WO2005061707A1 - Procede d'evaluation de la sensibilite d'une cellule cancereuse a l'inhibiteur eg5 - Google Patents

Procede d'evaluation de la sensibilite d'une cellule cancereuse a l'inhibiteur eg5 Download PDF

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WO2005061707A1
WO2005061707A1 PCT/JP2004/019783 JP2004019783W WO2005061707A1 WO 2005061707 A1 WO2005061707 A1 WO 2005061707A1 JP 2004019783 W JP2004019783 W JP 2004019783W WO 2005061707 A1 WO2005061707 A1 WO 2005061707A1
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substituted
unsubstituted
gene
inhibitor
sensitivity
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PCT/JP2004/019783
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Japanese (ja)
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Fumikazu Shinohara
Masaya Obayashi
Tetsuo Yoshida
Tetsuya Tsujita
Ryuichiro Nakai
Yoshinori Yamashita
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to JP2005516541A priority Critical patent/JPWO2005061707A1/ja
Publication of WO2005061707A1 publication Critical patent/WO2005061707A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • 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.
  • Cancer chemotherapy with various anticancer drugs is one of the most important methods in treating cancer.
  • 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.
  • many genes have been reported as determinants of sensitivity to multiple anticancer drugs.
  • the drug transporters MDR1 J, Biol. Chem., 265, 506-514, 1990
  • 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.
  • 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 ⁇ ⁇ ⁇ ⁇ 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).
  • 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.
  • MDR1 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.
  • 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.
  • the present invention relates to the following (1) to (54).
  • step (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);
  • step (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 is provided.
  • 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 ⁇ 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
  • Cell lines selected are A549 s Calu-1, Calu-3, NCI-H23, NCI-H69, NCI-H226 ⁇ 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 ⁇ 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.
  • step (a) (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.
  • 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 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 3 ⁇ 4 15D ) m2 — ⁇ wherein Q represents a single bond, substituted or unsubstituted phenylene or cycloalkylene, and ml and ⁇ 12 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.
  • 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 TM and R 8B together with an adjacent nitrogen atom; To form an unsubstituted
  • 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.
  • R 1 is hydrogen atom
  • R 6A wherein, R 6A represents a lower alkyl
  • R 4 is substituted lower alkyl
  • R 5 is phenyl.
  • 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.
  • 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)
  • 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,
  • cysteine derivative is an L-cysteine derivative.
  • R 24 is phenyl, 4-methylphenyl, 2-naphthyl, 4-pyridyl, or 1,3-benzodioxoyl-5-yl
  • 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.
  • 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.
  • step (b) measuring the expression level of the gene selected in step (a) for the test cancer cell
  • step (c) assigning, for each gene, the score determined in step (a) based on the expression level measured in step (b),
  • Adenine phosphoribosyl transcriptase APRT
  • BIRC3 Bakilowis IAP repeat-containing protein
  • TFAIP2 Tumor necrosis factor spike inducible protein 2
  • 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.
  • a reagent for quantifying mRNA comprising an oligonucleotide complementary to the mRNA, for use in the method according to (22).
  • the expression level of a gene is measured by immunizing a protein which is a gene product of the gene.
  • An agent for enhancing sensitivity to an Eg5 inhibitor comprising a gene exhibiting activity or a protein encoded by the gene.
  • the sensitivity enhancer according to (29), wherein the cell is a lung cancer cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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).
  • 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).
  • 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.
  • the Eg5 inhibitor is the thiadiazolin derivative according to any one of (6) to (9) or a pharmacologically acceptable salt thereof.
  • 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 is selected from the group consisting of CYP24A1, 0BRGRP, BF, APRT, BIRC3, SQSTMU TNFAIP2 S ITM2B, ABCC2, ANXA3, FAM3C and ABCC3 in the cells.
  • 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.
  • the method for identifying a gene involved in sensitivity to an Eg5 inhibitor of the present invention includes the following steps (a) to (c). .
  • step (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)
  • step (c) Step of selecting genes that were correlated in step (b)
  • 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.
  • 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. .
  • 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.
  • the Eg5 inhibitor used in the present invention is not particularly limited as long as it has Eg5 inhibitory activity.
  • WO04 / 034972 WO03 / 039460 s WO03 / 049527 s WO03 / 050122 WO03 / 050064,
  • 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.
  • 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. '
  • lower alkynyl examples include straight-chain or branched alkynyl having 2 to 8 carbon atoms, specifically ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and the like.
  • cycloalkyl examples include cycloalkyl having 3 to 8 carbon atoms, specifically, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, cycloheptyl and the like.
  • the aryl portion of aryl, aryloxy and arylamino includes, for example, phenyl, naphthyl and the like.
  • heterocyclic group examples include an aliphatic heterocyclic group and an aromatic heterocyclic group.
  • 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.
  • aromatic heterocyclic group examples 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 ⁇ 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.
  • aromatic heterocyclic group examples include the aromatic heterocyclic groups described in (vi) above.
  • heterocyclic group formed together with an adjacent nitrogen atom examples 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.
  • cycloalkylene examples include cycloalkylene having 3 to 8 carbon atoms, specifically, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene and the like.
  • Phenylene includes 1,4-phenylene, 1,3-phenylene or 1,2-phenylene.
  • Halogen means each atom of fluorine, chlorine, bromine and iodine.
  • 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
  • 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.
  • substituted aryl substituted or unsubstituted aryl
  • 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)
  • R aralkyloxycarbonyl, or R 31 and R 32 together with the adjacent nitrogen atom are substituted or unsubstituted He
  • substituted 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 ( ⁇ 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 alky
  • Heterocyclic group (the substituent in the substituted heterocyclic group "is synonymous with, - CONR 3 in 3 ⁇ 4 35 (wherein, R 34 and R 35 described below substitutions in substituted double heterocyclic group group ( ⁇ )) '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 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 C3 ⁇ 40) 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.
  • substituted or unsubstituted lower alkynyl 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.
  • R 51 is the R 47 as synonymous
  • 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.
  • 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
  • substituted or unsubstituted aryl 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.
  • 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.
  • 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.
  • 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.
  • substituent (d) in the substituted lower alkyl Is the same as the substituent (d) in the substituted lower alkyl).
  • 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 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 substituted or unsubstituted arylothio the substituent in the substituted arylothio is the same as the substituent (e) in the above substituted aryl)
  • 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).
  • 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), ( ⁇ ) and Q include, for example, inorganic salts such as hydrochloride, sulfate, phosphate, acetate, maleate, and fumarate.
  • 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.
  • salts of compounds (I), ( ⁇ ) and Q when it is desired to obtain salts of compounds (I), ( ⁇ ) 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 ⁇ 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.
  • R 1 R 2 R 3 R 4 R 6 Compound AH C0CH 3 C0C3 ⁇ 4 CH 3. O compound BH COC (CH 3) 3 COC (CH 3) 3 (C3 ⁇ 4) 2 C0N3 ⁇ 4 O compound c H COC (CH 3) 3 C0C ( C3 ⁇ 4) 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 (c3 ⁇ 4 ) 3 CH 2 NHS0 2 CH 2 G3 ⁇ 4 - ⁇ Compound GH COC (CH 3 ) 3 coc (c3 ⁇ 4) 3 CH 2 CH 2 N (C3 ⁇ 4) 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 c3 ⁇ 4 cr compound JH C0C (C3 ⁇ 4) 3 CO
  • 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.
  • PBS phosphate buffered saline
  • 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.
  • a blocking solution PBS containing 1% fetal bovine serum
  • the primary antibody solution (0.2% mouse monoclonal anti-tubulin antibody [Sigma-Aldrich] , Cat. No. T9026]
  • a blocking solution containing 0.2% Egret anti-tubulin antibody Sigma Aldrich, Cat. No. 3559
  • 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.
  • compound A and compound D 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.
  • 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.
  • 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 3 ⁇ 4 into cells.
  • 'Total protein content can be measured by the Sulfolodamine B Atsey method or the like.
  • 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.
  • RNA or protein can be extracted from the human cancer cell line of (1) by the following general method.
  • Trisol (TRIZ0L) reagent Trisol (TRIZ0L) reagent [Invitrogen
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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 are selected as described above. Examples include the following 12 genes.
  • CYP24A1 Cytochrome P450 'family 24 ⁇ subfamily A' polypeptide 1 ( ⁇ —0Q0782)
  • BIRC3 Baculovirus IAP repeat-containing protein— ( ⁇ —001165)
  • TNFAIP2 tumor necrosis factor-inducing protein 2 (NM_006291)
  • ITM2B integral membrane protein 2B ( ⁇ —021999)
  • ABCC2 ATP binding cassette 'Subfamily 1 C ⁇ Member 2 (Marauder-000392)
  • X ANXA3: Annexin A3 (Thigh-005139)
  • FAM3C Homologous sequence 3 containing family 1 'member 1 3 (NMJ14888)
  • BCO07436 hypothetical protein BC007436 (XM-037317)
  • MAP1B microtubule-associated protein 1B (band-005909)
  • HSPCA one heat shock 90 kDa protein (BC023006)
  • FLJ23269 Virtual protein F23269 (AK026922)
  • 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.
  • the present invention further provides a method for determining the sensitivity of a cancer cell to a 5-inhibitor.
  • 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
  • step (c) assigning, for each gene, the score determined in step (a) based on the expression level measured in step (b).
  • 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.).
  • 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 3 ⁇ 4 + ⁇ ⁇ ⁇ + a n X n + b (Equation 1),
  • n + 1 cell lines 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 ' ⁇ & ⁇ 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.
  • the statistics are used as an index for optimization and to select genes so that the index becomes smaller.
  • the score of each gene is expressed as the product of the partial regression coefficient obtained above and the expression level of the gene.
  • genes that are important for predicting the sensitivity to 5 inhibitors include the relationship between the expression level selected in (A) and the sensitivity to Eg5 inhibitors.
  • CYP24A1, 0BRGRP, APRT ⁇ NFAIP2, ITM2B, ABCC2, BC007436, MAP1B ⁇ 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.
  • 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.
  • 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)
  • 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.
  • the sensitivity of the cancer cell of the cancer patient to the Eg5 inhibitor can be determined.
  • 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.
  • 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.
  • 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.
  • the gene 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.
  • 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 Bio Science (Amersham
  • any method for introducing DNA into animal cells can be used.
  • 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. .
  • 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 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)).
  • 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
  • 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
  • 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
  • a drug that expresses 4400 lereotide should be used as a potent sensitizer for EEgg55 inhibitors.
  • 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.
  • 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.
  • a sequence having 19 bases following AA and a GC content of 30 to 70%, more preferably around 50% is selected.
  • 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.
  • pSilencer 1.0-U6 (Ambion)
  • pSUPER oligo engine
  • siRNA expression vector such as an siRNA expression vector
  • 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 ⁇ 3′-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
  • 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,
  • 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.
  • 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.
  • (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)
  • 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.
  • 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 ⁇ 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.
  • an Eg5 inhibitor such as CYP24A1, OBRGRPs BF, APRT, BIRC3, SQSTM1, TNFAIP2, Rinse B ⁇ 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
  • 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.
  • 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.
  • 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.
  • the repo overnight gene the human liziferiferase 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a carrier such as a salt solution, a pudose solution, or a mixture of a saline solution and a glucose solution.
  • a carrier such as a salt solution, a pudose solution, or a mixture of a saline solution and a glucose solution.
  • these parenteral preparations one selected from the excipients, disintegrants, lubricants, binders, surfactants, plasticizers and diluents, preservatives, flavors, etc.
  • more auxiliary components can be added.
  • 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.
  • 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.
  • the dose and the number of doses vary depending on the various conditions described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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 ⁇ ) 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.
  • 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.
  • NM.032290 127, 128 hyperthetical protein DKFZp761C 121) 761C121
  • Casein kinase 1 alpha 1 CSNK1A1 N _001892 131, 132 angiotensinogen AGT NM.000029 133,134 tubulin 2 (tubulin, beta, 2).
  • Intracellular retinol binding protein 1 Intracellular retinol binding protein 1
  • RBP1 NM— 002899 167, 168 retinol binding protein 1, cellular
  • Adrenergic 32 receptor (adrenergic, beta-2-, receptor, surface) ADRB2 NM— 000024 173, 174 Homologous sequence 3 containing family 'member 3
  • 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.
  • DEPC dimethyl pyrocarbonate
  • 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.
  • 10 jl of a 5-fold diluted solution was used.
  • 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.
  • 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.
  • ABI sequence detection system ABSI PRISM 7700, Applied Biosystems
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • Example 3 Correlation analysis of gene expression profile and sensitivity to Eg5 inhibitor
  • Table 4 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.
  • the Pearson correlation coefficient r calculated by the following formula was calculated. .
  • 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.
  • HNRPA2B1 0.29 0.15-0.26 0.04
  • 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.
  • 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.
  • 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
  • the resistance gene BIRC3 is known as a gene that suppresses cell death
  • the susceptibility genes CCNB1 and CCNB2 are genes related to the cell cycle.
  • 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.
  • 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 , ⁇ ⁇ and the constant term b.
  • 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.
  • 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 ⁇ PC-14 / CDDP ⁇ MRC-5, IMR-90, EBC-1, NCI-H292, NC band ⁇ NCI-H838, ⁇ , 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.
  • 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 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 initial model constructed in (i) is complicated because there are as many as 19 explanatory variables.
  • 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.
  • S_PLUS2000 User's Guide the statistical analysis software “S-Plus2000” (Mathematical Systems Co., Ltd.
  • 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.
  • Example 1 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.
  • 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.
  • Example 2 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.
  • SEQ ID NO: 1 Inventor: Fumi Shinohara; Masaya Obayashi; Tetsuro Yoshida

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Abstract

L'invention concerne un procédé qui consiste à mesurer les sensibilités à l'inhibiteur Eg5 de deux ou plusieurs lignées cellulaires cancéreuses humaines et les quantités d'expression d'un ou de plusieurs gènes humains et à identifier un gène présentant une corrélation entre sa quantité d'expression et la sensibilité à l'inhibiteur Eg5 en tant que gène se rapportant à la sensibilité à l'inhibiteur Eg5. L'invention concerne un procédé qui consiste à choisir un ou plusieurs gènes parmi ceux qui se rapportent à la sensibilité à l'inhibiteur Eg5 ainsi identifiés, à mesurer les quantités d'expression des gènes choisis dans des cellules cancéreuses, à attribuer des cotes en fonction des quantités d'expression et, par conséquent, à évaluer la sensibilité des cellules cancéreuses à l'inhibiteur Eg5.
PCT/JP2004/019783 2003-12-24 2004-12-24 Procede d'evaluation de la sensibilite d'une cellule cancereuse a l'inhibiteur eg5 WO2005061707A1 (fr)

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JP7478094B2 (ja) 2017-10-31 2024-05-02 エクセンシア ゲゼルシャフト ミット ベシュレンクテル ハフツング 試験化合物の選択性を決定する方法
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