) or a pharmacologically acceptable salt thereof.

 Hereinafter, the compound represented by the general formula (I) is referred to as a compound (I), and the compound represented by the general formula (II) is referred to as a compound (II). Further, the compound represented by the general formula (A) is referred to as compound A. The same applies to compounds of other formula numbers.

 Hereinafter, embodiments of the present invention will be described in detail.

 (A) Method for identifying a gene involved in sensitivity of a cancer cell to an Eg5 inhibitor The method for identifying a gene involved in sensitivity to an Eg5 inhibitor of the present invention includes the following steps (a) to (c). .

 (a) measuring the sensitivity to an Eg5 inhibitor and the expression level of one or more human genes in two or more human cancer cell lines

 (b) analyzing the correlation between the expression level of the gene in each cell line and the sensitivity of the cell line to the Eg5 inhibitor, for each gene whose amount of germ was measured in step (a)

(c) Step of selecting genes that were correlated in step (b)

 (1) Human cancer cell line

 The human cancer cell line used for the measurement in the present invention may be any cell line as long as it is a human-derived cancer cell line, but as the number of types of cell lines increases, the correlation analysis in the above step (b) becomes more difficult. It is preferable because the obtained correlation value is likely to be significant. Specific cell line panels include 26 cell lines derived from lung cancer, A549, Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226, NCI-H345, N417, NCI-Satsu, NCI-H596, NCI-H1299, SBC-3, PC-14, PC-14 / DTX, PC-14 / CDDP, MRC-5, IMR-90, EBC-1, NCI-H292, SHP-77, NCI- H441, NCI-H838, A427, NCI-H522, Calu-6 and PC-9, Colo205, HT-29, HCT116, SW480, DLD-1 and WiDr, 6 cell lines derived from colorectal cancer, from kidney cancer A panel consisting of four cell lines, PANC-1, MIA PaCa-2, AsPC-1, and BxPC-3, and two cell lines derived from ovarian cancer, SK-0V-3 and 0VCAR-3 Or some can be used.

Also, the importance of expressed genes and their functions to cells differs depending on the organ, and it is conceivable that genes involved in sensitivity to Eg5 inhibitors differ depending on the organ from which cancer cells are derived. Therefore, by using a cancer cell line derived from one specific organ as the cell line used for the measurement, it is possible to identify a gene that is more involved in the sensitivity of the cancer cells of that organ to the Eg5 inhibitor. . If one wants to specifically identify genes related to the sensitivity of lung cancer cells to Eg5 inhibitors, for example, when using Eg5 inhibitors for the treatment of lung cancer, only lung cancer cell lines, for example, A549, a lung cancer-derived 26 cell line, Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226, NCI-H345 _S N417, NCI-H460, NCI-H596, NCI-H1299 _N SBC-3, PC-14, PC-14 / DTX, PC-14 / CDDP _S MRC-5, IMR-90, EBC-1, NCI-H292, SHP-77, NCI-H441NCI-H838, A427, NCI-H522, Calu-6 and PC-9 or all Using some As a result, a gene related to the sensitivity of an Eg5 inhibitor to lung cancer cells can be identified.

 (2) Eg5 inhibitor

 The Eg5 inhibitor used in the present invention is not particularly limited as long as it has Eg5 inhibitory activity. For example, compound (I) described in WO2004 / 092147 or a pharmacologically acceptable salt thereof, compound (II) Or a pharmacologically acceptable salt thereof,

Compound Q (SB-715992) or a pharmaceutically acceptable salt thereof described in WO01 / 030768, WO01 / 098278, etc., WO02 / 057244 _s麵 / 094839, WO03 / 097053, WO03 / 103575 WO03 / 106426s WO04 / 006865 , WO04 / 009036, WO04 / 024086, picture 4/032840,

WO04 / 034972. WO03 / 039460 _s WO03 / 049527 _s WO03 / 050122 WO03 / 050064,

¥ O03 / 049678 WO03 / 049679, WO03 / 079973, 麵 / 099211, picture / 105855,

WO04 / 03717U WO04 / 039774 _s W004 / 058700, Orchid 4/058148, WO02 / 078639

Compounds described in, for example, Sec./079149, Reference / 079169, WO03 / 099286, WO04 / 078758, etc., and preferably Compound (I) or a pharmacologically acceptable salt thereof, compound (Π) or Examples thereof include a pharmacologically acceptable salt thereof, Compound Q and a pharmacologically acceptable salt thereof.

 In the definition of each group of the general formulas (I) and (Π),

 (i) The lower alkyl moiety of the lower alkyl, lower alkoxy, lower alkylaminodialkyl or lower alkylamino includes, for example, linear or branched alkyl having 1 to 10 carbon atoms, specifically methyl, ethyl, propyl, Examples include isopropyl, butyl, isoptyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, and decyl.

 The two lower alkyl moieties of the di-lower alkylamino may be the same or different.

 (ii) Examples of the lower alkenyl include straight-chain or branched alkenyl having 2 to 8 carbon atoms, specifically, vinyl, aryl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and the like. '

 (iii) Examples of lower alkynyl include straight-chain or branched alkynyl having 2 to 8 carbon atoms, specifically ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and the like.

 (iv) Examples of cycloalkyl include cycloalkyl having 3 to 8 carbon atoms, specifically, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, cycloheptyl and the like.

 (v) The aryl portion of aryl, aryloxy and arylamino includes, for example, phenyl, naphthyl and the like.

(vi) Examples of the heterocyclic group include an aliphatic heterocyclic group and an aromatic heterocyclic group. Examples of the aliphatic heterocyclic group include a 5- or 6-membered monocyclic aliphatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, and a 3- to 8-membered ring. Condensed bicyclic or tricyclic condensed aliphatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, and the like; Specific examples include pyrrolidinyl, imidazolidinyl, piberidinyl, piperazinyl, morpholinyl, thiomorpholinyl, biperidino, morpholino, oxazolinyl, dioxolanyl, tetrahydroviranyl, and 1-decahydroxyisoquinolinyl. Examples of the aromatic heterocyclic group include a 5- or 6-membered monocyclic aromatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur _λ atom, a 3- to 8-membered ring And a condensed aromatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, which is condensed with Nil, pyrrolyl, pyridyl, pyrazinyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, isothiazolyl, thiazi.azolyl, oxazolyl, oxaziazolyl, pyrimidinyl, indolyl, isoindolyl, quinazolinazolyl, benzoimidazolyl, benzoimidazolyl, benzoimidazolyl , 1, 3—Ben Zozoxo Lou 5-yl.

 (vii) Examples of the aromatic heterocyclic group include the aromatic heterocyclic groups described in (vi) above.

 (vii i) Examples of the heterocyclic group formed together with an adjacent nitrogen atom include, for example, an aliphatic heterocyclic group containing at least one nitrogen atom. The aliphatic heterocyclic group containing at least one nitrogen atom may contain an oxygen atom, a sulfur atom or another nitrogen atom, such as pyrrolidinyl, morpholino, thiomorpholino, birazolidinyl, piperidino, piperazinyl, homopiperazinyl, Aziridinyl, azetidinyl, perhydroazepinyl, perhydroazosinyl, succinimidyl, pyrrolidonyl, gluimidium, piperidonyl and the like.

 (ix) Examples of cycloalkylene include cycloalkylene having 3 to 8 carbon atoms, specifically, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene and the like.

 (x) Phenylene includes 1,4-phenylene, 1,3-phenylene or 1,2-phenylene.

 (xi) Halogen means each atom of fluorine, chlorine, bromine and iodine.

(xii) The substituents in the substituted lower alkyl, substituted lower alkoxy, substituted lower alkenyl, substituted lower alkynyl, substituted cycloalkyl, substituted lower alkylamino and substituted dilower alkylamino are the same or different, for example, having 1 to 3 substituents. Halogen, hydroxy, oxo, nitro, azido, cycloalkyl, substituted or unsubstituted aryl (the substituent in the substituted aryl is the same as the substituent (xiv) in the substituted aryl described below), A substituted or unsubstituted heterocyclic group · (the substituent in the substituted heterocyclic group is synonymous with the substituent (XV) in the substituted heterocyclic group described later), — CONR "R ²⁸ , wherein R ²⁷ and R ²⁸ is the same or different and is a hydrogen atom, hydroxy, substituted or unsubstituted lower alkyl (substituent in the substituted lower alkyl) And the same or different, for example, having 1 to 3 substituents, such as hydroxy, lower alkoxy, oxo, carboxy, lower alkoxycarbonyl, substituted or unsubstituted aryl (substituents in the substituted aryl are those described below) Replacement reel A substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (XV) in the substituted heterocyclic group described later), Alkanol, substituted or unsubstituted aryloyl (substituents in the substituted arylo have the same meaning as the substituent (xiv) in the substituted aryls described below), halogen, one CONR ²⁹ R ³ ° [wherein R ^2S and ^{3} is the same or different and is a hydrogen atom, a hydroxy group, a substituted or unsubstituted lower alkyl (substituent (a) in the substituted lower alkyl is the same or different and is, for example, a substituted or unsubstituted hydroxy, , Gen, lower alkoxy, oxo, carboxy, lower alkoxycarbonyl, aryl, heterocyclic group, amino, lower alkylamino, dilower alkylamino Lower alkenyl, lower alkyl, cycloalkyl, substituted or unsubstituted aryl (substituents in the substituted aryl are the same as the substituent (xiv) in the substituted aryl described below), substitution Or an unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (XV) in the substituted heterocyclic group described later); a substituted or unsubstituted arylo (the substituent in the substituted aryloyl) Is the same as the substituent (xiv) in the substituted aryl described below) or an aralkyloxy group, or represents a substituted or unsubstituted heterocyclic group in which R ²⁹ and R ^3e are taken together with the adjacent nitrogen atom. (The substituent in the substituted heterocyclic group formed together with the adjacent nitrogen atom is a substituted complex formed together with the adjacent nitrogen atom described below.) Forming the substituent (XV) as synonymous) in], - NR ³¹ R ³² [wherein, R ³¹ and R ³² same or different, a hydrogen atom,啬換or unsubstituted lower alkyl (the The substituent in the substituted lower alkyl is a substituent in the substituted lower alkyl.

(a), lower alkenyl, lower alkynyl, cycloalkyl, substituted or unsubstituted aryl (substituents in the substituted aryl are substituents in the following substituted aryl (xiv) A substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (XV) in the substituted heterocyclic group described below); a substituted or unsubstituted lower alkanol ( The substituent in the substituted lower alkanol is the same as the substituent (a) in the above-mentioned substituted lower alkyl, a substituted or unsubstituted arylo (the substituent in the substituted arylo is a substituent in the above-mentioned substituted lower aryl (xiv) Or R aralkyloxycarbonyl, or R ³¹ and R ³² together with the adjacent nitrogen atom are substituted or unsubstituted Heterocyclic group (the substituent in the substituted heterocyclic group formed together with the adjacent nitrogen atom is the substituent (XV) in the substituted heterocyclic group formed together with the adjacent nitrogen atom described later) Substituted or unsubstituted lower alkenyl (the substituent in the substituted lower alkenyl has the same meaning as the substituent (a) in the above-mentioned substituted lower alkyl). Or unsubstituted lower alkynyl (the substituent in the substituted lower alkynyl is the same as the substituent (a) in the above-mentioned substituted lower alkyl); substituted or unsubstituted cycloalkyl (the substituent in the substituted cycloalkyl is The same as the substituent (a) in the above-mentioned substituted lower alkyl), substituted or unsubstituted aryl (the substitution in the substituted aryl) The group has the same meaning as the substituent (xiv) in the substituted aryl described below. A substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (XV) in the substituted heterocyclic group described later), a substituted or unsubstituted lower alkoxy (the substituted lower alkoxy group) The substituent in alkoxy is the same as the substituent (a) in the above-mentioned substituted lower alkyl. The substituted or unsubstituted lower alkanol (the substituent in the substituted lower alkyl is the substituent in the above-mentioned substituted lower alkyl. A substituted or unsubstituted aryloyl (the substituent in the substituted aryl is the same as the substituent (xiv) in the substituted aryl below), a substituted or unsubstituted aryloxycarbonyl (The substituent in the substituted aryloxycarbonyl is synonymous with the substituent (xiv) in the substituted aryl below hereinafter) or Lal Kill O carboxymethyl or a carbonyl, or R ²⁷ and substituents R ²⁸ is to be formed is a nitrogen atom and one cord in contact connexion such together with the adjacent nitrogen atom a substituted or unsubstituted Hajime Tamaki (該隣substituents on the heterocyclic group has the same meaning as substituent (XV) in the substituted heterocyclic group connexion formed such with below the adjacent nitrogen atom) to form formed a>, _C0 ₂ R ³³ {wherein, R ³³ is a hydrogen atom, a substituted or unsubstituted lower alkyl [substituent in the substituted lower alkyl] is the same or different, for example, having 1 to 3 substituents, hydroxy, halogen, lower alkoxy, oxo, carboxy; A lower alkoxycarbonyl, a substituted or unsubstituted aryl (the substituent in the substituted aryl is the same as the substituent (xiv) in the substituted aryl described below), a substituted or unsubstituted aryl. Heterocyclic group (the substituent in the substituted heterocyclic group "is synonymous with, - CONR ³ in ¾ ³⁵ (wherein, R ³⁴ and R ³⁵ described below substitutions in substituted double heterocyclic group group (χν)) 'respectively R ²⁹ and R are as defined above), ³⁶ R ³⁷ per dish (in the formula, ^ and R ³⁷ are each as defined above as R ³¹ and R ³² ) and the like.] Substituted or unsubstituted lower Alkenyl (the substituent in the substituted lower alkenyl has the same meaning as the substituent (b) in the above-mentioned substituted lower alkyl), substituted or unsubstituted lower alkynyl (the substituent in the substituted lower alkenyl is the above-mentioned substituted lower alkynyl A substituent (b) in alkyl, a substituted or unsubstituted cycloalkyl (substituent in the substituted cycloalkyl is a substituent in the above-mentioned substituted lower alkyl)

a substituted or unsubstituted aryl (the substituent in the substituted aryl is the same as the substituent (xiv) in the substituted aryl below) or a substituted or unsubstituted heterocyclic group (Wherein the substituent in the substituted heterocyclic group has the same meaning as the substituent (xv) in the substituted heterocyclic group described below), —COR ³⁸ (wherein, R ³⁸ has the same meaning as R ³³ ), — NR ³⁹ R ^{4 (wherein} , R ³⁹ and R ^M are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl {substituent in the substituted lower alkyl,

(c) may be the same or different and is, for example, hydroxy, halogen, lower alkoxy, oxo, carboxy, lower alkoxycarbonyl, substituted or unsubstituted aryl having 1 to 3 substituents (substituted aryl) Is the same as the substituent (xiv) in the below-mentioned substituted aryl, a substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the substituent in the following substituted heterocyclic group) (Synonymous with (XV)), one 0 (CH ₂ C¾0) _n R "(where n represents an integer of 1 to 15 and R" represents a lower alkyl), and one CONR ⁴² R ⁴³ ( Wherein R ⁴² and R ⁴³ are the same as R ²⁹ and R ³ °, respectively. Is defined), -S0 ₂ ⁴ [wherein, R "is lower alkyl, lower alkenyl, lower alkynyl, substituted or unsubstituted Ariru (substituents in the substituted Ariru the substituent in the later replacement Ariru (xiv) synonymous) or a substituted or unsubstituted Hajime Tamaki (the substituent in the substituted heterocyclic group of the substituent in the later of the substituted heterocyclic璟基(XV) represents a synonymous)], - dish ⁴ ¥ ⁶ (Wherein, and R «are the same as defined above for R ³¹ and R ³² ), respectively; and substituted or unsubstituted lower alkenyl (the substituent in the substituted lower alkenyl is the above-mentioned substituted lower alkyl) Substituent (c) is the same as defined above, substituted or unsubstituted lower alkynyl (the substituent in the substituted lower alkynyl is the same as the substituent (c) in the above-mentioned substituted lower alkyl) A) substituted or unsubstituted cycloalkyl (the substituent in the substituted cycloalkyl has the same meaning as the substituent (c) in the substituted lower alkyl described above), a substituted or unsubstituted aryl (the substitution in the substituted aryl) The group is the same as the substituent (xiv) in the substituted aryl described below), the substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (XV) in the substituted heterocyclic group described later) A substituted or unsubstituted lower alkanol (the substituent in the substituted lower alkyl group is a substituent in the above-mentioned substituted lower alkyl)

(c) as synonymous), one COR ⁴⁷ "[wherein, ⁷ represents a hydrogen atom, a substituted or unsubstituted lower alkyl (the substituent in said substituted lower alkyl is that put the substituted lower alkyl substituent (c )), Substituted or unsubstituted lower alkenyl (the substituent in the substituted lower alkenyl is the substituent in the above-mentioned substituted lower alkyl)

a substituted or unsubstituted lower alkynyl (substituents in the substituted lower alkynyl are the same as the substituents (c) in the substituted lower alkyl). Alkyl (the substituent in the substituted cycloalkyl is the same as the substituent (c) in the above-mentioned substituted lower alkyl); substituted or unsubstituted aryl (the substituent in the substituted aryl is described below). The same as the substituent (xiv) in the substituted aryl, the substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (xv) in the substituted heterocyclic group described later) NR ⁴⁸ R "(wherein R ⁴⁸ and R ^M are each as defined above for R ³¹ and R ³² ) or — 0R ⁵ ° wherein R ^{5 ()} is a hydrogen atom, Substituted or unsubstituted lower alkyl (the substituted The substituent in lower alkyl is the same as the substituent (c) in the above-mentioned substituted lower alkyl), lower alkenyl, lower alkynyl, cycloalkyl, substituted or unsubstituted aryl (substituents in the substituted aryl are described later) Or a substituted or unsubstituted heterocyclic group (the substituent in the substituted heterocyclic group is the same as the substituent (xv) in the substituted heterocyclic group described below). (wherein, R ⁵¹ is the R ⁴⁷ as synonymous) represents} or a S0 ₂ R ⁵¹ or represents, or R ³⁹ and R ^w are properly even together such connexion substituted with the adjacent nitrogen atom of unsubstituted Heterocyclic group (The substituent in the substituted heterocyclic group formed together with the adjacent nitrogen atom is the substituent (xv) in the substituted heterocyclic group formed together with the adjacent nitrogen atom described below. Forming a righteous)>, in one ^{N + R 52 R 53 R 54} r ( wherein, R ⁵² and R ⁵³ are the same or different and each represents lower alkyl or R ⁵² and R ^53, is Together with an adjacent nitrogen atom forms a heterocyclic group, R ⁵⁴ represents lower alkyl, X represents chlorine, bromine or iodine atom), —OR ⁵⁵ {wherein R ⁵⁵ is substituted Or unsubstituted lower alkyl (substituent (c) in the substituted lower alkyl is the same as substituent (c) in the above-mentioned substituted lower alkyl). Substituted or unsubstituted lower alkenyl (the substituted lower alkenyl) The substituent in alkenyl has the same meaning as the substituent (c) in the above-mentioned substituted lower alkyl), substituted or unsubstituted lower alkynyl (the substituent in the substituted lower alkynyl is the substituent in the above-mentioned substituted lower alkyl) A substituted or unsubstituted cycloalkyl (the substituent in the substituted cycloalkyl is the same as the substituent (c) in the above-mentioned substituted lower alkyl) A substituted or unsubstituted aryl (the substituent in the substituted aryl is the same as the substituent (x iv) in the substituted aryl described below), a substituted or unsubstituted heterocyclic group (in the substituted heterocyclic group The substituent has the same meaning as the substituent (XV) in the later-described substituted heterocyclic group) or —COR ⁵⁶ (wherein R ⁵⁶ has the same meaning as above)}, -SR ⁵⁷ (wherein , R "has the same meaning as R ⁵⁵ described above; -S0 ₂ ^{68 wherein} R ⁵⁸ is a substituted or unsubstituted lower alkyl (the substituent in the substituted lower alkyl is the substituent (c in the above-mentioned substituted lower alkyl) )) Substituted or unsubstituted lower alkenyl (the substituent in the substituted lower alkenyl is the same as the substituent (c) in the above-mentioned substituted lower alkyl), and substituted or unsubstituted lower alkynyl (The substituted lower The substituent in rukinyl has the same meaning as the substituent (c) in the above-mentioned substituted lower alkyl), substituted or unsubstituted cycloalkyl (the substituent in the substituted cycloalkyl is the substituent in the above-mentioned substituted lower alkyl)

(substituted with (c)), substituted or unsubstituted aryl (substituents in the substituted aryl are the same as substituent (xi v) in the substituted aryl below), substituted or unsubstituted complex A cyclic group (the substituent in the substituted heterocyclic group has the same meaning as the substituent (XV) in the substituted heterocyclic group described below) or one NR ⁵⁹ R ^6D (wherein R ^{59 and 11 are each} as defined above) represents an R ³¹ and R ³² as synonymous)] - 0s0 in ₂ R ^sl (wherein, R ⁶¹ is the R ⁵⁸ as synonymous), and the like.

 Lower alkyl, lower alkoxy, lower alkoxycarbonyl, lower alkylamino, lower alkyl portion of di-lower alkylamino and lower alkynyl, lower alkenyl, lower alkynyl, cycloalkyl, aryl, aryl and arylo shown here The aryl moiety, the heterocyclic group, the heterocyclic group formed together with the adjacent nitrogen atom, and the halogen of the xycarbonyl are the lower alkyl (i), the lower alkenyl (ii) and the lower alkynyl, respectively. (Iii) synonymous with cycloalkyl (iv), aryl (V), heterocyclic group (vi), heterocyclic group (vi ii) formed together with adjacent nitrogen atom, and halogen (xi) is there. The aralkyl part (xiii) of the aralkyloxycarbonyl shown here includes, for example, aralkyl having 7 to 15 carbon atoms, specifically, penzyl, phenethyl, benzhydryl, naphthylmethyl and the like.

(xiv) a substituted aryl, a substituted aryloxy, a substituted arylamino, a substituted phenylene, a substituted aromatic heterocyclic group and a substituted aromatic heterocyclic group among the substituted heterocyclic groups; Examples of the substituents in the substituted or unsubstituted lower alkyl are the same or different, for example, halogen, hydroxy, nitro, cyano, carboxy, formyl, methylenedioxy, substituted or unsubstituted lower alkyl having 1 to 3 substituents. Examples of the same or different are, for example, halogen, oxo, carboxyl, lower alkoxycarbonyl, amino, lower alkylamino, di-lower alkylamino, hydroxy, lower alkoxy, aryl and heterocyclic groups having 1 to 3 substituents. Substituted or unsubstituted lower alkenyl (the substituent in the substituted lower alkenyl has the same meaning as the substituent (d) in the above-mentioned substituted lower alkyl), substituted or unsubstituted lower alkynyl (in the substituted lower alkynyl The substituent is as described above. A substituent (d) having the same meaning as the substituent (d) in lower alkyl, a substituted or unsubstituted cycloalkyl (the substituent in the substituted cycloalkyl has the same meaning as the substituent (d) in the above-mentioned substituted lower alkyl), Substituted or unsubstituted lower alkoxy (the substituent in the substituted lower alkoxy is the same as the substituent (d) in the above-mentioned substituted lower alkyl), substituted or unsubstituted lower alkylthio (the substituent in the substituted lower alkylthio) Is the same as the substituent (d) in the above-mentioned substituted lower alkyl), amino, substituted or unsubstituted lower alkylamino (the substituent in the substituted lower alkylamino is the substituent (d )), Substituted or unsubstituted di-lower alkylamino-(substituted di-lower alkyl) The substituent in amino is the same as the substituent (d) in the above-mentioned substituted lower alkyl), the substituted or unsubstituted lower alkanol (the substituent in the substituted lower alkyl is the substituent in the above-mentioned substituted lower alkyl ( substituted or unsubstituted lower alkanoyloxy (substituents in the substituted lower alkanoyloxy are the same as the substituents (d) in the above substituted lower alkyl), substituted or unsubstituted (Substituted in the substituted lower alkanoylamino is the same as the substituent (d) in the substituted lower alkyl), substituted or unsubstituted lower alkanoylthio (substituted lower alkanoylthio) Is the same as the substituent (d) in the above-mentioned substituted lower alkyl), Substituted or unsubstituted lower alkylaminocarbonyl (the substituent in the substituted lower alkylaminocarbonyl has the same meaning as the substituent (d) in the above substituted lower alkyl), substituted or unsubstituted lower alkylamino Carbonyloxy (the substituent in the substituted lower alkylaminocarbonyloxy has the same meaning as the substituent (d) in the above-mentioned substituted lower alkyl); substituted or unsubstituted di-lower alkylaminocarbonyl (the substituted lower alkylaminocarbonyl); The substituent in the di-lower alkylaminocarbonyl has the same meaning as the substituent (d) in the above-mentioned substituted lower alkyl), the substituted or unsubstituted lower alkoxycarbonyl (the substituent in the substituted lower alkoxycarbonyl) is as described above. Is the same as the substituent (d) in the substituted lower alkyl). Is an unsubstituted di-lower alkylaminocarbonyloxy (the substituent in the substituted di-lower alkylaminocarbonyloxy is the same as the substituent in the above-mentioned substituted lower alkyl), and the substituted or unsubstituted aryl is The substituent (e) in substitution or aryl is the same or Is different and includes, for example, halogen having 1 to 3 substituents, such as halogen, hydroxy, lower alkoxy, cyano, nitro, carboxy, lower alkoxycarbonyl, amino, lower alkylamino, di-lower alkylamino, etc. A heterocyclic group (the substituent in the substituted heterocyclic group has the same meaning as the substituent (e) in the above-mentioned substituted aryl), a substituted or unsubstituted arylsulfonyl (the substituent in the substituted arylsulfonyl is A substituent (e) having the same meaning as the substituent (e) in the substituted aryl, a substituted or unsubstituted aryloxy (the substituent in the substituted aryl is the same as the substituent (e) in the substituted aryl). Substituted or unsubstituted arylamino (substituents in the substituted arylamino are as defined above) Substituted or unsubstituted arylthio (substituted in the substituted aryl) (substituents in the substituted aryl are the same as the substituent (e) in the substituted aryl), substituted or unsubstituted Substituted aryl (the substituent in the substituted aryl is synonymous with the substituent (e) in the above-mentioned substituted aryl), substituted or unsubstituted arylooxy (the substituent in the substituted aryl is the above-mentioned substituent) The same as the substituent (e) in the substituted aryl, the substituted or unsubstituted arylothio (the substituent in the substituted arylothio is the same as the substituent (e) in the above substituted aryl), Unsubstituted arylamino (substituent in the substituted arylamino is the same as the substituent (e) in the above-mentioned substituted aryl) A) substituted or unsubstituted heterocyclic amino (the substituent in the substituted heterocyclic amino is the same as the substituent (e) in the above substituted aryl), and a substituted or unsubstituted heterocyclic oxy (the substituted The substituent in the heterocyclic oxy is the same as the substituent (e) in the above-mentioned substituted aryl, the substituted or unsubstituted heterocyclic thio (the substituent in the substituted heteroaryl is the same as the substituent in the above-mentioned substituted aryl) Group (e)).

Lower alkyl, lower alkoxy, lower alkylthio, lower alkylamino, di-lower alkylamino shown here (the two lower alkyl portions of the di-lower alkylamino may be the same or different), lower alkanol, lower alkanoyloxy , Lower alkanoylamino, lower alkanoylthio, lower alkylaminocarbon, lower alkylaminocarbonyloxy, di-lower alkylaminocarbonyl (the two lower alkyl moieties of the di-lower alkylaminocarbonyl may be the same or different Good), di-lower alkylaminocarbonyloxy (the two lower alkyl moieties of the di-lower alkylaminocarbonyloxy may be the same or different), lower alkoxycarbonyl and di-lower alkylaminocarboxy The lower alkyl portion of oxy (the two lower alkyl portions of the di-lower alkylaminoaminocarbonyloxy may be the same or different) has the same meaning as the lower alkyl (i) and is a lower alkenyl, lower alkynyl, Cycloalkyl and halogen are as defined above for lower alkenyl (ii), lower alkynyl (iii), cycloalkyl (iv) and halogen (xi), respectively. In addition, the aryl parts of aryl, arylsulfonyl, aryloxy, arylthio, arylamino, arylo, aryloyoxy, aryloythio and aryloyamino shown here The heterocyclic group, the heterocyclic amino, the heterocyclic oxy, and the heterocyclic group portion of the heterocyclic thio are the same as the above-mentioned aryl (V), and are the same as the above-mentioned heterocyclic group (vi).

 (xv) The substituted heterocyclic group of the substituted heterocyclic group and the substituted heterocyclic group formed by complexing the adjacent nitrogen atom are the same as the substituent (xiv In addition to the groups mentioned in the definition of), oxo and the like can be mentioned.

 The pharmacologically acceptable salts of compounds (I), (II) and Q are, for example, pharmacologically acceptable; acid addition salts, metal salts, ammonium salts, organic amine addition salts, caro salts with amino acids, etc. Is included. Examples of the pharmacologically acceptable caroate with an acid of the compounds (I), (Π) and Q include, for example, inorganic salts such as hydrochloride, sulfate, phosphate, acetate, maleate, and fumarate. And pharmacologically acceptable metal salts, for example, alkali metal salts such as sodium salt and potassium salt, and alkaline earth salts such as magnesium salt and calcium salt. Pharmacologically acceptable ammonium salts include, for example, salts of ammonium, tetramethylammonium, etc., and pharmacologically acceptable organics. Amine addition salts include, for example, carbohydrate salts such as morpholine and piperidine, and pharmacologically acceptable amino acid addition salts include, for example, lysine, glycine, Two Ruaranin, Asuparagin acid addition salts there up 'is such as glutamic acid.

 Compound (I) can be produced by the method described in WO03 / 051854 or WO2004 / 092147, or according to it.

 Compound (II) can be produced by the method described in J. Med. Cem., 13, 414-418, 1970 or J. Med. Chem., 15, 13-16, 1972, or according to them. it can. Compound Q can be produced, for example, by the method described in WO03 / 070701 or a method analogous thereto.

 Some of the compounds (I), (II) and Q may have stereoisomers such as positional isomers, geometric isomers, optical isomers and tautomers. All possible isomers, including these, and mixtures thereof can be used for identification.

 When it is desired to obtain salts of compounds (I), (Π) and Q, when compounds (I), (II) and Q are obtained in the form of a salt, they can be purified as they are, and in the free φ form When it is obtained, compounds (I), (II) and Q may be dissolved or suspended in an appropriate solvent, and then isolated and purified by forming a salt by adding an acid or a base.

 Compounds (I), (II) and Q and their pharmacologically acceptable salts may exist in the form of adducts with water or various solvents, and these adducts are also used in the present invention. It can be used for a method for identifying a gene and the like.

Specific examples of the compounds (I) and (II) are shown in Tables 1 and 2. Table 1

 (I)

^{R 1 R 2 R 3 R 4} R 6 Compound AH C0CH ₃ C0C¾ CH _3. O compound _{BH COC (CH 3) 3 COC} (CH 3) 3 (C¾) 2 C0N¾ O compound _{c H COC (CH 3) 3} C0C ( C¾) ₃ , (CH ₂ ) ₂ NHS0 ₂ CH ₃ O compound EH COCH (CH ₃ ) ₂ COCH (CH ₃ ) ₂ • (CH ₂ ) ₂ NHS0 ₂ CH ₃ O compound FH COC (CH ₃ ) ₃ coc (c¾ ) ₃ CH ₂ NHS0 ₂ CH ₂ G¾ -Ό Compound GH COC (CH ₃ ) ₃ coc (c¾) ₃ CH ₂ CH ₂ N (C¾) ₂ -O Compound HH COC (CH ₃ ) ₃ COC (CH ₃ ) ₃ CH ₂ CH ₂ CONHCH ₂ CH ₂ OH O compound IH C0CH ₃ COCH3 c¾ cr compound JH C0C (C¾) ₃ COC (CH ₃ ) ₃ CH ₂ NHS0 ₂ CH = CH ₂ O compound KH COC (CH ₃ ) ₃ COCHg CH ₂ thigh S0 ₂ C¾C¾N (CH ₃ ) ₂ O compound LH COC (CH ₃ ) ₃ C0C (C¾) ₃ C¾NHS0 ₂ C¾C¾NC¾CH ₃ O compound MH coc (c¾) ₃ COC (CH ₃ ) ₃ CH ₂ C¾NH ₂ O compound NH COC (CH ₃ ) ₃ COC (CH ₃ ) ₃ CH ₂ NHS0 ₂ (CH ₂ ) ₃ N¾ O compound 0 H COC (CH ₃ ) ₃ COC (CH ₃ ) ₃ CH ₂ NHS0 ₂ (CH ₂ ) ₃ N (CH ₃ ) ₂ O compound _{PH C0C (CH 3) 3 C0C} (CH 3) 3 CH 2 NHS0 2 CH 2 CH 2 N (CH 3) CH 2 C¾ O Table 2

 (ID

H H 次に、 化合物 （I) および化合物 （II) の Eg5阻害作用について、 試験例を用 いて説明する。 R ²⁴ R ²⁵ R ²⁶ Compound D

HH Next, the Eg5 inhibitory activity of compound (I) and compound (II) will be described using test examples.

Test Example 1 Phenotypic analysis of M-phase cells by immunocytochemistry

 M-phase phenotyping by immunocytochemical staining was performed with reference to the literature (Oncogene, 19, 5303-5313, 2000). A human lung cancer cell line A549 (ATCC number: CCL-185) was cultured with the test compound for 17 hours. After washing with phosphate buffered saline (PBS), the cells were fixed with methanol kept at -20 ° C for 1 minute to fix the cells. After washing with PBS, the cells were permeated with PBS containing 0.2% Triton-X for 15 minutes. After washing with PBS, the cells were blocked with a blocking solution (PBS containing 1% fetal bovine serum) for 30 minutes, and the primary antibody solution (0.2% mouse monoclonal anti-tubulin antibody [Sigma-Aldrich] , Cat. No. T9026] and a blocking solution containing 0.2% Egret anti-tubulin antibody (Sigma Aldrich, Cat. No. 3559) for 30 minutes. After washing with PBS, secondary antibody solution

 {0.025% Alexa Fluor 546 conjugated anti-mouse IgG antibody (Molecular Probes, Catalog No. A-11030), 0.5% Alexa フ ロ ー ow 488 conjugated anti-mouse IgG antibody (Molecular Microtubules, centrosomes, and chromosomes were visualized by reacting with Blocking solution containing 1-mol / l Hoechst 33342} for 30 minutes. The phenotype of M-phase cells was observed using an inverted fluorescence microscope. When compound A or compound D (Acros) was used as the test compound, cells that had accumulated in the M phase by compound A and compound D were converted into single-star microtubules (Monastral 'microtubule' array), single cells. It showed a characteristic phenotype of the polar centrosome (monopolar one-sbindle) and the localization of circularly distributed chromosomes. Such M-phase phenotypes have been demonstrated by neutralizing antibodies against ¾5 (Cell, M, 1159-1169, 1995) and the Eg5-specific inhibitor monastrol (Science, 286, 971-974, 1999) described in the literature. The phenotype was similar to the treated cells.

 These results suggest that Compound A and Compound D inhibit Eg5.c Test Example 2 Inhibition test on enzyme activity of Eg5

For preparation of full-length recombinant human Eg5, refer to the literature (Cell, 83> 1159-1169, 1995). It was carried out. Baculoviruses expressing full-length human Eg5 with His tag fused to the N-terminus were infected into Spodoptera frugiperda Sf9 insect cells, and after culturing, the culture was centrifuged to collect the cells. The collected cells were suspended in a buffer and the supernatant was collected by centrifugation. The supernatant was passed through a nickel agarose column, and Eg5 fused with a His tag at the N-terminal was affinity purified to obtain a partially purified sample.

ATPase activity of Eg5 was measured with reference to the literature (EMBO J., 13, 751-757, 1994; Proc. Natl. Acad. Sci. U.S..A., 89> 4884-4887, 1992). did. 25 mmol / 1 piperazine N, N, monobis (ethanesulfonate) (PIPES) / KOH (pH 6.8), 1 tol ol8 ethylene glycol bis (2 aminoethyl ether) ) Tetraacetic acid (EGTA), 2 mmol / 1 MgCl ₂ , 1 tmol ol dithiothreitol (DTT), 5 zmol / 1 paclitaxel, 100 ug / ml ゥ serum albumin, 25 〃g / ml tubulin [ Cytoskeleton, Cytoskeleton, TL238], 200 uml / l 2-amino-6-mercapto-7-methylpurine riboside (MESG substrate _N Molecular Props, catalog number E- 6646), 1 U / ml purine nucleoside phosphorylase (Molecular Props, Rikiyu Log No.E-6646), 1 mraol / 1 ATP, 12.5 μg / ml Eg5 An enzymatic reaction was performed on the solution. The enzyme reaction was performed at 30 ° C for 30 minutes. The absorbance at 360 wake, which is an indicator of ATPase activity, was measured using a plate reader (Molecular Devices, SpectraMax 340PC ³⁸⁴ ). The relative activity was calculated assuming that the absorbance in the absence of the compound in the presence of Eg5 was 100% and the absorbance in the absence of the compound in the absence of Eg5 was 0%, and the _IC5Q value was calculated.

When measured using compound A or compound D as the test compound, compound A and compound D inhibited the ATPase activity of Eg5 in a concentration-dependent manner, and the _IC5Q value of compound A was 2 / mol / l. 0.8 〃mol eight.

 As described above, the results of Test Example 1 and Test Example 2 showed that Compound A and Compound D had an Eg5 inhibitory action. That is, it was shown that compound (I) and compound (II) can be used as E5 inhibitors.

 (3) Method for measuring sensitivity to anticancer drugs

In the present invention, as a method for measuring the sensitivity of cancer cells to an anticancer agent, the method includes contacting an Eg5 inhibitor with the human cancer cell of (1) for a certain period of time, and then, regarding the cell, the number of living cells, the growth rate of XA or the total protein. There is a method of measuring the amount. Sensitivity can be determined, for example, by performing the above measurement for various concentrations of the Eg5 inhibitor, creating a calibration curve showing the correlation between the concentration of the Eg5 inhibitor and the measured value, and using the same method for cells that were not contacted with the Eg5 inhibitor. Eg5 inhibitor concentration (GI ₅ ) that gives a value equivalent to 50% when the measured value is taken as 100%, or its logarithm when the concentration of G is expressed in mol / 1

(l og ₁₀ GI ₅₀ ) or-l og _{1 ()} GI ₅ . Can be represented by The number of viable cells is measured by a method using a color reaction such as the MTT method (J. Immunol. Methods, 65> 55-63, 1983) and the XTT method (J. Immunol. Methods, 142, 257-265, 1991). can do. DNA synthesis rate can be measured by measuring the uptake of thymidine labeled with ¾ into cells. Can be measured. 'Total protein content can be measured by the Sulfolodamine B Atsey method or the like.

 (4) Measurement of gene expression level

 In the present invention, the expression level of a gene can be measured by quantifying mRNA that is a gene transcription product or protein that is a gene product.

 (i) RNA or protein extraction

 RNA or protein can be extracted from the human cancer cell line of (1) by the following general method. In the case of RNA, for example, Trisol (TRIZ0L) reagent [Invitrogen

 (Invitrogen)] may be used according to the attached operation method. In the case of proteins, it may be performed according to a written book such as, for example, The Protein Handbook, Humana Press (1996).

 (ii) mRNA quantification

 The quantification of mRNA can be performed by using techniques such as DNA microarray, Northern plot analysis, and RT-PCR using the RNA extracted in (1) as a measurement sample. Also, an RNA quantification reagent containing a polynucleotide complementary to the RNA for use in RT-PCR is included in the scope of the present invention.

 (i ii) Protein quantification

 Protein quantification can be performed by immunochemical quantification using specific antibodies. Monoclonal or polyclonal antibodies can be prepared according to the method described in, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988). When a commercially available antibody is available, it can be used after confirming its specificity. Using the obtained antibody, ELISA, RIA, and Western plotting can be performed to quantify the protein according to the method described in a written document (eg, “Enzyme Immunoassay”, Medical Shoin (1982)). In addition, an immunoassay reagent containing an antibody against the protein for use in the above method is also included in the scope of the present invention.

 The expression level of the gene is represented by the signal intensity per a certain amount of RNA or protein, or the logarithm thereof, measured by the method (i i) or (i ii) described above. When NA is quantified by real-time PCR, a type of RT-PCR, it can also be expressed as the number of reaction cycles (Ct value) at which the amplification reaction progresses logarithmically and reaches a certain signal intensity.

 (5) Gene selection

In the present invention, for each gene, the correlation between the expression level in each cell line and the sensitivity of the cell line to the Eg5 inhibitor is analyzed, and the correlation between the expression level and the sensitivity of the cell line to the Eg5 inhibitor is analyzed. To select genes with The correlation analysis performed here assumes that when the expression level of a gene and the sensitivity to an Eg5 inhibitor are expressed by logarithm of signal intensity, Ct value, logarithm of G, etc., the values are assumed to be normally distributed. This can be done by calculating the Son's correlation coefficient (hereinafter simply referred to as the correlation coefficient). The presence or absence of a correlation is determined by whether or not the correlation coefficient is 0. When the correlation coefficient is 0, there is no correlation, and when it is not 0, there is correlation. The correlation coefficient is It takes a value between 1 and -1. The closer the absolute value of the correlation coefficient is to 1, the higher the correlation, and the closer to 0 the lower the correlation. The significance of the obtained correlation coefficient can be tested by the P value (probability that the correlation coefficient of the population becomes 0) obtained from the number of samples based on the null hypothesis. For example,? If the value is <0.05, the correlation coefficient is significant at the 5% significance level. Genes whose expression levels are negatively correlated with the sensitivity to Eg5 inhibitors, ie, genes with lower expression levels in cells with higher sensitivity to Eg5 inhibitors (hereinafter also referred to as resistance genes) are selected as described above. Examples include the following 12 genes.

 (i) CYP24A1: Cytochrome P450 'family 24 · subfamily A' polypeptide 1 (顺 —0Q0782)

 (ii) 0BRGRP: lebutin receptor gene-related protein (丽 _017526)

 (i ii) BF: Factor B (丽 —001710)

 (iv) APRT: Adenine phosphoribosyltransferase (丽 —000485)

 (v) BIRC3: Baculovirus IAP repeat-containing protein— (丽 —001165)

 (vi) SQSTM1: Sequence 1 (O-003900)

 (vii) TNFAIP2: tumor necrosis factor-inducing protein 2 (NM_006291)

 (vii i) ITM2B: integral membrane protein 2B (丽 —021999)

 (ix) ABCC2: ATP binding cassette 'Subfamily 1 C · Member 2 (Marauder-000392) (X) ANXA3: Annexin A3 (Thigh-005139)

 (xi) FAM3C: Homologous sequence 3 containing family 1 'member 1 3 (NMJ14888)

 (xii) ABCC3: ATP-binding cassette 'subfamily-1C-member-13 (NM_003786) In addition, a gene with a positive correlation, that is, a gene having a higher expression level in a cell that is more sensitive to an Eg5 inhibitor (hereinafter, a susceptibility gene) The following 7 genes can be mentioned as examples.

 (i) BCO07436: hypothetical protein BC007436 (XM-037317)

 (ii) MAP1B: microtubule-associated protein 1B (band-005909)

 (iii) CCNB1: Cyclin Bl (丽 一 031966)

 (iv) HSPCA: one heat shock 90 kDa protein (BC023006)

 (V) CGNB2: Cyclin B2 (thigh _004701)

 (vii) FLJ23269: Virtual protein F23269 (AK026922)

 (vii) SLC12A2: Solute Carrier 1 · Family 1 12 · Member 2 (NMJ01046) These genes are defined as genes with a rooster sequence of the DNA sequence shown in parentheses based on the GenBank accession number. .

 Genes identified using one Eg5 inhibitor as genes whose expression levels correlate with sensitivity to the Eg5 inhibitor are correlated not only with the Eg5 inhibitor but also with the sensitivity to other Eg5 inhibitors. It is considered a certain gene.

 (B) Method for determining sensitivity of cancer cells to Eg5 inhibitor

The present invention further provides a method for determining the sensitivity of a cancer cell to a 5-inhibitor. As a determination method, a method including the following steps (a) to (e) is exemplified. (a) Select one or more genes from the genes identified by the method (A) above, and For determining the score corresponding to the expression level of

 (b) a step of measuring the expression level of the gene in cancer cells for determining sensitivity

 (c) assigning, for each gene, the score determined in step (a) based on the expression level measured in step (b).

 (d) A step of obtaining a value that is an index of sensitivity from the assigned score for all selected genes

 (e) determining the sensitivity to the Eg5 inhibitor by comparing the predetermined threshold with the value determined in (d).

 The above-mentioned score determination, calculation of a numerical value as an index of sensitivity, and comparison with a threshold value are performed by multiple regression analysis, discriminant analysis, SVM (support vector-machine) method, principal component analysis, self-organizing map method, etc. This can be done by using a multivariate analysis method (described in Experimental Data Analysis by SAS, Keisuke Takeuchi, Tokyo University Press, 1990, etc.).

Hereinafter, each step in the case of using the multiple regression analysis will be described. The selected genes are n, susceptibility Y for Eg5 inhibitors - in log _1Q GI ₅ ", when expressed the expression level of each gene in the logarithm of the Ct value or signal strength, the multiple regression analysis model, the expression level of a gene Using X as an explanatory variable, the sensitivity Y, which is the target variable, can be expressed by the following equation 1. Y = a ₁ X ₁ + a ₂ ¾ + · · · + a _n X _n + b (Equation 1),

Y: target variable (Sensitivity:-lo ^ GI

, X _2, ...: independent variables (gene expression level: Ct value or the logarithm of the signal _{intensity) a 1 a 2, -a n} : partial regression coefficients

b: Constant term

 Hereinafter, the case where the expression level of each gene is represented by a Ct value will be described.When the logarithm of the signal intensity is used, the determination can be similarly performed using the logarithm of the signal intensity instead of the Ct value. .

By substituting the measured values of the sensitivity Y (-log ₁₀ GI ₅₀ ) and the expression level of each gene, X ₂ ,... (Ct value) for n + 1 cell lines into this equation, respectively. , to create a (n + 1) first-order linear polynomial, by solving these primary linear polynomial, it is possible to obtain the partial regression coefficients _{a ls} a ₂ '·· & η and a constant term b for each gene . The gene used in this method can be selected arbitrarily from the group of genes identified by the method (A) above, but it can be used for criteria such as Cp statistic, adjusted coefficient of freedom, and Akaike's information criterion. It is desirable to use the statistics as an index for optimization and to select genes so that the index becomes smaller. In this way, by selecting genes important for predicting the sensitivity to an Eg5 inhibitor, it is possible to improve the prediction accuracy, that is, to perform optimization while suppressing complexity. In the case of the multiple regression analysis model, the score of each gene is expressed as the product of the partial regression coefficient obtained above and the expression level of the gene.

Examples of genes that are important for predicting the sensitivity to 5 inhibitors, which are selected when the multiple regression analysis model is optimized, include the relationship between the expression level selected in (A) and the sensitivity to Eg5 inhibitors. CYP24A1, 0BRGRP, APRT ヽ NFAIP2, ITM2B, ABCC2, BC007436, MAP1B ヽ HSPCA, selected from the 19 genes listed as correlated genes CCNB2 and FLJ23269. "The expression level (Ct value) of the gene selected as described above in cancer cells for which sensitivity is to be determined can be measured by the method described in (A) (3) above. From the Ct value of the above, the score of each gene described above, that is, the product of the partial regression coefficient a obtained above and the Ct value is assigned.Based on Equation 1, the constant term b is added to the sum of the assigned scores of each gene. by adding the value, sensitivity of the indicators and ing numerical -. 10§ _{1 (^ 1 5} is required therefore -. as the threshold log _1G GI _{5 (),} for example, selection of the gene of the (a) By setting an appropriate numerical value such as the average value of the sensitivity of each cancer cell (-log _{10 GI5O} ) measured at the time of Sensitivity to the agent can be determined.

 By using a cancer cell derived from a cancer tissue extracted from a cancer patient by a biopsy or the like as the cancer cell, the sensitivity of the cancer cell of the cancer patient to the Eg5 inhibitor can be determined. When the cancer cells derived from the cancer patient are determined to be highly sensitive to the Eg5 inhibitor, the treatment can be effectively performed by using the Eg5 inhibitor for the treatment.

 (C) Selection of genes that regulate cancer cell sensitivity.

 Whether the gene determined to be correlated with the sensitivity to the Eg5 inhibitor by the method (A) actually defines the sensitivity, that is, the sensitivity to the Eg5 inhibitor by changing the gene expression level The following method can be used to determine whether it can be changed.

 (1) Forced gene expression

 A cDNA containing a gene protein coding region selected from a group of genes having a positive correlation between the expression level selected in (A) and the sensitivity to an Eg5 inhibitor was directly expressed in an appropriate animal cell. By inserting the gene and inserting the resulting expression vector into cancer cells, the gene can be forcibly expressed and its expression level can be increased. An Eg5 inhibitor is added to the cancer cells, and the sensitivity to the Eg5 inhibitor is measured. If the sensitivity to the Eg5 inhibitor is higher than when the expression vector is not introduced, the gene is a gene that regulates the sensitivity of the cell to the Eg5 inhibitor. This indicates that the gene can enhance the sensitivity of cancer cells to Eg5 inhibitors.

 Examples of expression vectors include, for example, pEGFP-C2 (Clontech), PAGE107 (Japanese Patent Laid-Open No. 3-22979; Cytotecnology, 3, 133-140, 1990), pAS3-3 (Japanese Patent Laid-Open No. 2-227075). , PCDM8 (Nature, 329> 840-842, 1987), pGMV-Tagl (Stratagene), pcDNA3.1 (+) (Invitrogen), pREP4 (Invitrogen), pMSG (Amersham Bio Science (Amersham

 Biosciences), pAMo (J. Biol. Chem, 268. 222782-22787, 1993) and the like.

 • As a method for introducing a recombinant vector, any method for introducing DNA into animal cells can be used.

(Cytotechnology, 3, 133-140, 1990), the calcium phosphate method (Japanese Unexamined Patent Publication 222277007755)), Lilipopoff Fekkukushiyonyon method ((PPrroocc .. NNaattll .. AAccaadd .. SSccii .. UU .. SS .. AA .. ,, 88 >> 77441133--77441177 ,, 11998877 )) The area that raises things and the like can be completed. .

 ((22)) Suppression of the expression of the genetic gene

 '' There is a negative difference between the expression level selected in ((AA)) and the susceptibility to the EEgg55 inhibitor. Negative correlations correspond to the genetic genes selected from a group of genetic genes that have 55 negative genetic correlations. Introduce otstide, ssiiRRNNAAss or A. ssiiRRNNAAs or A. ssiiRRNNAA or ssiiRRNNAA to the cancer cell line. The method of suppressing the expression of the genetic gene and reducing the amount of the expression of the genetic gene is described below. Can be done with . An EEgg55 inhibitor is added to the cancer cell vesicles, and the susceptibility of the EEgg55 inhibitor to the EEgg55 inhibitor is measured. . Higher susceptibility to EEgg55 inhibitor compared to when not inhibiting the expression of the genetic gene In some cases, the genetic gene may be less sensitive to the susceptibility of cancer cell vesicles to EEgg55 inhibitors. EEgg55 of cancer cell vesicles, which is a genetic gene that regulates There is a distinction between here and here, which is an inherited gene that can be formed by increasing and enhancing the susceptibility of inhibitors to inhibitors. Take it. .

 The expression of ssiiRRNNAA, ssiiRRNNAA or ssiiRRNNAA or ssiiRRNNAA or A. Based on the description below ((EE)), Yuichi was engaged in the production and introduction and introduction of 1155 cysts into cancer cells. You can do it here and there. .

 ((DD)) EEgg55 Inhibitor Inhibitor, Sensitivity-enhancing Potent Drug ((ii))

 The positive and negative phases between the expression level selected in ((AA)) and the susceptibility to the EEgg55 inhibitor Genetic genes that have a correlation, ff BBCC000077443366, MMAAPP11BB, CCCC belly, HHSSPPCCAA, CCCCNNBB22, FF 2233226699, and SSLLCC1122AA22 The selected single or multiple selected genetic genes are the multiple genetic genes and are described in the above ((CC)).

2200 ((11)), which causes the cancer cells to express forced onset by the method described in (11). Genetic genes containing genetic genes that exhibit active activity to enhance and enhance susceptibility to inhibitory agents The therapeutic drug can be used as a susceptibility-enhancing potent drug for the EEgg55 inhibitor. . . Furthermore, as a result of the forced expression of the genetic gene, the genetic gene in the cancer cell vesicle is coded. As the amount of protein protein increases or decreases, the susceptibility to EEgg55 inhibitor increases. The genetic gene is coco

2255 ——Protein protein preparation containing dodosulin protein protein is a potent sensitizer for EEgg55 inhibitor You can use it as a medicine here and there. .

 The genetic gene that exhibits the activity of enhancing and enhancing the susceptibility of the EEgg55 inhibitor to the EEgg55 inhibitor described above is described above. The protein protein that the gene is coded for is singly used as a susceptibility-enhancing potentiator for the EEgg55 inhibitor. Although it is possible to use it in Germany, there is usually one that is usually acceptable in pharmacology and pharmacology. It ’s better than that

It is mixed with the 3300 carrier and mixed together, and is well-known to the public in the field of pharmaceutical technology. It is desirable that this method be used as a pharmaceutical and pharmaceutical preparation manufactured and manufactured by the method. . The details of such pharmaceutical preparations as described herein will be described later in ((GG)) in the present specification. .

 ((EE)) Sensitive sensitizing potentiator against EEgg55 inhibitor ((ii ii))

 There is a negative / negative phase between the expression level selected in ((AA)) and the susceptibility to the EEgg55 inhibitor. Genetic genes that have a correlation of 3355, such as CCYYPP2244AA11, 00BBRRGGRRPP ヽ ヽ BBFF, AAPPRRTT, BBIIRRCC33, SSQQSSTTMM11 ,, TTNNFFAAIIPP22 ,,

A single or multiple genetic gene selected from among IITTMM22BB, AABBCCCC22, AANNXXAA33 ,, FFAAMM33CC and AABBCCCC33. Thus, the present invention relates to inhibiting the expression of the genetic gene in cancer cell vesicles and suppressing the expression of the genetic gene. SsiiRRNNAA or Aan, which suppresses the expression of genetic genes that show active activity and that enhances susceptibility to ssiiRRNNAA Or ss iiRRNNAA

4400, a drug that expresses 4400 lereotide, should be used as a potent sensitizer for EEgg55 inhibitors. Can. Further, it is considered that the function of the protein encoded by the gene is involved in the enhancement of the sensitivity. Therefore, an antibody against the protein encoded by the gene, preferably a neutralizing antibody that suppresses the function of the protein is considered. Can be used as a sensitivity enhancer for the Eg5 inhibitor. .

 Next, the siRNA used in the present invention will be described. The siRNA is a short double-stranded RNA containing a partial sequence of the mRNA of a certain target gene and its complementary sequence, and can be expressed by RNAi (RNA interference) to suppress the expression of the gene. The sequence of sNA can be appropriately designed based on the conditions of the literature (Genes Dev., 13, 3191-3197, 1999) from the nucleotide sequence of mRNA. Preferably, in the region 50 to 100 bases downstream from the start codon in the translation region of the mRNA, a sequence having 19 bases following AA and a GC content of 30 to 70%, more preferably around 50%, is selected. Two RNAs each having a selected 19-base sequence and three complementary roosters each having a sequence with TT added to the 3 'end of each rooster are synthesized by a DNA synthesizer and annealed to produce siRNA. Also, pSilencer 1.0-U6 (Ambion), pSUPER (oligo engine

 (OligoEngine) Inc.), and inserting a DNA corresponding to the selected 19-base sequence into an siRNA expression vector, such as an siRNA expression vector, can produce a vector that expresses an siRNA capable of suppressing the expression of the gene. .

 The antisense polynucleotide used in the present invention is a polynucleotide having a nucleotide sequence complementary to a continuous sequence of 15 or more nucleotides in the nucleotide sequence of the target gene. Polynucleotides include DNA, RNA, and derivatives of polynucleotides. Examples of the polynucleotide derivative include a polynucleotide derivative in which a phosphodiester bond in a polynucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in a polynucleotide in which a phosphodiester bond is converted to a Ν3′-P5 ′ phosphoramidate bond. Polynucleotide derivative in which the ribose and phosphodiester bond in the polynucleotide has been converted to a peptide nucleic acid bond, polynucleotide derivative in which peracyl in the polynucleotide has been replaced by C-5 propynylperacyl, in polynucleotide Polynucleotide derivative in which peracyl is substituted with C-5 thiazoleperacyl, polynucleotide derivative in which cytosine in the polynucleotide is substituted with C-5 propynyl cytosine, and cytosine in the polynucleotide in which phenoxazine is modified A polynucleotide derivative substituted with a tosin, a polynucleotide derivative in which the report in the polynucleotide is substituted with 2'-ο-provyl ribose, or a ribose in the polynucleotide substituted with 2, -methoxyethoxy ribose Polynucleotide derivatives and the like.

 Antisense RNA / DNA technology (Bioscience and Industry, 50, 322, 1992; Chemistry, 46, 681, 1991; Biotechnology, 9, 358-362, 1992; Trends

Biotechnol., 10> 87-91, 1992; Trends Biotechnol., 10, 152-158, 1992; Cell Engineering, 16, 1463 (1997)), Triple'helix technology (Trends Biotechnol., 10, 132-136, 1992), by administering the above-mentioned antisense polynucleotide to cancer cells, transcription or translation of the target gene in the cancer cells can be suppressed, and as a result, the expression of the target gene can be suppressed. Especially polybepti of target gene A nucleotide sequence complementary to 15 to 100 bases including the initiation codon of the region encoding the code is preferred. Further, a polynucleotide derivative which is not subject to degradation by deoxyribonuclease or ribonuclease is preferable.

 Antisense polynucleotides are commercially available DNA synthesizers, WO93 / 20095,

It can be produced using the method described in WO0280185. In addition, a recombinant vector in which the target gene is connected in the reverse direction downstream of the promoter of the expression vector used for forced expression of the gene of (1) in (C) is prepared, and this recombinant vector is produced. Can be expressed in cancer cells by introducing it into cancer cells

 An antibody against the protein can be obtained by a method known to those skilled in the art as described in (A) (4) (iii). The neutralizing antibody that suppresses the function of the protein is obtained by binding the antibody to the protein to the protein and measuring the function of the protein, and comparing the case where the antibody is not bound. An antibody whose function is suppressed can be obtained by selecting from antibodies against the protein.

 SiRNAs or antisense polynucleotides exhibiting an activity of enhancing sensitivity to the various 5 inhibitors described above, vectors expressing the siRNAs or antisense polynucleotides, and antibodies are used as sensitivity enhancers for E & 5 inhibitors. Can be used alone, but is usually mixed with one or more pharmacologically acceptable carriers, and manufactured by any method well known in the pharmaceutical arts. It is desirable to use it as a pharmaceutical preparation. The details of such a pharmaceutical preparation will be described later in (G) in the present specification.

 (F) Screening method for substance that enhances sensitivity to Eg5 inhibitor A test substance is administered to cells, and a positive correlation is found between the expression level selected in (A) and the sensitivity to Eg5 inhibitor. Measure the expression level of one or more genes selected from certain genes, for example, BC007436, MAP1B, CCNB1, HSPCA, CCNB2, FLJ23269, and SLC12A2, and compare with the expression level when no test substance is administered. By selecting a substance whose expression level increases, a candidate substance that enhances sensitivity to an Eg5 inhibitor can be screened. In particular, if the gene is (C)

If the gene is one that regulates sensitivity to an Eg5 inhibitor selected by the method described in (1), a substance that enhances sensitivity to the Eg5 inhibitor can be screened by this method.

In addition, a test substance is administered to cells, and a gene having a negative correlation between the expression level selected in (A) and sensitivity to an Eg5 inhibitor, such as CYP24A1, OBRGRPs BF, APRT, BIRC3, SQSTM1, TNFAIP2, Rinse B ヽ Measure the expression level of one or more genes selected from ABCC2, AMA3, FAM3C and ABCC3, and decrease the expression level of the gene compared to the expression level when no test substance is administered By selecting a substance, a candidate substance that enhances sensitivity to an Eg5 inhibitor can be screened. In particular, when the gene is a gene that regulates the sensitivity to an Eg5 inhibitor selected by the method described in (2) of (C), a substance that enhances the sensitivity to an Eg5 inhibitor by this method is used. Can be screened. The fact that these substances enhance the sensitivity to Eg5 inhibitors means that these substances are administered to cancer cells, the sensitivity to the Eg5 inhibitor is measured, and compared to the sensitivity without the substance. Can be confirmed by

 The expression level of the gene was determined by the method described in (3) of (A) using * a cell, in particular, a cancer cell line selected from the human cancer cell line panel used in selecting the gene to be measured. Then, the mRNA of the gene can be measured by measuring the amount of protein encoded by the gene. Alternatively, a repo overnight expression cell containing an expression control region of a gene whose expression level is measured is prepared as follows, and the expression level of the repo overnight gene in the cell is defined as the expression level of the target gene. Can be measured. The repo-overexpressing cells are obtained by isolating the expression control region located upstream of the coding region of the gene whose expression level is to be measured from human genomic DNA, and promoting the expression of the exogenous gene in an appropriate expression vector for animal cells. Alternatively, the expression control region can be inserted instead, and a vector ligated to an appropriate repo overnight gene can be introduced into cells downstream of the expression control region. As the repo overnight gene, the human luluiferiferase gene, blue fluorescent protein (GFP) gene,? -Galactosidase gene and the like can be used. The expression level of the repo overnight gene is measured by measuring the amount of luminescence or color development using a substrate that emits or develops color by the enzymatic activity of luciferase or /?-Galactosidase, and the amount of fluorescence emitted by GFP. It can be carried out.

 Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, their modified forms and derivatives, and mammals (for example, mice, rats, and guinea pigs). Urine, body fluid, tissue extract, cell culture supernatant, and cell extract of hamsters, hamsters, bush, olive, olive, poma, dog, cat, monkey, human, etc., as well as non-peptide compounds, Examples include fermented products, extracts of plants and other organisms.

 The substance obtained by the above-described screening method of the present invention is useful as a sensitivity enhancer for an Eg5 inhibitor.

 (G) Pharmaceutical preparation of sensitivity enhancer for Eg5 inhibitor of the present invention

 Various genes, proteins, antibodies, substances and the like as described in (D), (E) and (F) above in the present specification can be used alone as a sensitivity enhancer for Eg5 inhibitors However, it is usually desirable to provide them as various pharmaceutical preparations. The pharmaceutical preparations are used for animals or humans.

 The pharmaceutical preparation according to the present invention can contain the above-mentioned active ingredient or a pharmaceutically acceptable salt thereof alone or as a mixture with any other active ingredient for treatment. In addition, these pharmaceutical preparations are prepared by mixing the active ingredient with one or more pharmacologically acceptable carriers and by any method well-known in the technical field of pharmaceuticals.

 Preferably, the route of administration is to use the most effective one for prevention or treatment, and it may be oral or parenteral, for example, intravenous.

 Examples of the dosage form include tablets and injections.

For example, tablets suitable for oral administration include excipients such as lactose, mannitol, and starch. It can be produced using additives such as a disintegrant, a lubricant such as magnesium stearate, a binder such as hydroxypropyl pill cellulose, a surfactant such as S-fatty acid ester, and a plasticizer such as glycerin.

 Formulations suitable for parenteral administration preferably comprise a sterile aqueous preparation containing the active compound which is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier such as a salt solution, a pudose solution, or a mixture of a saline solution and a glucose solution. Also, in these parenteral preparations, one selected from the excipients, disintegrants, lubricants, binders, surfactants, plasticizers and diluents, preservatives, flavors, etc. exemplified for the oral preparations Alternatively, more auxiliary components can be added.

 When the above active ingredient or a pharmaceutically acceptable salt thereof is used for the above purpose, it is usually administered systemically or locally, in an oral or parenteral form. The dosage and frequency of administration vary depending on the form of administration, the age and weight of the patient, the nature or severity of the condition to be treated, etc., but in the case of oral administration, usually 0.01 to 1 dose per adult per dose; L000 mg, preferably in the range of 0.05 to 500 mg, administered once or several times a day. In the case of parenteral administration such as intravenous administration, G.001 to 1000 mg, preferably 0.01 to 300 mg per adult is administered once or several times a day, or in the range of 1 to 24 hours a day. Administer intravenously continuously. However, the dose and the number of doses vary depending on the various conditions described above.

 '' In addition, in the case of a gene therapy agent utilizing a nucleic acid such as DM or RNA as an active ingredient for enhancing sensitivity to an Eg5 inhibitor, the DNA or RNA may be used alone or in a retrovirus vector, adenovirus vector, adenovirus vector, After insertion into an appropriate vector such as an associated virus vector, a method of formulating, prescribing and administering according to the above-described conventional methods, or a method of administering by a non-viral gene transfer method can be used.

 The recombinant virus vector can be prepared according to the method described below. Prepare a fragment of DNA (hereinafter also referred to as target DNA) to be used as an active ingredient. The recombinant virus vector is constructed by inserting the DNA fragment downstream of the promoter in the virus vector.

 In the case of an RNA virus vector, a recombinant virus is constructed by preparing RNA fragments homologous to the target DNA and inserting them into the downstream of a promoter in the virus vector. For the RNA fragment, in addition to the double strand, either the sense strand or the antisense strand is selected depending on the type of the virus vector. For example, in the case of a retrovirus vector, an RNA homologous to the sense strand is selected, and in the case of a sense virus vector, an RNA homologous to the antisense strand is selected.

The recombinant virus vector is introduced into a packaging cell compatible with the vector. As the packaging cell, any cell can be used as long as it can supply the protein deficient in a recombinant virus vector deficient in at least one gene encoding a protein necessary for virus packaging. For example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3, and the like can be used. Packaging thread The protein to be supplied by HI vesicles In the case of gag, pol, env, etc. derived from mouse retrovirus, and in the case of lentivirus 夕, in the case of gad ヽ pol, env ヽ vpr ヽ vpu, vif ヽ tat ;, rev, nef etc. derived from HIV In the evening, proteins such as E1A and E1B derived from adenovirus, and in the case of adeno-associated virus, proteins such as Rep (p5, pl9s p40) and Vp (Cap).

 As the virus vector, those containing a promoter at a position where a recombinant virus can be produced in the above-mentioned packaging cell and the target DNA can be transcribed in the target cell are used. Plasmid vectors include MFG (Pro Natl. Acad .: Sci. USA, 92, 6733-6737, 1995), pBabePuro (Nucleic Acids Res., 18> 3587-3596, 1990), LL-CG, CL-CG, CS -CG ヽ CLG (J. Virol., 72, 8150-8157, 1998), pAdexl (Nucleic Acids Res., 23, 3816-3821, 1995) and the like are used. Any promoter can be used as long as it functions in human tissues. For example, the promoter of the cytomegalovirus (CMV) IE (i-ediate early) gene and the early stage of SV40 can be used. Promoters, retrovirus promoters, meta-mouth thionein promoters, heat shock protein promoters, SR promoters and the like can be mentioned. Alternatively, the human CMV IE gene enhancer may be used together with the promoter.

 Methods for introducing a recombinant virus vector into packaging cells include, for example, the calcium phosphate method (Japanese Unexamined Patent Application Publication No. 2-227075) and the lipofection method (Proc. Natl. Acad. Sci. USA, 8; 7413-7417, 1987). Can be given.

 In addition, the above-described method of administering the recombinant viral vector may be combined with direct in vivo gene transfer using ribosome delivery in addition to the above-described method, so that it can be administered to a patient's cancer tissue. The virus vector may be directed. That is, a complex is prepared by combining target DNA of an appropriate size with a polylysine-conjugate antibody specific for adenovirus / hexon protein, and the resulting complex is bound to adenovirus vector. Thus, a wireless vector can be prepared. The virus vector reaches the target cell stably, is taken into the cell by endosome, is degraded in the cell, and can efficiently express the gene.

 In the present invention, the vector expressing the target DNA or the siRNA or the antisense polynucleotide can also be delivered to the lesion by a non-viral gene transfer method.

 Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation (Virology, 52, 456-467, 1973; Science, 209, 1414-1422, 1980), microinjection (Proc. Natl. Acad. Sci. USA, 77> 5399-5403, 1980;

Proc. Natl. Acad. Sci. USA, 77, 7380-7384, 1980; Cell, 27, 223-231, 1981; Nature, 291. 92-94, 1981), ribosome-mediated membrane fusion-mediated transfer method ( Proc. Natl. A.cad, Sci. USA, 84, 7413-7417, 1987; Biochemistry, 28, 9508-9514, 1989; J. Biol. Chem., 264, 12126-12129, .1989; Hum. Gene Ther , 3, 267-275, 1992; Science, 249, 1285-1288, 1990; Circulation,, 2007- (2011, 1992) Alternatively, direct DNA uptake and receptor-mediated DNA transfer

 (Science, l_, 1465-1468, 1990; J. Biol. Chem., 266, 14338-14342, 1991; Proc. Natl. Acad. Sci. USA, 87, 3655-3659, 1990; J. Biol. Chem. , 264, 16985-16987, 1989; BioTechniques, U, 474-485, 1991; Proc. Natl. Acad. Sci. USA, 87, 3410-3414, 1990; Proc. Natl. Acad. Sci. USA, 88, 4255. -4259, 1991; Proc. Natl. Acad. Sci. USA, 87. 4033-4037, 1990; Proc. Natl. Acad. Sci. USA, 88> 8850-8854, 1991; Hum. Gene Ther., 3, 147. -154, 1991).

 In the ribosome-mediated membrane fusion-mediated transfer method, direct administration of a ribosome preparation to the target tissue enables local gene uptake and expression in the tissue. For direct targeting of DNA to the cancerous tissue of a patient, a direct DNA uptake technique is preferred. Receptor-mediated DNA transfer is accomplished, for example, by conjugating the DNA (usually in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligando DNA conjugate, if desired, can be injected directly into a blood vessel and directed to a target tissue where receptor binding and localization of the DNA-protein complex occur. Adenovirus can also be co-infected to disrupt endosomal function to prevent intracellular destruction of DNA.

 The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. BEST MODE FOR CARRYING OUT THE INVENTION

 [Example 1] Sensitivity test for Eg5 inhibitor consisting of a panel of 38 human cell lines. The six cell lines derived from lung cancer, six cell lines derived from colon cancer, and four cell lines derived from kidney cancer shown in Table 3 Thirty-eight 38 strains, two strains derived from ovarian cancer, were used. The ATCC is obtained from the American Type Culture Collection, while the JCRB is the Japanese Collection of Research.

Bioresources) Cell Bank, Nishio is a grant from Dr. Nishio of the National Cancer Center.

All cell lines RPMI 1640 (manufactured by Nissui Seiyaku) medium (10% © shea calf serum, 100 units / ml penicillin, 100 〃G / ml streptomycin耷) in, 37 ° C, 5 C0 ₂ presence in Cultured.

 Each cultured cell was dispensed in 0.05 ml portions into each well of a 96-well microphone mouth plate. After culturing at 37 ° C for 24 hours in a carbon dioxide gas incubator, 0.05 ml of the drug appropriately diluted with the above medium was added to each well, and cultured at 37 ° C for 72 hours in a carbon dioxide gas incubator overnight. As drugs, compound A and compound D were used as Eg5 inhibitors, and paclitaxel and vinorelbine were used as microtubule acting drugs as controls. .

The number of cells in each cell was measured using Cell Proliferation Kit II Gel Proliferation Kit II, Roche Diagnostics (Roche Diagnostics). After adding 50 of the kit's color reaction reagent to each well, keep at 37 ° C for 3 hours in an acid gas incubator overnight, measure the wavelength using a microplate reader (M-Spmax250, manufactured by Wako Pure Chemical Industries). The absorbance difference obtained by subtracting the absorbance at the control wavelength of 655 from the absorbance of the 490 dishes was measured and used as an index of the cell number. The ratio of the difference in absorbance at each drug concentration to the difference in absorbance when no drug was added was calculated and defined as the cell viability. Using a microplate reader and the attached analysis software Softmax.Pro (SOFTmax PRO, Wako Pure Chemical Industries, Ltd.), a 4-parameter-one-stop-distic calibration is performed using the following formula based on the drug concentration and cell viability studied. A line was created and the GI _5Q value (X value when Y = 50%, unit mol / 1) was calculated.

Y2 {(AD) pa1 + (X / C) ^B )} + D, X: Drug concentration (mol / 1), Y: Cell viability (%) Each of the 38 cell lines of the four drugs used -Log _{1 () for} GI ₅ . Table 4 shows the values of (up to two decimal places).

Table 4

 Drug

 Compound A Compound D No. Taritaxel vinorelbine cell line

 A549 5.24 8.90 8.30-

Calu-1 5.55 8.92 8.04 8.28

Calu-3 5.34 8.64 8.22 7.91

NCI-H23 5.74 9.13 8.40 8.30

NCI-H69 6.07 9.51 8.52 8.70

NCI-H226 5.55 8.93 8.74 8.47

NCI-H345 5.49 8.91 7.92 8.40

N417 5.56 8.93 8.65 8.27

NCI-H460 5.65 9.06 8.55 8.41

NCI-H596 5.61 8.97 8.21 8.17

NCI-H1299 5.78 9.15 8.05 8.32

SBC-3 5.72 9.16 8.53 8.71

PC-14 5.90 9.28 8.53 8.36

PC-14 / DTX 5.92 9.23 7.70 7.82

PC-14 / CDDP 5.90 9.30 8.38 8.30

MRC-5 5.89 9.32 8.09 8.16

MR- 90 5.98 9.34 8.14 8.30

EBC-1 5.65 8.98 8.35 8.34

NCI-H292 5.53 8.91 8.38 8.24

SHP-77 5.74 8.98 7.70 7.82

NCI-H441 5.12 8.20 7.85 8.15

NCI-H838, 5.00 8.52 8.22 8.15

A427 5.52 8.96 8.05 8.30

NCI-H522 5.58 9.03 8.54 8.86

Calu-6 5.92 9.31 8.50 8.71

PC-9 5.92 9.26 8.34 8.20

COLO 205 5.59 8.98 8.38 8.50

HT-29 5.39 8.81 8.58 8.53

HCT116 5.92 9.32 8.53 8.44

SW480 5.45 8.90 8.13 8.28

DLD-1 5.69 9.05 7.70 7.82

WiDr 5.39 8.78 8.38 8.55

PANC-1 5.61 8.98 8.24 8.15

MIA PaCa-2 5.74 9.17 8.49 8.57

AsPC-1 5.29 8.59 8.18 7.94

BxPC-3 5.60 9.01 8.58 8.35

SK-OV-3 6.04 9.40 8.51 8.56

OVCAR-3 5.55 8.95 8.47 8.70- 4019783

[Example 2] Acquisition of gene expression profile of human 38 cell line panel

The expression of the 119 human genes shown in Tables 5-1 to 5-4 in the human 38 cancer cell lines shown in Table 3 was examined by quantitative RT-PCR according to the following method. The abbreviations of the gene names in Tables 5-1 to 5-4 are based on the gene symbol recognized by the Human Genome Analysis Organization (HUGO) Gene Nomenclature Committee, if any. Was used.

' ¹ 第 5— 3表

'' Table ¹ —Table 3

 Gene name GenBank ¾ Bra Gene name

 (Abbreviation) Imaichi

9kD signal recognition particle 9kD SRP9 NM-1 003133 123,124 Contains baculovirus IAP repeat 3

 BIRC3 N _001165 125, 126 (baculoviral IAP repeat-containing o)

Virtual protein DKFZp761C121 DKFZp

 NM.032290 127, 128 (hypothetical protein DKFZp761C 121) 761C121

Phosphoribosyl pyrophosphate amide transferase

 PPAT N .002703 129, 130 (phosphoribosyl pyrophosphate amidotransferase)

Casein kinase 1, alpha 1 CSNK1A1 N _001892 131, 132 angiotensinogen AGT NM.000029 133,134 tubulin 2 (tubulin, beta, 2). TUBB2 NM_006088 135, 136 MAP7 NM-1 003980 137, 138 activating transcription factor 4 ATF4 NM.001675 139, 140

Glu / Asp rich C-terminal domain containing Cbp / p300 binding transcriptional activator 1

(Cbp / p300— interacting transactivator, with Glu / Asp-rich CITED 1 NM.004143 141, 142 carboxy-terminal domain, 1)

Frizzled-related protein FRZB Hire— 001463 143, 144 Aquaporin 1 (aquaporin 1) AQP1 NM-1 000385 145, 146 Sequestosome 1 (sequestosome 1) SQST 1 N _003900 147, 148 Heat shock factor binding protein 1

 HSBP1 NM 001537 149, 150

(heat shock factor binding protein 1)

Adenine'phosphoribosyltransferase

 APRT NM— 000485 151, 152 (adenine phosphoribosyltransferase)

Phosphoserine '' aminotransferase

 PSAT1 NM—02.1154 153, 154 (phosphoserine aminotransferase)

Transgelin TAGLN NM— 003186 155, 156 coagulation factor II receptor F2R NM— 001992 157,158 cyclin Bl (cyclin Bl) CCNB1 'NM— 031966 159, 160 cyclin B2 (cyclin B2 ) CCNB2 NM— 004701 161, 162 Villin 2 (villin 2) VIL2 003379 163, 164 Heterogeneous liponucleoprotein A2 / B1 HNRPA2B

 NM one 002137 165, 166

(heterogeneous nuclear ribonucle oprotem A2 / B1) 1

Intracellular retinol binding protein 1

 RBP1 NM— 002899 167, 168 (retinol binding protein 1, cellular)

Syntrophin beta 1 SNTB1 NM— 021021 169, 170 Carcinoembryonic antigen-related cell adhesion molecule 5

 CEACA 5 NM 004363 171, 172

(carcinoembryonic antigen-related cell adhesion molecule 5)

Adrenergic) 32 receptor (adrenergic, beta-2-, receptor, surface) ADRB2 NM— 000024 173, 174 Homologous sequence 3 containing family 'member 3

 FA 3C NM 014 888 175, 176

(family with sequence similarity 3, member C)

Dual specificity phosphatase 6 DUSP6 NM 001946 177, 178 cell division cycle 25B CDC25B Sonoichi 004358 179, 180 syndecan 2 (Syndecan 2) SDC2 NM.002998 181, 182 Phospholipase C β 4 (phospholipase C, beta 4) P and CB4 NM— 000933 183, 184 Ribosomal protein L13 RP and 13 NM— 000977 185, 186 Solute carrier family 12

SLC12A2 NM_001046 187, 188 (solute carrier family 12, member 2) Table 5-4

(1) cDNA synthesis

 The 38 human cell lines shown in Table 3 were cultured in the same manner as in Example 1, cells were collected during the logarithmic growth phase, and total RNA was extracted using RNeasy [; RNeasy, QIAGEN] .

cDNA was synthesized using a Superscript First-strand Synthesis System for RT-PCR (Invitrogen) using RT-PCR. According to the attached manual, the procedure was as follows. 10 mmol / 1 dNTPs 1〃1 and 0.5 g / 〃l oligo (dT) ₁₂ — ₁₈ 1 JLLI were added to the obtained total RNA 5 and 9〃1 of water treated with dimethyl pyrocarbonate (DEPC). Filled up with 1. After heat denaturation at 65 ° C for 5 minutes, quench on ice and allow to stand for 1 minute or more.10XRT buffer 1 2 j 25 t ol / l MgCl ₂ 4 j 0,1 mol / 1 DTT 2 〃 1 and ribonuclease 1 ゥ 1 (RNaseOUT) was added, and the mixture was kept at 42 ° C for 2 minutes. In addition, 1〃1 (50 units) of Superscript II RTase is added 42. A reverse transcription reaction was performed at 50 ° C. for 50 minutes and heated at 70 ° C. for 15 minutes to inactivate the enzyme. Thereafter, the reaction was carried out at 37 ° C for 20 minutes with the addition of ribonuclease H1-1, and the mixture was diluted with water to a total volume of 1 ml. In the following quantitative RT-PCR reaction system, 10 jl of a 5-fold diluted solution was used.

 (2) Measurement of gene expression by quantitative RT-PCR

All reagents used were For Real Time PCR TaKaRa Ex Taq R-PCR Version (Takara Shuzo). Per sample, 10XR-PCR buffer (without Mg ²⁺ ) 2.0 u 250 glut / l Mg ²⁺ solution 0.2 JUL I 10 mmol / 1 dNTPs 0.6 〃1, 1/2500 dilution Cyber 'Green (SYBR Green ) I 1.0 〃1, 4 mol / 1 gene-specific primers each 1 · 5 U ExTaq R-PCR 0.2 〃 1, H ₂ 0 3 〃 reaction after the preparation of one, not a 10 1 to 96 Ueru PCR plate min Noted. Primers specific to each gene were those shown in SEQ ID NOs in Tables 5-1 to 5-4. Further, the cDNA prepared in (1) was added in the form of 10, and the mixture was stirred by pipetting. The plate was sealed without gaps and subjected to real-time PCR.

 For real-time PCR, use an ABI sequence detection system (ABI PRISM 7700, Applied Biosystems) according to the manufacturer's instructions, heat at 95 ° C for 5 minutes, and then heat at 95 ° C for 1 minute (denaturation). 45 cycles of 1 minute at 60 ° C (annealing) and 1 to 2 minutes at 72 ° C (extension) are repeated, and heating is performed at 72 ° C for 5 minutes (annealing temperature and extension time vary depending on the gene) Perform PCR with 7 cells.

第 6 _ 2表 From the obtained results, the number of cycles (Ct value) at which the signal intensity after background correction reached 1,000 for each gene was determined and used as an index of the expression level. This signal intensity was set as a condition that the amplification reaction proceeded logarithmically and did not reach the plateau in any sample. A smaller Ct value indicates a higher expression level, and a difference of 1 in Ct value can be interpreted as having a theoretically approximately 2 times difference in expression. Tables 6-1 to 6-9 show the background-corrected Ct values (up to two decimal places) for 119 genes of 38 cell lines obtained as described above as gene expression profiles. Indicated. “Average” in Tables 6-7 to 6-9 indicates the average Ct value of each gene among 38 cell lines. Genes are represented by abbreviations in Tables 5-1 to 5-4.

Table 6_2

 Cell strain Calu Calu NCI- NCI- NCI- NCI— NCI- NCI- NCI- SBC-Michiden \ A549 -1 -3 H23 H69 H226 H345 N417 H460 H596 H1299 3 PC-14

ABCB1 29.78 27.09 28.76 29.96 28.46 31.07 26.42 29.11 28.02 28.71 27.86 27.74 28.07

PLAU 22.97 22.16 20.62 24.69 27.87 29.82 26.76 29.81 24.48 22.06 20.60 27.20 19.42

CD24 20.49 20.80 18.49 22.67 19.74 23.13 16.19 20.90 22.06 22.09 23.07 18.92 19.70

FN1 24.19 19.59 23.26 28.78 31.17 27.93 24.12 31.20 19.14 21.96 26.14 23.92 26.29

CYP24A1 23.82 24.85 25.34 25.91 32.36 24.26 28.89 32.32 27.35 30.18 28.53 29.68 30.30

PLOD2 22.00 21.65 22.95 24.07 25.02 20.04 22.94 26.78 24.89 21.21 23.19 25.02 22.58

CCND2 31.92 24.85 31.43 19.85 29.62 22.97 26.43 26.87 33.13 33.07 25.56 29.01 28.64

NPTX1 35.20 33.21 35.74 32.57 33.62 35.19 30.04 34.02 35.48 34.15 33.82 32.06 32.19

DNAJA3 27.07 26.03 25.58 26.08 26.02 25.67 24.97 26.50 24.35 25.14 24.53 25.53 26.05

MOX2 32.22 27.17 34.97 26.73 29.71 20.74 24.26 28.48 33.93 30.75 24.87 28.37 29.14

NDRG2 31.23 31.22 30.42 30.38 31.51 29.95 29.44 30.56 30.17 29.10 29.69 27.83 28.21

ZDHHC3 24.68 24.66 24.23 24.65 25.69 24.80 24-28 26.40 24.73 24.70 24.15 23.83 24.70

CASK 25.97 24.81 26.85 28.84 26.41 28.28 22.93 25.20 24.38 26.60 27.54 25.57 26.19

D then C1 27.77 24.34 29.69 27.79 27.79 25.28 25.06 27.97 27.06 27.67 26.01 26.41 26.79

SF3A3 30.20 25.40 29.21 29.64 29.01 31.00 24.33.27.32 29.75 28.93 26.70 26.68 27.92

TNFAIP2 22.33 23.28 25.85 29.66 31.21 26.45 28.24 31.69 21.58 23.55 33.18 24.87 27.36

ANTXRl 25.89 25.50 32.23 27.28 30.27 25.56 27.01 29.21 24.13 27.15 27.70 28.28 29.25

PPID 24.32 '21 .85 27.01 25.81 21.49 25.67 17.44 21.43 23.85 25.21 25.79 20.16 20.94

CDK6 26.20 24.51 27.04 27.32 26.40 25.87 26.43 26.97 24.14 26.90 25.10 25.75 26.72

SC5DL 27.21 24.50 26.96 27.58 24.15 27.07 21.93 24.65 25.19 26.64 27.22 26.14 26.34

ING4 27.28 23.47 29.01 26.62 24.93 27.88 21.72 26.38 26.59 26.18 26.28 26.10 26.43

SRP9 20:63 19.04 23.37 22.14 20.15 23.06 18.33 21.48 19.08 21.40 22.06 20.13 22.14

BIRC3 24.23 25.41 26.84 25.04 34.67 28.37 27.37 32.33 28.77 26.18 24.55 33.21 26.63

DKFZp761

 25.17 24.51 30.35 26.64 22.99 27.49 23.08 26.31 21.50 27.22 27.07 24.83 26.61 cm

 PPAT 24.75 23.87 27.45 24.83 24.03 26.36 23.20 23.17 23.17 26.05 25.07 23.11 25.12

CS K1A1 23.98 21.43 25.52 25.06 23.53 24.92 21.72 24.05 22.39 24.22 23.14 23.09 22.91

AGT 33.01 31.16 25.02 34.16 30.40 36.89 27.07 29.38 40.00 34.49 40.00 29.10 30.47

TUBB2 19.49 19.15 22.90 20.94 20.47 21.47 18.49 21.44 19.68 21.47 20.05 21.20 20.93 AP7 24.16 26.50 27.10 24.81 23.81 28.73 22.45 26.66 22.06 28.09 27.06 24.88 26.10

ATF4 21.68 19.20 21.29 21.24 20.21 22.42 19.22 21.36 18.24 21.24 21.22 20.19 21.26

CITED 1 33.27 31.60 35.92 31.87 27.18 34.50 27.73 26.49 31.53 33.82 31.74 30.34 32.14

FRZB 32.41 28.42 33.41 33.70 27.15 34.03 26.31 30.27 32.19 32.18 33.13 28.06 28.52

AQP1 32.45 31.35 31.09 32.95 30.01 31.88 26.12 31.15 33.03 30.63 34.51 29.53 31.56

SQSTMl- 22.82 22.05 25.08 25.15 26.57 23.31 24.42 29.11 22.22 23.64 25.44 27.78 26.26

HSBPl 22.15 21.77 22.86 22.14 22.36 21.37 20.07 22.14 21.42 23.34 22.39 22.43 22.24

APRT 23.67 24.75 24.75 24.53 24.44 24.49 24.40 23.80 24.08 24.07 23.93 24.33 24.61

PSATl 22.06 22.12 21.37 22.17 22.53 22.60 21.54 23.88 19.20 21.24 19.02 20.24 21.64

TAGLN 32.21 31.63 30.92 32.17 32.21 33.09 29.76 32.97 33.11 31.89 32.15 30.63 30.80

F2R 23.99 21.60 24.35 22.31 29.30 24.25 27.02 29.57 25.43 22.36 21.03 25.51 21.10

CCNBl 20.42 19.42 23.09 18.26 20.21 19.40 20.41 22.00 19.64 20.05 18.24 19.56 19.57 9

 Halla ε-9

第 6— 5表 SL6l0 / P00Zdr / 13d .0.T90 / S00Z OAV

Table 6-5

6

6

 Halla 9-1 9

£ 8L6l0 / 00ZdT / 13d .0.T90 / S00Z OAV OS

Halla

 9

£ 8Z.6l0 / ^ 00Zdf / X3d .0.T90 / S00Z OAV Table 6-8

 Cell line COLO HT HCT DLD PANC MIA AsPC BxPC SK- OVCA

 SW48C WiDr average transmission message "205 -29 116-1 -1 PaCa-2-1-3 OV-3 R-3

 ABCBl 29.82 27.76 31.82 29.34 25.29 28.65 29.71 30.92 31.10 31.05 26.69 27.61 28.22

PLAU 27.16 29.23 25.45 26.38 26.10 29.18 21.74 22.15 26.55 23.13 21.17 24.10 24.22

CD24 19.41 19.20 22.97 24.20 20.96 19.14 21.33 23.43 26.50 20.85 16.76 19.04 20.78

FN1 33.52 30.57 28.75 24.55 35.46 31.73 30.90 32.17 36.80 26.75 18.10 23.24 25.88

CYP24A1 28.43 28.18 26.07 26.21 30.40 28.64 29.68 31.88 31.45 29.39 26.76 30.14 28.59

P and OD2 24.27 25.28 25.3'3 31.71 24.08 24.25 22.70 31.59 34.47 24.18 22.00 23.46 24.19

CCND2 31.33 30.41 32.21 28.73 31.99 34.26 30.75 25.77 34.57 29.08 28.92 32.32 28.90

NPTXl 33.82 33.61 34.83 35.82 36.12 34.82 34.00 34.69 35.68 35.35 30.53 34.05 33.90

DNAJA3 25.74 26.74 26.32 26.30 26.97 25.97 26.45 26.02 29.93 26.67 25.03 25.45 25.92

MOX2 29.48 25.97 30.82 32.09 33.43 27.76 30.22 31.94 34.96 32.80 22.93 22.83 29.33

NDRG2 27.81 29.86 29.12 30.58 30.99 28.66 30.48 31.07 31.53 29.33 28.49 28.92 29.71

ZDHHC3 23.86 23.93 24.19 24.01 24.52 23.03 25.07 24.64 28.47 24.56 23.97 23.99 24.43

CASK 26.84 25.82 40.00 26.44 25.55 26.02 27.69 28.75 29.16 29.18 24.27 25.33 26.36

D CI 29.04 27.91 31.45 26.58 27.48 29.13 29.16 30.39 29.22 28.63 23.59 27.53 26.95

SF3A3 29.83 26.73 28.80 28.18 26.70 25.84 28.27 29.31 31.12 29.05 26.06 26.24 27.84

TNFAIP2 30.87 23.35 28.84 24.81 29.74 23.58 24.01 27.87 27.81 25.68 23.84 22.05 26.20

ANTXRl 31.41 30.32 30.89 28.41 31.61 31.83 26.85 29.95 32.71 30.00 26.79 26.01 28.00

PPID 22.96 21.09 27.67 23.86 23.28 23.07 23.09 25.85 25.97 27.39 18.34 21.92 23.00

CDK6 26.77 25.73 27.51 26.02 26.77 26.09 26.75 29.49 26.99 26.94 25.43 22.62 25.51

SC5D 27.93 24.05 28.36 25.24 23.83 23.76 26.21 29.72 29.12 28.74 22.57 23.93 25.53

ING4 28.24 25.02 30.11 26.08 27.38 25.14 28.87 28.45 29.26 28.53 25.84 23.72 25.96

SRP9 22.06 19.78 23.11 20.29 20.63 19.45 21.79 23.61 23.26 23.78 20.06 19.29 20.82

BIRC3 30.03 28.22 34.16 26.03 26.94 28.18 25.47 29.02 28.59 30.18 27.35 28.94 27.53

DKFZp

 27.08 25.39 28.16 24.77 25.28 25.08 27.21 29.24 31.02 28.47 25.03 24.11 25.69 761C121

 PPAT 27.04 24.58 26.09 23.98 24.59 23.17 26.28 27.04 28.54 27.48 24.24 23.11 24.68

CSNKIAI 24.98 22.23 25.72 21.96 22.59 21.90 23.79 23.86 25.11 25.32 22.50 22.95 23.09

AGT 28.82 30.24 40.00 36.86 34.29 28.63 31.47 38.56 35.07 35.84 28.05 32.26 32.12

TUBB2 21.22 19.80 21.18 18.51 19.72 19.33 21.02 22.76 23.44 21.12 19.61 18.56 20.42 AP7 24.58 22.51 28.36 24.59 24.25 22.13 27.65 31.34 28.30 27.20 24.07 22.79 25.32

ATF4 22.40 19.60 22.9620.27 19.20 19.08 22.14 21.31 20.90 21.75 21.30 19.12 20.52

CITED 1 34.42 31.99 32.81 30.56 32.84 31.78 32.03 35.22 35.89 34,53 30.22 32.29 31.84

FRZB 30.69 29.40 34.30 31.95 30.44 31.34 30.81 33.91 33.79 34.74 27.01 27.67 30.44

AQP1 26.86 30.33 33.54 33.10 33.11 29.71 32.26 34.54 34.99 33.55 29.15 33.16 31.16

SQSTMl 27.09 24.30 27.93 24.04 23.72 23.25 23.59 24.44 25.16 27.06 25.32 23.84 24.45

HSBPl 24.44 22.16 23.26 22.58 22.80 21.86 23.12 24.02 26.34 23.36 21.32 21.46 21.95

APRT 24.26 23.39 24.47 23.42 23.86 22.65 24.08 23.75 27.59 25.14 23.45 23.75 24.31

PSATl 22.21 22.93 22.14 21,96 21.38 20.40 22.97 19.87 28.13 21.44 20.89 19.54 21.72

TAGLN 32.65 32.79 33.94 34.79 33.26 34.95 32.74 33.70 35.87 33.88 29.12 31.38 31.95

F2R 29.90 22.12 25.02 27.50 24.96 21.56 22.63 24.39 25.36 25.92 20.40 22.67 24.34

CCNBl 19.85 20.96 19.35 19.98 21.68 20.47 20.04 22.83 26.92 20.73 18.72 19.52 2.0.43

£ L6l0 / t00Zd £ / ∑Jd .0.T90 / S00Z OAV [Example 3] Correlation analysis of gene expression profile and sensitivity to Eg5 inhibitor To search for genes related to sensitivity to Eg5 inhibitor, data on sensitivity to Eg5 inhibitor obtained in Example 1 and Example 2 The data of the gene expression profile obtained in the above was integrated and the correlation analysis was performed. We thought that it was important to analyze each cell line derived from the same tissue, and we analyzed the expression profiles of 119 genes shown in Tables 6-1 to 6-6 in Example 2 for 26 cell lines derived from lung cancer. Using the data of the sensitivity to four drugs including the Eg5 inhibitor shown in Table 4 of Example 1, the Pearson correlation coefficient r calculated by the following formula was calculated. .

() Here, Xi is the Ct value (Ct _x ) of the cell i after the background correction of the gene X, and indicates the expression level of the gene X. ^ Is the GI _5Q value of drug y in cell i

(GI ₅ .y) logarithmic multiplied one 1 - a (log _1G GI ₅ .y), shows a sensitivity against a drug y cell i. x _m denotes the mean value of the cell lines the expression level of gene X (C), y _m denotes the average value of the cell lines sensitive _{(-log 1 () GI 5Q y} ) to the drug y. The correlation coefficient r between the expression level of each gene and the sensitivity to each drug is shown in Tables 7-1 to 7.3. Each gene is abbreviated from Table 5-1 to Table 5-4.

第 7— 2表

Table 7-2

 Compound A Compound D Paclitaxel Vinorelenolevin Genes ^^^^

 ABCB1 0.09 -0.02-0.67 one 0.36

PLAU 0.04-0.08 '-0.42

 CD24 '-0.30 -0.27 -0.14

 FN1 -0.14 -0.17 -0.30 -0.25

CYP24A1 -0.62 -0.50 0.01 -0.19

PLOD2 -0.26 .-0.32 -0.35 -0.48

CCND2 0.13 0.14 0.10 0.05

NPTX1 0.11 0.13-0.20 0.14

DNAJA3 -0.05-0.18-0.16 0.03

MOX2 0.08 0.08 -0.02 0.04

NDRG2 -0.06 0.00 0.15 0.13

ZDHHC3 0.06 0.00-0.34 -0.07

CASK -0.24 -0.24 -0.22 0.14

D then C1 0.09 0.11-0.26 0.06

SF3A3 -0.04 -0.11-0.35 0.10

TNFAIP2 -0.38 -0.34 0.12

 ANTXRl -0.13 0.02 -0.06 -0.06

PPID 0.09 0.12-o0 o.11 0.26

CDK6 0.03 -0.01 -0.18 o -0.05

SC5D -0.05-0.15 0.04

ING4-0.09 -0.10 -0.24 0.18

SRP9 -0.09 -0.09 -0.21 0.29

BIRC3 -0.42 -0.40-0.35-0.31

DKFZp761C121 -0.06 -0.01 -0.07 0 o o o.35 o dimensions

PPAT 0.01 0.02-0.10 0.2 o8

CSNKIAI 0.17 0.13 0.11

AGT -0.18 -0.24 -0.39 -0.40

TUBB2 0.18 0.22 -0.37 0.11

MAP7 -0.31 -0.27 -0.11 -0.04

ATF4 -0.20 -0.26-0.19 0.11

CITED 1 0.14 0.22 0.11 0.43

FRZB 0.22 0.23 -0.17 0.22

AQP1 -0.22 -0.18 -0.24 0.01

SQSTMl -0.39-0.36 -0.14 0.04

HSBPl 0.04 -0.03 -0.30 -0.05

APRT -0.42 -0.42 0.02 0.12

PSATl 0.23 0.18 -0.12 0.14

TAGLN 0.18 0.18 -0.38 -0.20

F2R 0.23 0.33 0.00 0.04

CCN'Bl 0.37 0.33 -0.11 0.11 Table 7-3

 Compound A Compound D Paclitaxel Vinolenolevin

CCNB2 0.35 0.26 0.06 0.23

VIL2-0.21 -0.30-0.15-0.11

HNRPA2B1 0.29 0.15-0.26 0.04

RBP1 0.08 0.08 0.04 0.33

SNTB1 -0.26 -0.38-0.51

 CEACAM5 -0.10 -0.30 -0.45 -0.24

ADRB2 0.02-0.01 -0.29 -0.43

FAM3C -0.34 -0.51-0.29 .-0.14

DUSP6 -0.03 -0.15-0.38 -0.04

CDC25B 0.01 0.02 -0.17 0.02

SDC2 0.10 0.16 0.03 0.13

PLCB4 0.27 0.40-0.02 0.04

RPL13 0.13 0.05 -0.33 -0.24

SLC12A2 0.33 0.33 0.01 0.15

HMGBl 0.28 0.20 -0.07 0.16

ITGA5 0.03 0.03 -0.12 -0.24

TXNRDl 0.23 0.14-o o 0.21 -0.20 o

 TRIP12 0.26 0.26 0.0 αι1 0.17

LMANl 0.27 0.41 -0.02 0.04

MAP IB 0.38 0.42-0.19 -0.06

IC T -0.01 -0.05 -0.26 0.06

HIP-55 0.01 -0.06-0.27 0 ο.04

MAPRE2 0.27 0.31-0.18 -0.10 inch

OBRGRP -0.52 -0.54-0.32 0.21

KIAA0449 0.07 0.08-0.02 0.25

BC007436 0.52 0.48 -0.13 0.04

PDIP46 0.16 0.07-0.21 0.09

DNCHl -0.10 -0.08 -0.11 0.11

KIAA1389 -0.05-0.02 -0.01-0.01

MGC34646 -0.02-0.08 -0.41

FLJ23269 0.33 0.44 0.09 0.29

IMP-1 0.27 0.37-0.04 0.17

C13orf7 0.23 0.22-0.12 0.14

OK / SW-cl.56 0.22 0.21 -0.28 0.11

HSPCA 0.37 0.41 -0.17 0.08

ESDN-0.19 -0.20-0.23

4512944 -0.08 -0.02 0.03 .0.25

FLJ90798 0.17 0.19-0.03 0.26 '

PGK1 -0.02 -0.14 -0.25 -0.14 The correlation coefficient r can take on values from -1 to 1.However, the farther away from 0 and closer to 1, the positive correlation between the expression level of gene X and the sensitivity to drug y, and the closer to -1 the negative phase. Shows that a battle is going on. As a gene involved in sensitivity to an Eg5 inhibitor, the absolute value of the correlation coefficient between the expression level and the sensitivity to compound A] is 0.33 or more (where “is equal to or less than 0.33” or “ 19 genes were selected that displayed a value greater than or equal to 0.33. Table 8 shows the correlation coefficients of the 19 genes thus selected for each drug. In the column of sensitivity and resistance, the correlation coefficient r for compound A is a negative number, and the lower the sensitivity to compound A, that is, the more resistant the cell line, the higher the expression level. "Sensitivity" was described for genes with a higher expression level as the relation number r was a positive number and the sensitivity to compound A was higher. Table 8

第 8表に示す通 、 7種の遺伝子が感受性遺伝子 （感受性の高い細胞株で高発 現している遺伝子） 、 12種の遺伝子が耐性遺伝子 （感受性の低い細胞株で高発 現している遺伝子） として選抜された。 これら全ての遺伝子が、 化合物 Dにおい ても相関係数の値の符号が一致しており、 うち 17種 （感受性 7種中 6種、 耐性 12種中 11種） については、 化合物 D-においても相関係数の絶対値が 0 . 33 以上 であった。 以上の結果と、 化合物 Aと化合物 Dの構造に共通性はないことを考え 合せると、 第 8表に示した選抜された 19遺伝子は、 Eg5阻害剤という共通の作 用機構を有する化合物群の薬剤感受性に一般的に関与する遺伝子であると考えら れる。 一方、 Eg5阻害活性を有さないパクリ夕キセルおよびビノレルビンについ ては BIRC3のみが共通に高い相関係数の絶対値を示したのみであり、 Eg5阻害剤 とは異なる薬剤感受性決定機構が存在することが示唆された。

As shown in Table 8, 7 genes are susceptible genes (genes highly expressed in sensitive cell lines) and 12 genes are resistant genes (genes highly expressed in less sensitive cell lines) Was selected as All of these genes have the same sign of the correlation coefficient value in Compound D, and 17 of them (6 out of 7 sensitive and 11 out of 12 resistant) have the same sign in Compound D-. The absolute value of the correlation coefficient was 0.33 or more. Considering the above results and the fact that there is no commonality between the structures of Compound A and Compound D, the selected 19 genes shown in Table 8 are shared by Eg5 inhibitors. It is considered to be a gene generally involved in the drug sensitivity of a group of compounds having a mechanism of use. On the other hand, for paclitaxel and vinorelbine, which do not have Eg5 inhibitory activity, only BIRC3 showed a high absolute value of the correlation coefficient in common, and a different drug sensitivity determining mechanism from that of Eg5 inhibitor exists. Was suggested.

 Among these genes, the resistance gene BIRC3 is known as a gene that suppresses cell death, and the susceptibility genes CCNB1 and CCNB2 are genes related to the cell cycle. Also included were the drug transposable genes such as ABCC2 and ABCC3, and CYP24A1, which belongs to the cytochrome P450 family reported to be highly expressed in breast cancer.

 These genes, including those that can be used as markers for predicting susceptibility to Eg5 inhibitors, are also considered to actually define susceptibility to Eg5 inhibitors.

[Example 4] Construction of sensitivity prediction model using Eg5 sensitivity correlation gene

 (1) Apply statistical model

A statistical model was constructed from the relationship between the expression level of genes correlated with sensitivity to Eg5 inhibitor and the sensitivity of lung cancer-derived cell lines to Eg5 inhibitor, and used as a model for predicting sensitivity from gene expression level. A multiple regression model was used as a statistical model. The general multiple regression model equation is shown as Equation 1. The partial regression coefficient a! For each term is solved by solving (n + 1) first-order linear polynomials that introduce the explanatory and objective variables of (n + 1) samples and their actual values into Equation 1. , A ₂ , · Ά and the constant term b. By substituting the value of the explanatory variable, Χ ₂ , '' Χ _η into Equation 1 using the obtained partial regression coefficients a ₂ , Ά _η and the constant term b, the predicted value 目的You can calculate ί ^ you can ο

Y = a ₁ X ₁ + a ₂ ¾ + ...- + a _n X „+ b (Equation 1)

 Y: target variable

, -X _n : explanatory variate

2 ヽ · a _n : Partial regression coefficient

b: Constant term A sensitivity prediction model was obtained by applying the sensitivity of the lung cancer cell line to the Eg5 inhibitor to the objective variable in the above multiple regression model equation and the expression level of a gene correlated with the sensitivity to the Eg5 inhibitor to the explanatory variable. Specifically, the Ct value was used as the gene expression level, and the -log _1Q GI _5Q value was used as the sensitivity to the Eg5 inhibitor.

 (2) Construction and evaluation of susceptibility prediction model based on susceptibility correlation gene in lung cancer-derived cells

 (i) Construction of initial model

a₂Ct₂、 ― a₁₉Ct₁₉の合計と bの値から、 該細胞の Eg5阻害剤に対する感受性の予測値- log_1QGI₅。を計算できる。 第 9表 Lung cancer 20 cell lines A549, NCI-H23, NCI-H69, NCI-H226, NCI-H345, NCI-H460 of 19 genes (Table 8) correlated with the sensitivity of the lung-derived cell lines selected in Example 3 , NCI-H596, NCI-H1299, PC-14 / DTX ヽ PC-14 / CDDP ヽ MRC-5, IMR-90, EBC-1, NCI-H292, NC band ヽ NCI-H838, 圆, NCI-H522, Calu - 6 and the Ct value in PC- 9, the same lung 20 cell lines - l og _1Q GI _5. The values were applied to the multiple regression model of (1). Each partial regression coefficient term and constant term were determined by solving the obtained 20 first-order linear polynomials. Table 9 shows the determined partial regression coefficients for each gene. The value of the constant term b was 0. The obtained equation was used as the initial multiple regression model A. The score assigned to gene i is represented by the product aiCti of the partial regression coefficient ai of the i term and the Ct value (Cti) of gene i. For a certain cell, the score of each of the above 19 genes

a ₂ Ct ₂ , —predicted value of sensitivity of the cells to Eg5 inhibitor—log _1Q GI ₅ from the sum of a ₁₉ Ct ₁₉ and the value of b. Can be calculated. Table 9

(ii) Construction of optimization model

The initial model constructed in (i) is complicated because there are as many as 19 explanatory variables. By optimizing the multiple regression model for this initial model, explanatory variables that are important for predicting the objective variable can be selected, and the prediction accuracy can be improved while suppressing the complexity of the model. The Cp statistic (“Quick and understandable statistical terms” by Sadao Ishimura, Tokyo Books) was used as an index for model optimization. Specifically, by executing the stepwise linear regression method (S_PLUS2000 User's Guide) on the statistical analysis software “S-Plus2000” (Mathematical Systems Co., Ltd.), one explanatory variable is added to the initial model at a time. When excluded Optimizing the model by repeatedly determining each partial regression coefficient term for the model, calculating the Cp statistic from the results, and selecting the model with the lower Cp statistic as the better model Was done. As a result, a lung cancer optimization model A ′ consisting of 11 genes with 8 fewer explanatory variables was completed. Table 10 shows the genes used as explanatory variables and the values of the partial regression coefficients selected in the optimization model A '. The value of the constant term b was 0. Table 10

( i i i ) モデルの評価

(iii) Model evaluation

Among the lung cancer cell lines of Example 1, the gene expression levels of six cell lines (Calu-1, Cain-3, N417, SBC-3, PC-14 and SHP-77) which were not used for model construction were determined by (ii) ) Is applied to the optimization model constructed in (2) to calculate the predicted value of the sensitivity of the six cell lines to the Eg5 inhibitor, and to evaluate the optimization model A 'by looking at the residual (measured value-predicted value) Was done. From the partial regression coefficient a of each of the 11 genes determined by the optimization model A 'and the expression level (Ct value) of each gene measured in Example 2, the score (product of a and Ct) of each gene was calculated. The predicted value of sensitivity (-Log _1Q GI _{5 ()} value) was calculated from the scores of (Table 11). This prediction is directly shown in Table 4 of Example 1-Log _{1 ()} GI ₅ . When the measured values were compared, the absolute value of the residual of _{-log 1 ()} GI _{5fl was less than 0.3} in 4 out of 6 strains (Calu-1, Calu-3, N417 and SBC-3). Below, the average of the absolute values of the residuals of the six stocks was 0.22, and good predictions with little residuals were obtained (Table 11). Therefore, this optimized model A '

It was shown to be effective in predicting sensitivity to the drug. Table 11

(3) Application to other cancer types

Of the 38 human cell lines of Example 1, 12 other carcinoma cells other than lung cancer (6 colon cancer lines: C0LO 205, HT-29, HCT116, SW480, DLD-1 and WiDr, 4 pancreatic cancer lines: PANC-1 , MIA PaCa-2, AsPC-l, BxPC-3, Ovarian cancer 2 strains: Apply the gene expression levels of SK-0V-3 and OVCAR-3) to the lung cancer optimization model constructed in Example 4 (2) Thus, the predicted value of the sensitivity of the 12 cell lines to the Eg5 inhibitor was calculated, and the optimized modenole A 'was evaluated by looking at the residual. Based on the partial regression coefficient a of each of the 11 genes determined by the lung cancer optimization model A 'and the expression level (Ct value) of each gene measured in Example 2, the score (product of a) of each gene was calculated. The predicted value of sensitivity (-Log ^ GIso value) was calculated from the score of. This predicted value is shown in Table 4 of Example 1-Log _1Q GI ₅ . 'Comparing the measured values, the absolute value of the residual of the _{-log 1Q} GI _5Q value was _0.3 or less in 7 out of 12 shares, and the average of the absolute value of the residual value of 12 shares = 0. The prediction accuracy was close to that of a lung cancer cell line (Tables 12-1 and 12-2). Therefore, this model, which was created based on lung cancer, was shown to be effective not only in predicting the sensitivity of lung cancer but also other carcinomas to Eg5 inhibitors. Table 12-1

Table 1 2—2

[Example 5] Expression of selected Eg5 susceptibility-related genes in human clinical cancer tissues 14 out of 19 genes (CYP24A1 OBRGRP BF) selected from the experimental results of correlation analysis between gene expression profile and sensitivity shown in Example 3 (APRT BIRC3 TNFAIP2, Rinse B ABCC2 ANXA3 FAM3C ABCC3 BC007436 MAP IB, CCNB1 HSPCA CCNB2 and FLJ23269) and real-time expression of GAPD, a house-kiving gene, as a positive control in human cancer tissues Analyzed by RT-PCR method.

 Using Clontech's commercially available cDNA CMatched cDNA Pairs for cancer and normal tissues from the same patient, human ovaries 1-5, A-E (total 10 samples), human breast 4, A-E (total 6 samples)] Analysis was performed. The real-time PCR was performed by the method described in Example 2. As a control, water was used instead of type I instead.

 The results were shown as Ct values obtained in the same manner as in Example 1. The results for ovarian cDNA are shown in Table 13 and the results for breast cDNA are shown in Table 14. The upper row of each gene shows the expression level in normal tissues, and the lower row shows the expression level in cancer tissues. No.

第 1 4表

Table 14

Thirteen genes, except ABCC2, showed Ct values of 40 or less in almost all ovarian and breast cancer tissues examined. This means that the genes examined in these cancer tissues are expressed.

Based on the results of this analysis, 13 of 14 genes were expressed in cancer tissues. Therefore, examining the expression of the selected 19 genes shown in Table 8 in human cancer tissues can be used to predict the sensitivity to Eg5 inhibitors. Industrial applicability P2004 / 019783 By the present invention, it is possible to select genes for predicting the sensitivity to Eg5 inhibitors in cancer cells and cancer cell lines extracted from cancer patients, and to use those genes for cancer cells against Eg5 inhibitors. Sensitivity can be determined. In addition, it is possible to enhance the sensitivity of cancer cells to an Eg5 inhibitor by using these genes or controlling the expression of those genes. As a result, cancer can be effectively treated with an Eg5 inhibitor.

"Sequence List Free Text"

 SEQ ID NO: 1—Inventor: Fumi Shinohara; Masaya Obayashi; Tetsuro Yoshida

 Inventor: Tetsuya Tsujita; Ryuichiro Nakai

 Inventor: Y. Yamashita,

Claims

The scope of the claims  1. The following steps (a) to (c)  (a) measuring the sensitivity to an Eg5 inhibitor and the expression level of one or more human genes in two or more human cancer cell lines,  (b) analyzing the correlation between the expression level of the gene in each cell line and the sensitivity of the cell line to the Eg5 inhibitor for each gene whose expression level was measured in step (a); (c) selecting a gene that was correlated in step (b), A method for identifying a gene involved in sensitivity of a cancer cell to an Eg5 inhibitor. 2. The human cancer cell lines used for measurement in step (a) are A549, Calu-1, Calu-3, NCI-H23, NCI-H69, NGI-H226, NCI-H345, N417, NC-translated NCI-H596 NCI-H1299, SBC-3, PC-14, PC-14 / DTX ヽ PC-14 / CDDP, MRC-5, IMR-90, EBC-1, NCI-H292, SHP-77, NCI-H441 NCI-H838, A427, NCI -A lung cancer cell line consisting of H522, Calu-6 and PC-9, a colon cancer cell line consisting of Colo205, HT-29, HCT116, SW480, DLD-1 and WiDr, PANC-1, MIA PaCa- 2.All or some cell lines selected from the group consisting of cell line derived from ovarian cancer consisting of AsPC-1 and BxPC-3 and cell line derived from ovarian cancer consisting of SK-0V-3 and 0VCAR-3 The method of claim 1, wherein 3. The cell lines selected are A549, Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226, NCI-H345, N417, NC H460, NCI-H596, NCI-H1299, SBC -3, PC-14, PC-14 / DTX PC-14 / CDDP ヽ MRG-5, IMR-90, EBC-1, NCI-H292, SHP-77, NCI-H441. NCI-H838, A427, NCI- 3. The method according to claim 2, wherein the cell line is all or a part of a cell line selected from a lung cancer-derived cell line consisting of H522, Calu-6 and PC-9.  4. The method according to claim 1, wherein the human cancer cell line used for the measurement in step (a) is a cancer cell line derived from one specific organ.  5. The method according to claim 4, wherein one specific organ is a lung. 6. The Eg5 inhibitor, which measures the sensitivity, has the general formula (I)

In the formula, R ¹ and R ⁴ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkyl, Represents an unsubstituted aryl or a substituted or unsubstituted heterocyclic group, Is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl A ring group, -C (= W) R ⁶ [wherein W represents an oxygen atom or a sulfur atom, R ⁶ is a hydrogen atom, Or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle A group, — NR ⁷ R ⁸ , wherein and R ⁸ are the same or different and are each a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or Represents an unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted heterocyclic ring wherein and and ^ are taken together with an adjacent nitrogen atom forming a group), in -OR ⁹ (wherein, R ³ is substitution or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or Unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, a substituted or unsubstituted Ariru or substituted or unsubstituted heterocyclic group) or -SR ¹⁰ (wherein, R ^1Q has the same meaning as above R ⁹ ) —, NR "R" (wherein R ¹¹ and R ¹² are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted Lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic group or —C (= 0) R ¹³ wherein R ¹³ is a hydrogen atom, substituted or unsubstituted Unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl Le, a substituted or unsubstituted heterocyclic group, - NR ^7A R ^8A (wherein, R ^7A and R ^8A are the same as those that the R ⁷ and R ^8), in one 0R ^9A (wherein, R ^9A is as defined above for R ⁹ ) or _SR ^1QA (wherein, R ^1QA is as defined above for R ⁹ )] or one S0 ₂ R "(wherein is or represents R ⁹ as synonymous), or R ¹ and R ² form a form a heterocyclic group of the connexion substituted or unsubstituted, such together with the adjacent nitrogen atom, Represents a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group. Or R ⁴ and R ⁵ are combined (CR ^15A R ^15B ) _ml — Q— (CR ¹⁵ ¾ ^15D ) _m2 — {wherein Q is a single bond, substituted or unsubstituted Represents methylene or cycloalkylene; ml and m2 are the same or different and each represents an integer of 0 to 4; however, ml and m2 are not simultaneously 0 and R ^15A , R ^15B , R and R ^15D are the same or different. Differently, a hydrogen atom, a halogen, a substituted or unsubstituted lower alkyl, one OR ¹⁶ [wherein R ¹⁶ is a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted Lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, — C0NR ^7B R ^8B (wherein R ^7B and R ^8B are the same or different and are a hydrogen atom, a substituted or unsubstituted lower alkyl Represents a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group; Or R ^7B and R ^SB together with an adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group), — S0 ₂ NR ⁷ ¾ ^8e (wherein ^ and R ^8e are each the aforementioned R ^7B and R ^8B Or COR ¹⁷ (wherein R ¹⁷ is a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted cyclo Represents an alkyl, substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group)], — NR ¹⁸ R ^{19 wherein} R ¹⁸ and R ¹⁹ are the same or different and each represents a hydrogen atom, Substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, — C0R ² ° (wherein R ^2D is hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted Substituted lower alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy, amino, substituted or unsubstituted lower Arukiruamino, a substituted or unsubstituted di-lower alkylamino, or substituted or unsubstituted § Li one Ruamino) or - S0 ₂ R ²¹ (wherein, R ²¹ is substituted if Ku is unsubstituted lower alkyl Represents a substituted or unsubstituted lower alkenyl, a substituted or unsubstituted lower alkynyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroradical.] Or C 0 ₂ R ²² (wherein ² is as defined above for R ¹⁷ ), or R ^15A and R ^15B Or R ^15G and R ^15D together represent an oxygen atom, and when ml or m2 is an integer of 2 or more, R ¹ R ^15B , R ^15C and R ^15D may be the same or different. And R ^15A , R ^15B , R ^15G and R ^{15D which} are bonded to two adjacent carbon atoms may each together form a bond. R ³ represents a hydrogen atom or one C (= W ^A ) R ²³ (wherein W ^A and R ²³ have the same meanings as W and R ⁶ above), and a thiadiazoline derivative represented by> or a drug thereof. The method according to any one of claims 1 to 5, which is a physiologically acceptable salt. 7. R ¹ is a hydrogen atom, R ² is -C (= 0) R ^6A (where R ^6A represents lower alkyl), and R ³ is -C (= 0) R (wherein R is lower alkyl), is substituted lower alkyl, and R ⁵ is phenyl. . 8 _{- (CH 2) na NHS0 2} R a ( wherein, na is 1 or 2, R ^a represents a substituted or unsubstituted lower alkyl) The method according to claim 6 or 7 is. 9. The claim wherein the thiadiazoline derivative represented by the general formula (I) is a compound selected from the group consisting of compounds represented by the following formulas (A) to (C) and (E) to (P). 6. The method according to 6.

10. The five inhibitors that measure the sensitivity are represented by the general formula (II)

 (Π) (In the formula, represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group, and R ²⁵ and R ²⁶ are the same or different and each represents a hydrogen atom, a halogen, a lower alkyl, a lower alkoxy, a hydroxy, a carboxy. , Or hydroxymethyl, or R ²⁵ and R ^{26 taken} together to represent an oxygen atom, a sulfur atom or a bond) or a pharmacologically acceptable salt thereof. The method according to any one of claims 1 to 5, wherein  11. The method according to claim 10, wherein the cysteine derivative is an L-cysteine derivative. 1 2. R ²⁴ is phenyl, 4-methylphenyl, 2 — naphthyl, 4 — pyridyl, or 1, 3 — benzodioxole-1-yl, and R ²⁵ and R ²⁶ are the same or different, The method according to claim 10 or 11, wherein the method is a hydrogen atom, a halogen, a lower alkyl, a lower alkoxy, a hydroxy, a carboxy, or a hydroxymethyl. 13. The method according to any one of claims 1 to 5, wherein the Eg5 inhibitor for measuring the sensitivity is a compound represented by the following formula (Q) or a pharmacologically acceptable salt thereof. the method of.

14. Steps (a) to (e) below  (a) selecting one or more genes from the genes identified by the method according to any one of claims 1 to 13, and determining a score according to the expression level;  (b) measuring the expression level of the gene selected in step (a) for the test cancer cell;  (c) assigning the score determined in step (a) to each gene based on the expression level measured in step (b),  (d) obtaining a numerical value indicative of the sensitivity of the test cancer cell to the Eg5 inhibitor from the assigned scores for all the selected genes,  (e) comparing a predetermined threshold value with the value obtained in (d), a method for determining sensitivity of a cancer cell to an Eg5 inhibitor.  15. The method according to claim 14, wherein the gene selected in the step (a) is one or more genes selected from the group consisting of the following genes (i) to (xix).  (1) Cytochrome P450 'Family 24 · Subfamily A · Polypeptide 1 ■ (CYP24A1)  (ii) Lebutin receptor gene-related protein (OBRGRP)  (iii) Factor B (BF)  (iv) Adenine phosphoribosyltransferase (APRT)  (V) Baculovirus IAP repeat-containing protein (BIRC3)  (vi) Sequence Zome 1 (SQSTM1)  (vii) Tumor necrosis factor α-inducing protein 2 (TNFAIP2)  (viii) integral membrane protein 2B (ITM2B)  (ix) ATP-coupled cassette 'Supfamily 1 C · Member 1 2 (ABCC2)  (X) Annexin A3 (ANXA3)  (xi) Homologous sequence 3 containing family. 'Member 3 (FAM3C)  (xii) ATP binding cassette 'Subfamily 1 C · Member 1 3 (ABCC3) (xiii) hypothetical protein BC007436 (BC007436) (xiv) Microtubule-associated protein IB (MAP1B)  (xv) Cyclin Bl (CCNB1)  (xvi) Heat shock 90 kDa protein 1 h (HSPCA)  (xvii) Cyclin B2 (CCNB)  (xviii) Virtual protein FLJ23269 (FLJ23269)  (xix) Solute Carrier 1 'Family 1 12Member 2 (SLC12A2)  16. The method according to claim 15, wherein the gene selected in the step (a) is', CYP24A1, 0BR (¾P, APRT, TNFAIP2, ITM2B ヽ ABCC2, BC007436, MAP IB, HSPCA ヽ CCNB2, and FLJ23269.  17. The Eg5 inhibitor according to any one of claims 6 to 9, wherein the thiadiazoline derivative or the pharmaceutically acceptable salt thereof according to any one of claims 6 to 9 The method of paragraph 1.  18.5 The method according to any one of claims 14 to 16 wherein the inhibitor is a cysteine derivative according to any one of claims 10 to 12 or a pharmacologically acceptable salt thereof. .  19. The method according to any one of claims 14 to 16, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof.  0. The method according to any one of claims 14 to 19, wherein the cancer cells are lung cancer cells. 21. The method according to any one of claims 1 to 20, wherein the expression level of the gene is measured by quantifying mRNA, which is a transcription product of the gene, using a DNA microarray. ·, 22. The method according to any one of claims 1 to 20, wherein the expression level of the gene is measured by quantifying mRNA, which is a transcription product of the gene, by quantitative PCR.  23. The method according to any one of claims 1 to 20, wherein the expression level of the gene is measured by quantifying mRNA, which is a transcription product of the gene, by Northern plot.  24. A reagent for quantifying an mRNA, comprising an oligonucleotide complementary to the mRNA, for use in the method according to claim 22.  25. The method according to any one of claims 1 to 20, wherein the expression level of the gene is measured by quantifying a protein which is a gene product of the gene by an immunochemical method.  26. The method according to claim 25, wherein the expression level of the gene is measured by quantifying a protein which is a gene product of the gene by ELISA.  27. The method according to claim 25, wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by Western plot.  2.8. An immunoassay containing an antibody against the protein for use in the method according to any one of claims 25 to 27. 29. BC007436, MAP1B, CCNB1 ヽ HSPCA, CCNB2, FLJ23269 and SLC12A2 A gene or a plurality of genes selected from the group consisting of a gene exhibiting an activity of enhancing sensitivity to an inhibitor by forcibly expressing it in a cancer cell or a protein encoded by the gene; Drugs that enhance sensitivity to drugs. 30. The sensitivity enhancer according to claim 29, wherein the cell is a lung cancer cell. 31. The sensitizer according to claim 29 or 30, wherein the Eg5 inhibitor is the thiadiazoline derivative according to any one of claims 6 to 9 or a pharmacologically acceptable salt thereof. . ' 32. The susceptibility according to claim 29 or 30 wherein the Eg5 inhibitor is the cysteine derivative according to any one of claims 10 to 12 or a pharmacologically acceptable salt thereof. Enhancer.  33. The sensitivity enhancer according to claim 29 or 30, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof.  34. Add test substance to cells, and add BC007436, MAP1B, CCNB1, A substance that measures the expression level of at least one gene selected from the group consisting of HSPCA, CCNB2, FLJ23269, and SLC12A2, and increases the expression level of the gene compared to the expression level of the gene without the addition of a test substance A method for screening a substance that enhances sensitivity to an Eg5 inhibitor, wherein the method selects a candidate substance that enhances sensitivity to an Eg5 inhibitor.  35. The screening method according to claim 34, wherein the cell is a lung cancer cell. 36. The screening method according to claim 34 or 35, wherein the Eg5 inhibitor is the thiadiazoline derivative according to any one of claims 6 to 9 or a pharmacologically acceptable salt thereof.  37. The screening method according to claim 34 or 35, wherein the Eg5 inhibitor is the cysteine derivative according to any one of claims 10 to 12 or a pharmacologically acceptable salt thereof. .  38. The screening method according to claim 34 or 35, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof.  39. CYP24AU 0BRGRP ヽ BF, APRT ヽ BIRC3, SQSTMK TNFAIP2, ITM2B ヽ A single or multiple genes selected from the group consisting of ABCC2, ANXA3, FAM3C and ABCC3, and suppress the expression of the genes in cancer cells An siRNA or an antisense polynucleotide that suppresses the expression of a gene exhibiting an activity to enhance sensitivity to an Eg5 inhibitor.  40. The siRNA or antisense polynucleotide according to claim 39, wherein the cancer cell is a lung cancer cell. 41. The sRNA or antisense according to claim 39 or 40, wherein the Eg5 inhibitor is the thiadiazoline derivative according to any one of claims 6 to 9 or a pharmacologically acceptable salt thereof. Polynucleotide. 42. The siRNA or anti-virus according to claim 39 or 40, wherein the Eg5 inhibitor is the cysteine derivative according to any one of claims 10 to 12 or a pharmacologically acceptable salt thereof. Sense polynucleotide. 43. The siRNA or antisense polynucleotide according to claim 39 or 40, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof.  44. A vector that expresses the siRNA or the antisense polynucleotide described in any one of claims 39 to 43.  45. An agent for enhancing sensitivity to an Eg5 inhibitor, comprising the siRNA or the antisense polynucleotide according to any one of claims 39 to 43 or the antisense polynucleotide according to claim 44.  46. CYP24A1 ヽ 0BRGRP ヽ BF ヽ APRT ヽ BIRC3, SQSTMU TNFAIP2, Rinse B ヽ Any gene selected from ABCC2, ANXA3, FAM3C and ABCC3, by suppressing the expression of the gene in cancer cells. A sensitivity enhancer for an Eg5 inhibitor, comprising an antibody against a protein encoded by a gene exhibiting an activity of enhancing sensitivity to an Eg5 inhibitor.  47. The sensitivity enhancer according to claim 46, wherein the Eg5 inhibitor is the thiadiazoline derivative according to any one of claims 6 to 9 or a pharmacologically acceptable salt thereof. 48. The sensitivity enhancer according to claim 46, wherein the Eg5 inhibitor is the cysteine derivative according to any one of claims 10 to 12, or a pharmacologically acceptable salt thereof. 49. The sensitivity enhancer according to claim 46, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof. 50. A test substance is added to the cells, and at least one gene selected from the group consisting of CYP24A1, 0BRGRP, BF, APRT, BIRC3, SQSTM1, TNFAIP2 _S ITM2B2 ABCC2, ANXA3, FAM3C and ABCC3 in the cells is added. The expression level is measured, and a substance that decreases the expression of the gene compared to the expression level of the gene when no test substance is added is selected as a candidate substance that enhances sensitivity to the Eg5 inhibitor. A method for screening a substance that enhances sensitivity to a substance.  51. The screening method according to claim 50, wherein the cell is a lung cancer cell.  52. The screening method according to claim 50 or 51, wherein the Eg5 inhibitor is the thiadiazoline derivative according to any one of claims 6 to 9 or a pharmacologically acceptable salt thereof.  53. The screening method according to claim 50 or 51, wherein the Eg5 inhibitor is the cysteine derivative according to any one of claims 10 to 12 or a pharmacologically acceptable salt thereof. .  54. The screening method according to claim 50 or 51, wherein the Eg5 inhibitor is the compound according to claim 13 or a pharmacologically acceptable salt thereof.