JP2016069348A - Therapeutic compositions for chronic myelogenous leukemia - Google Patents
Therapeutic compositions for chronic myelogenous leukemia Download PDFInfo
- Publication number
- JP2016069348A JP2016069348A JP2014202529A JP2014202529A JP2016069348A JP 2016069348 A JP2016069348 A JP 2016069348A JP 2014202529 A JP2014202529 A JP 2014202529A JP 2014202529 A JP2014202529 A JP 2014202529A JP 2016069348 A JP2016069348 A JP 2016069348A
- Authority
- JP
- Japan
- Prior art keywords
- imatinib
- cells
- pharmaceutical composition
- met
- resistant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 title claims abstract description 25
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 title claims abstract description 25
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 title claims abstract description 24
- 239000000203 mixture Substances 0.000 title claims abstract description 15
- 230000001225 therapeutic effect Effects 0.000 title claims abstract description 8
- 239000005517 L01XE01 - Imatinib Substances 0.000 claims abstract description 168
- 229960002411 imatinib Drugs 0.000 claims abstract description 168
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 claims abstract description 167
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 31
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000002067 L01XE06 - Dasatinib Substances 0.000 claims abstract description 21
- 229960002448 dasatinib Drugs 0.000 claims abstract description 21
- 239000005536 L01XE08 - Nilotinib Substances 0.000 claims abstract description 19
- 229960001346 nilotinib Drugs 0.000 claims abstract description 19
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000002137 L01XE24 - Ponatinib Substances 0.000 claims abstract description 18
- 229960001131 ponatinib Drugs 0.000 claims abstract description 18
- PHXJVRSECIGDHY-UHFFFAOYSA-N ponatinib Chemical compound C1CN(C)CCN1CC(C(=C1)C(F)(F)F)=CC=C1NC(=O)C1=CC=C(C)C(C#CC=2N3N=CC=CC3=NC=2)=C1 PHXJVRSECIGDHY-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000003840 Bafetinib Substances 0.000 claims abstract description 12
- 229950002365 bafetinib Drugs 0.000 claims abstract description 12
- ZGBAJMQHJDFTQJ-DEOSSOPVSA-N bafetinib Chemical compound C1[C@@H](N(C)C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=NC=3)C(C)=CC=2)C=C1C(F)(F)F ZGBAJMQHJDFTQJ-DEOSSOPVSA-N 0.000 claims abstract description 12
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 claims abstract description 6
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 claims abstract description 6
- 102000004441 bcr-abl Fusion Proteins Human genes 0.000 claims abstract description 6
- 108010056708 bcr-abl Fusion Proteins Proteins 0.000 claims abstract description 6
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 claims description 11
- -1 foatinib Chemical compound 0.000 claims description 11
- 230000019491 signal transduction Effects 0.000 claims description 10
- 229940124647 MEK inhibitor Drugs 0.000 claims description 8
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims description 8
- RWEVIPRMPFNTLO-UHFFFAOYSA-N 2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-3-pyridinecarboxamide Chemical compound CN1C(=O)C(C)=CC(C(=O)NOCCO)=C1NC1=CC=C(I)C=C1F RWEVIPRMPFNTLO-UHFFFAOYSA-N 0.000 claims description 4
- RFWVETIZUQEJEF-UHFFFAOYSA-N GDC-0623 Chemical compound OCCONC(=O)C=1C=CC2=CN=CN2C=1NC1=CC=C(I)C=C1F RFWVETIZUQEJEF-UHFFFAOYSA-N 0.000 claims description 4
- 229940126313 avutometinib Drugs 0.000 claims description 4
- ACWZRVQXLIRSDF-UHFFFAOYSA-N binimetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1F ACWZRVQXLIRSDF-UHFFFAOYSA-N 0.000 claims description 4
- HDAJDNHIBCDLQF-RUZDIDTESA-N SCH772984 Chemical compound O=C([C@@H]1CCN(C1)CC(=O)N1CCN(CC1)C=1C=CC(=CC=1)C=1N=CC=CN=1)NC(C=C12)=CC=C1NN=C2C1=CC=NC=C1 HDAJDNHIBCDLQF-RUZDIDTESA-N 0.000 claims description 3
- 229950009580 merestinib Drugs 0.000 claims description 3
- RDSACQWTXKSHJT-NSHDSACASA-N n-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2s)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide Chemical compound C1CC1(C[C@H](O)CO)S(=O)(=O)NC=1C(OC)=CC(F)=C(F)C=1NC1=CC=C(I)C=C1F RDSACQWTXKSHJT-NSHDSACASA-N 0.000 claims description 3
- 229950008933 refametinib Drugs 0.000 claims description 3
- 229960004066 trametinib Drugs 0.000 claims description 3
- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical compound CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 claims description 3
- VMJFTOSOFDEKTM-UHFFFAOYSA-N 1-[2-(1-methylpyrazol-4-yl)-11-oxobenzo[1,2]cyclohepta[2,4-b]pyridin-9-yl]-n-(pyridin-2-ylmethyl)methanesulfonamide Chemical compound C1=NN(C)C=C1C1=CN=C(C=CC=2C(=CC(CS(=O)(=O)NCC=3N=CC=CC=3)=CC=2)C2=O)C2=C1 VMJFTOSOFDEKTM-UHFFFAOYSA-N 0.000 claims description 2
- ORRNXRYWGDUDOG-UHFFFAOYSA-N 4-[2-fluoro-4-[(2-phenylacetyl)carbamothioylamino]phenoxy]-7-methoxy-n-methylquinoline-6-carboxamide Chemical compound C1=CN=C2C=C(OC)C(C(=O)NC)=CC2=C1OC(C(=C1)F)=CC=C1NC(=S)NC(=O)CC1=CC=CC=C1 ORRNXRYWGDUDOG-UHFFFAOYSA-N 0.000 claims description 2
- JRWCBEOAFGHNNU-UHFFFAOYSA-N 6-[difluoro-[6-(1-methyl-4-pyrazolyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]methyl]quinoline Chemical compound C1=NN(C)C=C1C1=NN2C(C(F)(F)C=3C=C4C=CC=NC4=CC=3)=NN=C2C=C1 JRWCBEOAFGHNNU-UHFFFAOYSA-N 0.000 claims description 2
- JGEBLDKNWBUGRZ-HXUWFJFHSA-N 9-[[[(2r)-1,4-dioxan-2-yl]methyl-methylsulfamoyl]amino]-2-(1-methylpyrazol-4-yl)-11-oxobenzo[1,2]cyclohepta[2,4-b]pyridine Chemical compound C=1C=C2C=CC3=NC=C(C4=CN(C)N=C4)C=C3C(=O)C2=CC=1NS(=O)(=O)N(C)C[C@@H]1COCCO1 JGEBLDKNWBUGRZ-HXUWFJFHSA-N 0.000 claims description 2
- UQRCJCNVNUFYDX-UHFFFAOYSA-N Golvatinib Chemical compound C1CN(C)CCN1C1CCN(C(=O)NC=2N=CC=C(OC=3C=C(F)C(NC(=O)C4(CC4)C(=O)NC=4C=CC(F)=CC=4)=CC=3)C=2)CC1 UQRCJCNVNUFYDX-UHFFFAOYSA-N 0.000 claims description 2
- 239000002146 L01XE16 - Crizotinib Substances 0.000 claims description 2
- 239000002176 L01XE26 - Cabozantinib Substances 0.000 claims description 2
- UCEQXRCJXIVODC-PMACEKPBSA-N LSM-1131 Chemical compound C1CCC2=CC=CC3=C2N1C=C3[C@@H]1C(=O)NC(=O)[C@H]1C1=CNC2=CC=CC=C12 UCEQXRCJXIVODC-PMACEKPBSA-N 0.000 claims description 2
- VNBRGSXVFBYQNN-UHFFFAOYSA-N N-[4-[(2-amino-3-chloro-4-pyridinyl)oxy]-3-fluorophenyl]-4-ethoxy-1-(4-fluorophenyl)-2-oxo-3-pyridinecarboxamide Chemical compound O=C1C(C(=O)NC=2C=C(F)C(OC=3C(=C(N)N=CC=3)Cl)=CC=2)=C(OCC)C=CN1C1=CC=C(F)C=C1 VNBRGSXVFBYQNN-UHFFFAOYSA-N 0.000 claims description 2
- BCZUAADEACICHN-UHFFFAOYSA-N SGX-523 Chemical compound C1=NN(C)C=C1C1=NN2C(SC=3C=C4C=CC=NC4=CC=3)=NN=C2C=C1 BCZUAADEACICHN-UHFFFAOYSA-N 0.000 claims description 2
- 229960001292 cabozantinib Drugs 0.000 claims description 2
- ONIQOQHATWINJY-UHFFFAOYSA-N cabozantinib Chemical compound C=12C=C(OC)C(OC)=CC2=NC=CC=1OC(C=C1)=CC=C1NC(=O)C1(C(=O)NC=2C=CC(F)=CC=2)CC1 ONIQOQHATWINJY-UHFFFAOYSA-N 0.000 claims description 2
- KTEIFNKAUNYNJU-GFCCVEGCSA-N crizotinib Chemical compound O([C@H](C)C=1C(=C(F)C=CC=1Cl)Cl)C(C(=NC=1)N)=CC=1C(=C1)C=NN1C1CCNCC1 KTEIFNKAUNYNJU-GFCCVEGCSA-N 0.000 claims description 2
- 229960005061 crizotinib Drugs 0.000 claims description 2
- 229950007540 glesatinib Drugs 0.000 claims description 2
- 229950010662 golvatinib Drugs 0.000 claims description 2
- KSERXGMCDHOLSS-LJQANCHMSA-N n-[(1s)-1-(3-chlorophenyl)-2-hydroxyethyl]-4-[5-chloro-2-(propan-2-ylamino)pyridin-4-yl]-1h-pyrrole-2-carboxamide Chemical compound C1=NC(NC(C)C)=CC(C=2C=C(NC=2)C(=O)N[C@H](CO)C=2C=C(Cl)C=CC=2)=C1Cl KSERXGMCDHOLSS-LJQANCHMSA-N 0.000 claims description 2
- AHYMHWXQRWRBKT-UHFFFAOYSA-N tepotinib Chemical compound C1CN(C)CCC1COC1=CN=C(C=2C=C(CN3C(C=CC(=N3)C=3C=C(C=CC=3)C#N)=O)C=CC=2)N=C1 AHYMHWXQRWRBKT-UHFFFAOYSA-N 0.000 claims description 2
- 229950005976 tivantinib Drugs 0.000 claims description 2
- ODIUNTQOXRXOIV-UHFFFAOYSA-N 1-[6-[[6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]sulfanyl]-1,3-benzothiazol-2-yl]-3-(2-morpholin-4-ylethyl)urea Chemical compound C1=CC(F)=CC=C1C1=NN2C(SC=3C=C4SC(NC(=O)NCCN5CCOCC5)=NC4=CC=3)=NN=C2C=C1 ODIUNTQOXRXOIV-UHFFFAOYSA-N 0.000 claims 1
- PDMUGYOXRHVNMO-UHFFFAOYSA-N 2-[4-[3-(6-quinolinylmethyl)-5-triazolo[4,5-b]pyrazinyl]-1-pyrazolyl]ethanol Chemical compound C1=NN(CCO)C=C1C1=CN=C(N=NN2CC=3C=C4C=CC=NC4=CC=3)C2=N1 PDMUGYOXRHVNMO-UHFFFAOYSA-N 0.000 claims 1
- UFICVEHDQUKCEA-UHFFFAOYSA-N N-[[3-fluoro-4-[[2-(1-methyl-4-imidazolyl)-7-thieno[3,2-b]pyridinyl]oxy]anilino]-sulfanylidenemethyl]-2-phenylacetamide Chemical compound CN1C=NC(C=2SC3=C(OC=4C(=CC(NC(=S)NC(=O)CC=5C=CC=CC=5)=CC=4)F)C=CN=C3C=2)=C1 UFICVEHDQUKCEA-UHFFFAOYSA-N 0.000 claims 1
- 108091000080 Phosphotransferase Proteins 0.000 claims 1
- 102000020233 phosphotransferase Human genes 0.000 claims 1
- 206010059866 Drug resistance Diseases 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 2
- 102000001291 MAP Kinase Kinase Kinase Human genes 0.000 abstract 1
- 108060006687 MAP kinase kinase kinase Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 177
- 230000003833 cell viability Effects 0.000 description 54
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 20
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 20
- 239000003112 inhibitor Substances 0.000 description 18
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 16
- 239000003814 drug Substances 0.000 description 16
- 230000001965 increasing effect Effects 0.000 description 15
- OYONTEXKYJZFHA-SSHUPFPWSA-N PHA-665752 Chemical compound CC=1C(C(=O)N2[C@H](CCC2)CN2CCCC2)=C(C)NC=1\C=C(C1=C2)/C(=O)NC1=CC=C2S(=O)(=O)CC1=C(Cl)C=CC=C1Cl OYONTEXKYJZFHA-SSHUPFPWSA-N 0.000 description 13
- 238000003119 immunoblot Methods 0.000 description 13
- 230000037361 pathway Effects 0.000 description 11
- 230000030833 cell death Effects 0.000 description 10
- 230000003247 decreasing effect Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 108010007457 Extracellular Signal-Regulated MAP Kinases Proteins 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 229940124597 therapeutic agent Drugs 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 101000876610 Dictyostelium discoideum Extracellular signal-regulated kinase 2 Proteins 0.000 description 7
- 101001052493 Homo sapiens Mitogen-activated protein kinase 1 Proteins 0.000 description 7
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 7
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 238000010998 test method Methods 0.000 description 7
- 238000003211 trypan blue cell staining Methods 0.000 description 7
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 6
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 6
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical compound NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 description 5
- 229940124290 BCR-ABL tyrosine kinase inhibitor Drugs 0.000 description 5
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 4
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 4
- 239000012828 PI3K inhibitor Substances 0.000 description 4
- 229940124639 Selective inhibitor Drugs 0.000 description 4
- 238000012790 confirmation Methods 0.000 description 4
- 229940043441 phosphoinositide 3-kinase inhibitor Drugs 0.000 description 4
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 4
- 238000000035 BCA protein assay Methods 0.000 description 3
- 229940121730 Janus kinase 2 inhibitor Drugs 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- HEAIZQNMNCHNFD-UHFFFAOYSA-N AMG-208 Chemical compound C=1C=NC2=CC(OC)=CC=C2C=1OCC(N1N=2)=NN=C1C=CC=2C1=CC=CC=C1 HEAIZQNMNCHNFD-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102000001301 EGF receptor Human genes 0.000 description 2
- 108060006698 EGF receptor Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 229940122661 MET tyrosine kinase inhibitor Drugs 0.000 description 2
- 208000014767 Myeloproliferative disease Diseases 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000012830 cancer therapeutic Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 229960002271 cobimetinib Drugs 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940121647 egfr inhibitor Drugs 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 229940124303 multikinase inhibitor Drugs 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 210000004214 philadelphia chromosome Anatomy 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229950010746 selumetinib Drugs 0.000 description 2
- CYOHGALHFOKKQC-UHFFFAOYSA-N selumetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1Cl CYOHGALHFOKKQC-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- GLBZSOQDAOLMGC-UHFFFAOYSA-N 1-(2-hydroxy-2-methylpropyl)-n-[5-(7-methoxyquinolin-4-yl)oxypyridin-2-yl]-5-methyl-3-oxo-2-phenylpyrazole-4-carboxamide Chemical compound C=1C=NC2=CC(OC)=CC=C2C=1OC(C=N1)=CC=C1NC(=O)C(C1=O)=C(C)N(CC(C)(C)O)N1C1=CC=CC=C1 GLBZSOQDAOLMGC-UHFFFAOYSA-N 0.000 description 1
- RCLQNICOARASSR-SECBINFHSA-N 3-[(2r)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3-d]pyrimidine-4,7-dione Chemical compound FC=1C(=O)N(C)C=2N=CN(C[C@@H](O)CO)C(=O)C=2C=1NC1=CC=C(I)C=C1F RCLQNICOARASSR-SECBINFHSA-N 0.000 description 1
- RNCNPRCUHHDYPC-UHFFFAOYSA-N 6-[[6-(1-methylpyrazol-4-yl)imidazo[1,2-b]pyridazin-3-yl]methyl]quinoline Chemical compound C1=NN(C)C=C1C1=NN2C(CC=3C=C4C=CC=NC4=CC=3)=CN=C2C=C1 RNCNPRCUHHDYPC-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 230000007730 Akt signaling Effects 0.000 description 1
- LQVXSNNAFNGRAH-QHCPKHFHSA-N BMS-754807 Chemical compound C([C@@]1(C)C(=O)NC=2C=NC(F)=CC=2)CCN1C(=NN1C=CC=C11)N=C1NC(=NN1)C=C1C1CC1 LQVXSNNAFNGRAH-QHCPKHFHSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000034951 Genetic Translocation Diseases 0.000 description 1
- 101000997832 Homo sapiens Tyrosine-protein kinase JAK2 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 150000004926 Imatinib derivatives Chemical class 0.000 description 1
- 239000002145 L01XE14 - Bosutinib Substances 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- CXQHYVUVSFXTMY-UHFFFAOYSA-N N1'-[3-fluoro-4-[[6-methoxy-7-[3-(4-morpholinyl)propoxy]-4-quinolinyl]oxy]phenyl]-N1-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide Chemical compound C1=CN=C2C=C(OCCCN3CCOCC3)C(OC)=CC2=C1OC(C(=C1)F)=CC=C1NC(=O)C1(C(=O)NC=2C=CC(F)=CC=2)CC1 CXQHYVUVSFXTMY-UHFFFAOYSA-N 0.000 description 1
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000005228 aryl sulfonate group Chemical group 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 229960003736 bosutinib Drugs 0.000 description 1
- UBPYILGKFZZVDX-UHFFFAOYSA-N bosutinib Chemical compound C1=C(Cl)C(OC)=CC(NC=2C3=CC(OC)=C(OCCCN4CCN(C)CC4)C=C3N=CC=2C#N)=C1Cl UBPYILGKFZZVDX-UHFFFAOYSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- BSMCAPRUBJMWDF-KRWDZBQOSA-N cobimetinib Chemical compound C1C(O)([C@H]2NCCCC2)CN1C(=O)C1=CC=C(F)C(F)=C1NC1=CC=C(I)C=C1F BSMCAPRUBJMWDF-KRWDZBQOSA-N 0.000 description 1
- RESIMIUSNACMNW-BXRWSSRYSA-N cobimetinib fumarate Chemical compound OC(=O)\C=C\C(O)=O.C1C(O)([C@H]2NCCCC2)CN1C(=O)C1=CC=C(F)C(F)=C1NC1=CC=C(I)C=C1F.C1C(O)([C@H]2NCCCC2)CN1C(=O)C1=CC=C(F)C(F)=C1NC1=CC=C(I)C=C1F RESIMIUSNACMNW-BXRWSSRYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229950008692 foretinib Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-M hydrogensulfate Chemical compound OS([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 1
- 229960003685 imatinib mesylate Drugs 0.000 description 1
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- PDYXPCKITKHFOZ-UHFFFAOYSA-N n-[4-(2-amino-3-chloropyridin-4-yl)oxy-3-fluorophenyl]-5-(4-fluorophenyl)-4-oxo-1h-pyridine-3-carboxamide Chemical compound NC1=NC=CC(OC=2C(=CC(NC(=O)C=3C(C(C=4C=CC(F)=CC=4)=CNC=3)=O)=CC=2)F)=C1Cl PDYXPCKITKHFOZ-UHFFFAOYSA-N 0.000 description 1
- YRCHYHRCBXNYNU-UHFFFAOYSA-N n-[[3-fluoro-4-[2-[5-[(2-methoxyethylamino)methyl]pyridin-2-yl]thieno[3,2-b]pyridin-7-yl]oxyphenyl]carbamothioyl]-2-(4-fluorophenyl)acetamide Chemical compound N1=CC(CNCCOC)=CC=C1C1=CC2=NC=CC(OC=3C(=CC(NC(=S)NC(=O)CC=4C=CC(F)=CC=4)=CC=3)F)=C2S1 YRCHYHRCBXNYNU-UHFFFAOYSA-N 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
本発明は、慢性骨髄性白血病の治療用組成物に関するもので、特に従来の治療薬に対して耐性を有する患者に対しても有効な治療用組成物を提供するものである。 The present invention relates to a therapeutic composition for chronic myelogenous leukemia, and particularly provides a therapeutic composition that is effective even for patients who are resistant to conventional therapeutic agents.
慢性骨髄性白血病（Ｃｈｒｏｎｉｃ Ｍｙｅｌｏｇｅｎｏｕｓ Ｌｅｕｋｅｍｉａ：ＣＭＬ）は、造血幹細胞を発症起源とする骨髄増殖性疾患である。フィラデルフィア染色体として知られる染色体転座による造血幹細胞の遺伝子の後天的異常が原因とされる。造血幹細胞が分化・成熟能を保ったまま自律的な増殖をし、白血球や血小板が増加する血液腫瘍である。この疾患は成人白血病の約１５％を占めるといわれている。 Chronic myelogenous leukemia (CML) is a myeloproliferative disease that originates from hematopoietic stem cells. It is caused by an acquired abnormality of hematopoietic stem cell genes due to a chromosomal translocation known as the Philadelphia chromosome. Hematopoietic stem cells proliferate autonomously while maintaining differentiation and maturation ability, and are blood tumors in which white blood cells and platelets increase. This disease is said to account for about 15% of adult leukemia.
慢性骨髄性白血病の治療薬としては、フィラデルフィア染色体の遺伝子産物であるＢｃｒ−Ａｂｌに対するチロシンキナーゼ阻害薬（Ｔｙｒｏｓｉｎｅ Ｋｉｎａｓｅ Ｉｎｈｉｂｉｔｏｒ：ＴＫＩ）であるメシル酸イマチニブ（以後単に「イマチニブ」ともいう。）が開発され、骨髄増殖性疾患患者の治療成績は著しく改善した。この薬は、Ｂｃｒ−Ａｂｌを標的とするため、分子標的薬と呼ばれている。 As a therapeutic agent for chronic myelogenous leukemia, imatinib mesylate (hereinafter also simply referred to as “imatinib”), a tyrosine kinase inhibitor (TKI) for Bcr-Abl, which is a gene product of the Philadelphia chromosome, was developed. As a result, treatment outcomes for patients with myeloproliferative disorders have improved significantly. This drug is called a molecular target drug because it targets Bcr-Abl.
しかし、イマチニブを用いた治療を行うと、その患者がイマチニブに対する耐性獲得することが臨床上の重大な問題となっている。このため、耐性を獲得した患者に対しても、効果を発揮するダサチニブ、ニロチニブ、ボスチニブといった第二世代のＡｂｌチロシンキナーゼ阻害剤が開発されている。 However, when treated with imatinib, it has become a serious clinical problem for the patient to acquire resistance to imatinib. For this reason, second generation Abl tyrosine kinase inhibitors such as dasatinib, nilotinib, and bosutinib have been developed that are effective even for patients who have acquired tolerance.
一方、癌治療薬において分子標的薬耐性が生じることは他の疾患でも知られている。例えば、上皮成長因子受容体（ＥＧＦＲ）遺伝子変異を有した肺がんに対するＥＧＦＲチロシンキナーゼ阻害剤（ＥＧＦＲ−ＴＫＩ）やＥＭＬ４−ＡＬＫ融合遺伝子を有する肺がんに対するＡＬＫチロシンキナーゼ阻害剤（ＡＬＫ−ＴＫＩ）は、きわめて高い著効性を示す。しかし、ほとんど例外なく１年から数年で耐性が獲得され再発するとされる（非特許文献１）。 On the other hand, the occurrence of molecular target drug resistance in cancer therapeutics is also known in other diseases. For example, an EGFR tyrosine kinase inhibitor (EGFR-TKI) for lung cancer having an epidermal growth factor receptor (EGFR) gene mutation and an ALK tyrosine kinase inhibitor (ALK-TKI) for lung cancer having an EML4-ALK fusion gene are extremely Highly effective. However, with almost no exception, resistance is acquired and recurs in 1 to several years (Non-Patent Document 1).
非特許文献１では、分子標的薬耐性獲得の原因として、標的自身の二次的変異、側副経路の活性化といった原因が開示されている。特に側副経路として肝細胞増殖因子（ＨＧＦ）がリガンドとなるＭｅｔタンパク質が活性化することで耐性を誘導することが紹介されている。 Non-Patent Document 1 discloses causes such as secondary mutation of the target itself and activation of the collateral pathway as the cause of acquiring molecular target drug resistance. In particular, as a collateral pathway, it has been introduced that resistance is induced by the activation of a Met protein whose ligand is hepatocyte growth factor (HGF).
また、特許文献１では、癌治療のための新規なｃ−Ｍｅｔ阻害剤が開示されている。特許文献１は、このｃ−Ｍｅｔ阻害剤にさらに第２の化学療法剤を組み合わせた医薬組成物が記載されている。その一群の第２の化学療法剤の一つとしてイマチニブが挙げられている。 Patent Document 1 discloses a novel c-Met inhibitor for cancer treatment. Patent Document 1 describes a pharmaceutical composition in which a second chemotherapeutic agent is further combined with this c-Met inhibitor. Imatinib is mentioned as one of the group of second chemotherapeutic agents.
特許文献１は、イマチニブとｃ−Ｍｅｔ阻害剤を組み合わせた医薬組成物が記載されているものの、どのような症例に対して用いることができたのか、またその効果についての記載は何もない。 Although Patent Document 1 describes a pharmaceutical composition in which imatinib and a c-Met inhibitor are combined, there is no description about what kind of case it can be used and its effect.
また、非特許文献１も分子標的薬の耐性獲得について一般的な知見の開示はあるものの、慢性骨髄性白血病に対するイマチニブの耐性獲得について言及されたものではない。つまり、現在なお、慢性骨髄性白血病に対するイマチニブの耐性獲得という問題に対して、第二世代Ａｂｌチロシンキナーゼ阻害剤以外の薬剤は存在していない。 In addition, Non-Patent Document 1 does not mention imatinib resistance acquisition against chronic myelogenous leukemia, although there is disclosure of general knowledge about acquisition of resistance of molecular target drugs. In other words, there are currently no drugs other than second-generation Abl tyrosine kinase inhibitors for the problem of acquiring resistance to imatinib against chronic myelogenous leukemia.
本発明の発明者らは、上記の課題を解決すべく鋭意検討した結果、ＨＧＦ−Ｍｅｔ受容体系阻害物質を有する医薬組成物、またはＭＥＫ／ＥＲＫ経路阻害物を含有する医薬組成物は、分子標的薬に耐性を示す慢性骨髄性白血病の治療薬であるイマチニブに対する感受性を増強させ、耐性を克服することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the inventors of the present invention have found that a pharmaceutical composition having an HGF-Met receptor system inhibitor or a pharmaceutical composition containing a MEK / ERK pathway inhibitor is a molecular target. The inventors have found that the sensitivity to imatinib, a therapeutic agent for chronic myeloid leukemia that is resistant to the drug, is enhanced to overcome the resistance, and the present invention has been completed.
より具体的に本発明に係る慢性骨髄性白血病の治療用組成物は、
Ｂｃｒ−Ａｂｌチロシンキナーゼを阻害する第１医薬組成物と、
Ｍｅｔチロシンキナーゼを阻害する第２医薬組成物で構成されることを特徴とする。
More specifically, the composition for treatment of chronic myeloid leukemia according to the present invention comprises:
A first pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase;
It comprises a second pharmaceutical composition that inhibits Met tyrosine kinase.
本発明によれば、イマチニブやダサチニブ、バフェチニブ、ポナチニブなどの分子標的薬に耐性を示す骨髄性慢性白血病の当該分子標的薬に対する感受性を増強若しくは、耐性の克服が可能な医薬組成物を提供することができる。 According to the present invention, there is provided a pharmaceutical composition capable of enhancing the sensitivity of a myelogenous chronic leukemia showing resistance to molecular target drugs such as imatinib, dasatinib, bafetinib, ponatinib, etc., or overcoming the resistance. Can do.
また、イマチニブやダサチニブ、バフェチニブ、ポナチニブなどの分子標的薬に耐性を示す慢性骨髄性白血病に対して有効な癌治療薬を提供することができる。本発明は、致命的なＴＫＩ耐性の治療方法を提案し、社会的意義は極めて大きなものである。 In addition, an effective cancer therapeutic agent can be provided for chronic myelogenous leukemia that is resistant to molecular target drugs such as imatinib, dasatinib, bafetinib, and ponatinib. The present invention proposes a method for treating fatal TKI resistance, and its social significance is extremely great.
以下に本発明に係る慢性骨髄性白血病の治療薬について説明を行う。なお、以下の説明は本発明の一実施の形態および一実施例についての例示であって、本発明は以下の説明に限定されるものではない。本発明の趣旨を逸脱しない限りにおいて、以下の実施の形態は変更することができる。 The therapeutic agent for chronic myeloid leukemia according to the present invention will be described below. In addition, the following description is the illustration about one embodiment and one Example of this invention, Comprising: This invention is not limited to the following description. The following embodiments can be modified without departing from the spirit of the present invention.
本発明に係る慢性骨髄性白血病の治療薬は、慢性骨髄性白血病の原因と言われるＢｃｒ−Ａｂｌチロシンキナーゼ活性を選択的に阻害する第１医薬組成物と、肝細胞増殖因子（ＨＧＦ）をリガンドとする受容体チロシンキナーゼのＭｅｔを阻害する第２医薬組成物を混合したものである。 The therapeutic agent for chronic myelogenous leukemia according to the present invention comprises a first pharmaceutical composition that selectively inhibits Bcr-Abl tyrosine kinase activity, which is said to be a cause of chronic myelogenous leukemia, and hepatocyte growth factor (HGF) as a ligand. And a second pharmaceutical composition that inhibits Met of the receptor tyrosine kinase.
また、第２医薬組成物としては、Ｍｅｔ／ＭＥＫ／ＥＲＫのシグナル経路を阻害するものでもよい。 Moreover, as a 2nd pharmaceutical composition, you may inhibit the signal pathway of Met / MEK / ERK.
慢性骨髄性白血病の治療薬としては、イマチニブが知られている。ところが、イマチニブを服用していると、やがて薬剤耐性が生じる。これは、イマチニブが結合していたチロシンキナーゼドメインが変位することで、イマチニブが結合しにくくなるためと考えられた。そのため、第２世代の慢性骨髄性白血病の治療薬として開発されたニロチニブは、Ｂｃｒ−Ａｂｌチロシンキナーゼにより強く選択的に結合し、活性を阻害する。 Imatinib is known as a therapeutic agent for chronic myelogenous leukemia. However, taking imatinib eventually leads to drug resistance. This is thought to be because imatinib becomes difficult to bind due to displacement of the tyrosine kinase domain to which imatinib was bound. Therefore, nilotinib developed as a therapeutic agent for second-generation chronic myelogenous leukemia binds strongly and selectively through Bcr-Abl tyrosine kinase and inhibits its activity.
しかし、イマチニブ耐性細胞についてより詳しく調べてみると、イマチニブ耐性を持たない細胞（以下「通常細胞」という）では、活性化していないＭｅｔチロシンキナーゼが活性状態にあることがわかった。つまり、イマチニブ耐性細胞では、Ｂｃｒ−Ａｂｌによるシグナル経路以外に、Ｍｅｔによるシグナル経路によって、細胞が存続している結果を得た。 However, when examining imatinib-resistant cells in more detail, it was found that non-activated Met tyrosine kinase is in an active state in cells not having imatinib resistance (hereinafter referred to as “normal cells”). That is, in imatinib-resistant cells, the result that the cells survived by the signal pathway by Met in addition to the signal pathway by Bcr-Abl was obtained.
そこで、本発明は、慢性骨髄性白血病の治療薬として、Ｂｃｒ−Ａｂｌチロシンキナーゼの阻害剤と、Ｍｅｔ阻害剤を併用することで、Ｂｃｒ−Ａｂｌチロシンキナーゼ阻害剤の耐性を克服しようとするものである。 Therefore, the present invention is intended to overcome the resistance of a Bcr-Abl tyrosine kinase inhibitor by using a Bcr-Abl tyrosine kinase inhibitor in combination with a Met inhibitor as a therapeutic agent for chronic myeloid leukemia. is there.
より具体的に、Ｂｃｒ−Ａｂｌチロシンキナーゼ阻害剤（第１医薬組成物）としては、イマチニブ、ニロチニブ、ダサチニブ、ポナチニブ、バフェチニブ等が好適に利用される。 More specifically, as a Bcr-Abl tyrosine kinase inhibitor (first pharmaceutical composition), imatinib, nilotinib, dasatinib, ponatinib, bafetinib and the like are preferably used.
また、Ｍｅｔチロシンキナーゼ阻害剤（第２医薬組成物）としては、カボザンチニブ（：Ｃａｂｏｚａｎｔｉｎｉｂ）（１）、クリゾチニブ（：Ｃｒｉｚｏｔｉｎｉｂ）（２）、フォアチニブ（：Ｆｏｒｅｔｉｎｉｂ）（３）、ＢＭＳ７７７６０７（４）、ゴルバチニブ（：Ｇｏｌｖａｔｉｎｉｂ）（５）、ＭＫ−２４６１（６）、ＭＧＣＤ−２６５（７）、ＭＫ−８０３３（８）、アムバチニブ（：Ａｍｕｖａｔｉｎｉｂ）（９）、ＴＡＳ−１１５（１０）、Ｓ４９０７６（１１）、ＢＭＳ−７５４８０７（１２）、ＢＭＳ−７９４８３３（１３）、ＬＹ２８０１６５３（１４）、ＣＫＩ２７／ＲＧ７３０４／ＲＯ５１２６７６６／ＣＨ５１２６７６６（１５）等のマルチ・キナーゼ阻害剤が利用できる。以下のそれぞれの分子構造を示す。また、ＢＩ８４７３２５と呼ばれるマルチ・キナーゼ阻害剤も用いることができる。 In addition, as a Met tyrosine kinase inhibitor (second pharmaceutical composition), cabozantinib (1), crizotinib (2), foretinib (3), BMS777607 (4), golbatinib (: Golvatinib) (5), MK-2461 (6), MGCD-265 (7), MK-8033 (8), Ambatinib (9), TAS-115 (10), S49076 (11), A multi-kinase inhibitor such as BMS-754807 (12), BMS-794833 (13), LY2801653 (14), CKI27 / RG7304 / RO5126766 / CH5126766 (15) can be used. Each molecular structure is shown below. A multi-kinase inhibitor called BI847325 can also be used.
また、ＰＨＡ−６６５７５２（１６）、ＥＭＤ１２１４０６３（１７）、ＪＮＪ−３８８７７６０５（１８）、ＰＦ−４２１７９０３（１９）、ＳＧＸ５２３（２０）、ＩＮＣＢ−０２８０６０（２１）、チバンチニブ（：Ｔｉｖａｎｔｉｎｉｂ）（２２）、ＳＡＲ１２５８４４（２３）、ボリチニブ（：Ｖｏｌｉｔｉｎｉｂ）（２４）、ＡＭＧ−４５８（２５）、ＮＶＰ−ＢＶＵ９７２（２６）、ＳＵ１１２７４（２７）、ＡＭＧ２０８（２８）等のＭｅｔ選択阻害剤が利用できる。以下のそれぞれの分子構造を示す。また、ＡＭＧ３３７と呼ばれるＭｅｔ選択阻害剤も用いることができる。 In addition, PHA-666552 (16), EMD1214063 (17), JNJ-38877605 (18), PF-4219793 (19), SGX523 (20), INCB-028060 (21), Tivantinib (22), SAR125844 Met selective inhibitors such as (23), voritinib (24), AMG-458 (25), NVP-BVU972 (26), SU11274 (27), AMG208 (28) can be used. Each molecular structure is shown below. A Met selection inhibitor called AMG337 can also be used.
また、第２医薬組成物は、Ｍｅｔ／ＭＥＫ／ＥＲＫのシグナル経路を阻害するものであればよく、ＭＥＫ阻害剤であってもよい。より具体的には、セルメチニブ（：ｓｅｌｕｍｅｔｉｎｉｂ）（２９）、レファメチニブ（：ｒｅｆａｍｅｔｉｎｉｂ）（３０）、ピマセルチブ（：ｐｉｍａｓｅｒｔｉｂ）（３１）、ＭＥＫ１６２／ＡＲＲＹ−１６２（３２）、ＡＺＤ８３３０／ＡＲＲＹ−４２４７０４（３３）、コビメチニブ（：ｃｏｂｉｍｅｔｉｎｉｂ）（３４）、ＧＤＣ−０６２３／ＲＧ７４２１／ＸＬ５１８（３５）、ＣＩＦ／ＲＧ７１６７／ＲＯ４９８７６５５（３６）、Ｅ６２０１（３７）、ＴＡＫ−７３３（３８）、ＰＤ−０３２５９０１（３９）、ＣＩ−１０４０／ＰＤ１８４３５２（４０）、ＡＳ７０３０２６（４１）、ＭＥＫ ｉｎｈｉｂｉｔｏｒ（４２）、ＰＤ３１８０８８（４３）、ＰＤ９８０５９（４４）、ＳＬ３２７（４５）、トラメチニブ（：Ｔｒａｍｅｔｉｎｉｂ）（４６）、Ｕ０１２６（４７）等のＭＥＫ選択阻害剤を用いることができる。以下それぞれの分子構造を示す。また、ＡＳ７０３９８８／ＭＳＣ２０１５１０３Ｂ、ＷＸ−５５４、ＲＲＹ−３００と呼ばれるＭＥＫ選択阻害剤を用いることもできる。 Moreover, the 2nd pharmaceutical composition should just inhibit the signal pathway of Met / MEK / ERK, and may be a MEK inhibitor. More specifically, selumetinib (: selumetinib) (29), refametinib (: refametinib) (30), pimasertiv (31), MEK162 / ARRY-162 (32), AZD8330 / ARRY-424704 (33) , Cobimetinib (34), GDC-0623 / RG7421 / XL518 (35), CIF / RG7167 / RO497655 (36), E6201 (37), TAK-733 (38), PD-0325901 (39), CI -1040 / PD184352 (40), AS703026 (41), MEK inhibitor (42), PD318088 (43), PD98059 (44), SL327 (45), trametinib : Trametinib) (46), can be used MEK selection inhibitors such as U0126 (47). Each molecular structure is shown below. In addition, a MEK selective inhibitor called AS703888 / MSC2015103B, WX-554, RRY-300 may be used.
また、より下流シグナル経路を阻害するものであってもよい。具体的には、ＢＶＤ−５２３（４８）、ＳＣＨ７７２９８４（４９）、ＶＴＸ１１ｅ（５０）、ＡＥＺＳ−１３１／ＡＥＺＳ−１３４（５１）、ＦＲ１８０２０４（５２）といったＥＲＫ１／２選択阻害剤を用いることができる。以下それぞれの分子構造を示す。また、ＭＫ−８３５３／ＳＣＨ９０００３５３と呼ばれるＥＲＫ１／２選択阻害剤を用いることもできる。 Moreover, you may inhibit a more downstream signal pathway. Specifically, ERK1 / 2 selective inhibitors such as BVD-523 (48), SCH772984 (49), VTX11e (50), AEZS-131 / AEZS-134 (51), and FR180204 (52) can be used. Each molecular structure is shown below. An ERK1 / 2 selective inhibitor called MK-8353 / SCH9000353 can also be used.
これらの医薬組成物（第１医薬組成物および第２医薬組成物）は、いずれも、薬学的に許容される塩を含んでもよい。より具体的には、塩酸塩、臭化水素酸塩、リン酸、硫酸塩、硫酸水素塩、アルキルスルホン酸塩、アリールスルホン酸塩、酢酸塩、安息香酸塩、クエン酸塩、マレイン酸塩、フマル酸塩、コハク酸塩、乳酸塩および酒石酸塩を含む酸付加塩；Ｎａ＋、Ｋ＋、Ｌｉ＋などのアルカリ金属カチオン、ＭｇもしくはＣａなどのアルカリ土類金属塩または有機アミン塩を含むことができる。 Any of these pharmaceutical compositions (first pharmaceutical composition and second pharmaceutical composition) may contain a pharmaceutically acceptable salt. More specifically, hydrochloride, hydrobromide, phosphoric acid, sulfate, hydrogen sulfate, alkyl sulfonate, aryl sulfonate, acetate, benzoate, citrate, maleate, Acid addition salts including fumarate, succinate, lactate and tartrate; alkali metal cations such as Na + , K + , Li + , alkaline earth metal salts such as Mg or Ca, or organic amine salts Can do.
以下に実施例を示しながら本発明に係る慢性骨髄性白血病用の治療用組成物について説明する。 The therapeutic composition for chronic myeloid leukemia according to the present invention will be described below with reference to examples.
（実施例１）＜イマチニブ耐性獲得因子の検討＞
Ｋ５６２細胞（白血病細胞株、ＪＣＲＢ細胞バンクでの細胞番号：ＪＣＲＢ００１９）とイマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、イマチニブを最終濃度が０、０．０５、０．１、０．５、１、５、１０、２０μＭになるように添加した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌ（０μＭ イマチニブ）に対する細胞生存率として評価した。
(Example 1) <Examination of imatinib resistance acquisition factor>
K562 cells (leukemia cell line, cell number in JCRB cell bank: JCRB0019) and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 0, 0.05, 0.1, 0.5, 1, 5, 10, 20 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control (0 μM imatinib).
イマチニブ添加による細胞生存率の結果を図１に示す。横軸はイマチニブ（Ｉｍａｔｉｎｉｂ）の添加量（μＭ）を示し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はＫ５６２細胞を表し、黒棒はイマチニブ耐性細胞を表す。 The results of cell viability with imatinib addition are shown in FIG. The horizontal axis indicates the amount of imatinib added (μM), and the vertical axis indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.
イマチニブの添加量が多くなるに従い、Ｋ５６２細胞の生存率は低下した。また、イマチニブが１０μＭ以上添加された場合は、Ｋ５６２細胞の細胞生存率はゼロであった。しかし、イマチニブ耐性細胞では、イマチニブの添加量が増加しても、細胞生存率はほぼ１００％であった。なお、以後のグラフを含め、グラフ中「Ｐ」は有意確率を示す。 As the amount of imatinib added increased, the survival rate of K562 cells decreased. When imatinib was added at 10 μM or more, the cell survival rate of K562 cells was zero. However, in imatinib resistant cells, the cell viability was almost 100% even when the amount of imatinib added increased. In addition, including the subsequent graphs, “P” in the graph indicates a significance probability.
以上のことから、Ｋ５６２細胞においてイマチニブによって細胞死を誘導する濃度において、イマチニブ耐性細胞に細胞死が誘導されないことを認めた。この結果から、作製したイマチニブ耐性細胞はイマチニブ耐性を示すことが明らかとなった。 From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by imatinib in K562 cells. From this result, it was revealed that the prepared imatinib resistant cells showed imatinib resistance.
（実施例２）＜イマチニブ耐性細胞におけるニロチニブ耐性の確認＞
Ｋ５６２細胞とイマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、ニロチニブを最終濃度が０、０．０１、０．０５、０．１、０．２、０．５、１、２ μＭになるように添加した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌ（０μＭ ニロチニブ）に対する細胞生存率として評価した。
(Example 2) <Confirmation of nilotinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Nilotinib was then added to a final concentration of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control (0 μM nilotinib).
ニロチニブ添加による細胞生存率の結果を図２に示す。横軸はニロチニブ（Ｎｉｌｏｔｉｎｉｂ）の添加量（μＭ）を示し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はＫ５６２細胞を表し、黒棒はイマチニブ耐性細胞を表す。 The results of cell viability by adding nilotinib are shown in FIG. The abscissa indicates the amount of nilotinib added (μM), and the ordinate indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.
ニロチニブの添加量が多くなるに従い、Ｋ５６２細胞の生存率は低下した。また、ニロチニブが０．２μＭ以上添加された場合は、Ｋ５６２細胞の細胞生存率はゼロであった。しかし、イマチニブ耐性細胞では、ニロチニブの添加量が増加しても、細胞生存率はほぼ１００％であった。なお、イマチニブ耐性細胞は、ニロチニブの添加量が２μＭまで増加した時に細胞生存率が約６０％まで低下した。 As the amount of nilotinib added increased, the survival rate of K562 cells decreased. When nilotinib was added in an amount of 0.2 μM or more, the cell survival rate of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of nilotinib added increased. The cell viability of imatinib resistant cells decreased to about 60% when the amount of nilotinib added was increased to 2 μM.
以上のことから、Ｋ５６２細胞においてニロチニブによって細胞死を誘導する濃度においても、イマチニブ耐性細胞に細胞死が誘導されないことを認めた。この結果から、作製したイマチニブ耐性細胞はニロチニブ耐性をも示すことが明らかとなった。 From the above, it was confirmed that cell death was not induced in imatinib-resistant cells even at a concentration at which cell death was induced by nilotinib in K562 cells. From this result, it was clarified that the prepared imatinib resistant cells also showed nilotinib resistance.
（実施例３）＜イマチニブ耐性細胞におけるダサチニブ耐性の確認＞
Ｋ５６２細胞とイマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、ダサチニブを最終濃度が０、０．０１、０．０５、０．１、０．２、０．５、１、２μＭになるように添加した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌ（０μＭ ダサチニブ）に対する細胞生存率として評価した。
(Example 3) <Confirmation of dasatinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, dasatinib was added to a final concentration of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control (0 μM dasatinib).
ダサチニブ添加による細胞生存率の結果を図３に示す。横軸はダサチニブ（Ｄａｓａｔｉｎｉｂ）の添加量（μＭ）を示し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はＫ５６２細胞を表し、黒棒はイマチニブ耐性細胞を表す。 The result of cell viability by adding dasatinib is shown in FIG. The horizontal axis represents the amount of dasatinib (Dasatinib) added (μM), and the vertical axis represents the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.
ダサチニブの添加量が多くなるに従い、Ｋ５６２細胞の生存率は低下した。また、ダサチニブが０．２μＭ以上添加された場合は、Ｋ５６２細胞の細胞生存率はゼロであった。しかし、イマチニブ耐性細胞では、ダサチニブの添加量が増加しても、細胞生存率はほぼ１００％であった。なお、イマチニブ耐性細胞は、ダサチニブの添加量が２μＭまで増加した時に細胞生存率が約８０％まで低下した。 As the amount of dasatinib added increased, the survival rate of K562 cells decreased. When dasatinib was added in an amount of 0.2 μM or more, the cell viability of K562 cells was zero. However, in imatinib resistant cells, the cell viability was almost 100% even when the amount of dasatinib added increased. The cell viability of imatinib resistant cells decreased to about 80% when the amount of dasatinib added was increased to 2 μM.
以上のことから、Ｋ５６２細胞においてダサチニブによって細胞死を誘導する濃度において、イマチニブ耐性細胞に細胞死が誘導されないことを認めた。この結果から、作製したイマチニブ耐性細胞はダサチニブ耐性をも示すことが明らかとなった。 From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by dasatinib in K562 cells. From this result, it was revealed that the prepared imatinib resistant cells also show dasatinib resistance.
（実施例４）＜イマチニブ耐性細胞におけるポナチニブ耐性の確認＞
Ｋ５６２細胞とイマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、ポナチニブを最終濃度が０、５、１０、２０、１００、２５０、５００、１０００ｎＭになるように添加した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌ（０μＭ ポナチニブ）に対する細胞生存率として評価した。
(Example 4) <Confirmation of ponatinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, ponatinib was added to a final concentration of 0, 5, 10, 20, 100, 250, 500, 1000 nM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control (0 μM ponatinib).
ポナチニブ添加による細胞生存率の結果を図４に示す。横軸はポナチニブ（Ｐｏｎａｔｉｎｉｂ）の添加量（μＭ）を示し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はＫ５６２細胞を表し、黒棒はイマチニブ耐性細胞を表す。 The results of cell viability by adding ponatinib are shown in FIG. The horizontal axis represents the amount of ponatinib (Ponatinib) added (μM), and the vertical axis represents the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.
ポナチニブの添加量が多くなるに従い、Ｋ５６２細胞の生存率は低下した。また、ポナチニブが５００ｎＭ以上添加された場合は、Ｋ５６２細胞の細胞生存率はゼロであった。しかし、イマチニブ耐性細胞では、ポナチニブの添加量が増加しても、細胞生存率はほぼ１００％であった。 As the amount of ponatinib added increased, the survival rate of K562 cells decreased. When ponatinib was added at 500 nM or more, the cell viability of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of ponatinib added was increased.
以上のことから、Ｋ５６２細胞においてポナチニブによって細胞死を誘導する濃度において、イマチニブ耐性細胞に細胞死が誘導されないことを認めた。この結果から、作製したイマチニブ耐性細胞はポナチニブ耐性をも示すことが明らかとなった。 From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by ponatinib in K562 cells. From this result, it was clarified that the prepared imatinib resistant cells also exhibited ponatinib resistance.
（実施例５）＜イマチニブ耐性細胞におけるバフェチニブ耐性の確認＞
Ｋ５６２細胞とイマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、バフェチニブを最終濃度が０、５、１０、５０、１００、５００、１０００、２５００ｎＭになるように添加した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌ（０μＭ バフェチニブ）に対する細胞生存率として評価した。
(Example 5) <Confirmation of resistance to buffetinib in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, buffetinib was added to a final concentration of 0, 5, 10, 50, 100, 500, 1000, 2500 nM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and the measured value was evaluated as the cell viability against control (0 μM buffetinib).
バフェチニブ添加による細胞生存率の結果を図５に示す。横軸はバフェチニブ（Ｂａｆｅｔｉｎｉｂ）の添加量（μＭ）を示し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はＫ５６２細胞を表し、黒棒はイマチニブ耐性細胞を表す。 FIG. 5 shows the results of cell viability by adding bafetinib. The horizontal axis indicates the amount of addition of bafetinib (Bafetinib) (μM), and the vertical axis indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.
バフェチニブの添加量が多くなるに従い、Ｋ５６２細胞の生存率は低下した。また、バフェチニブが２５００ｎＭ以上添加された場合は、Ｋ５６２細胞の細胞生存率はゼロであった。しかし、イマチニブ耐性細胞では、バフェチニブの添加量が増加しても、細胞生存率はほぼ１００％であった。なお、バフェチニブの添加量が２５００ｎＭになると、イマチニブ耐性細胞の細胞生存率は、約８０％まで低下した。 As the amount of bafetinib added increased, the survival rate of K562 cells decreased. Further, when vifetinib was added to 2500 nM or more, the cell viability of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of buffetinib added increased. In addition, when the addition amount of bafetinib reached 2500 nM, the cell viability of imatinib resistant cells decreased to about 80%.
以上のことから、Ｋ５６２細胞においてバフェチニブによって細胞死を誘導する濃度において、イマチニブ耐性細胞に細胞死が誘導されないことを認めた。この結果から、作製したイマチニブ耐性細胞はバフェチニブ耐性をも示すことが明らかとなった。 From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by buffetinib in K562 cells. From this result, it was clarified that the prepared imatinib-resistant cells also showed resistance to buffetinib.
（実施例６）＜イマチニブ耐性獲得因子の検討＞
作製したイマチニブ耐性細胞を用いて、耐性に関与する因子をイムノブロッティング解析にて検討した。
(Example 6) <Examination of imatinib resistance acquisition factor>
Using the prepared imatinib-resistant cells, factors involved in resistance were examined by immunoblotting analysis.
Ｋ５６２細胞、イマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔＫ５６２）を１５０ｃｍ２フラスコに播種した後、７２時間培養したものから細胞溶解液にてタンパク質を抽出し、サンプルとした。また、タンパク定量はＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ （Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ； Ｗａｌｔｈａｍ， ＭＡ， ＵＳＡ）を用いて行った。 After seeding K562 cells and imatinib-resistant cells (Imatinib-resistant K562) in a 150 cm 2 flask, protein was extracted from the cells cultured for 72 hours with a cell lysate. Protein quantification was performed using BCA Protein Assay (Thermo Fisher Scientific; Waltham, MA, USA).
各サンプルをＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜に転写し、抗ｐｈｏｓｐｈｏ−Ｍｅｔ （Ｔｙｒ１２３４／１２３５）抗体、抗ｐｈｏｓｐｈｏ−Ｍｅｔ （Ｔｙｒ１３４９）抗体、抗Ｍｅｔ抗体 （Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ； Ｂｅｖｅｒｌｙ， ＭＡ， ＵＳＡ）、及び抗β−ａｃｔｉｎ抗体 （Ｓｉｇｍａ）を用いてＭｅｔのリン酸化を検討した。 Each sample was transferred to PVDF membrane after SDS-PAGE, anti-phospho-Met (Tyr1234 / 1235) antibody, anti-phospho-Met (Tyr1349) antibody, anti-Met antibody (Cell Signaling Technology; Beverly, MA, USA), and Phosphorylation of Met was examined using an anti-β-actin antibody (Sigma).
イムノブロッティングの結果を図６に示す。図６では、各抗体毎に、Ｋ５６２細胞とイマチニブ耐性細胞のイムノブロッティング結果の写真を示す。発光は写真上黒い影として映っている。抗ｐｈｏｓｐｈｏ−Ｍｅｔ（Ｔｙｒ１２３４／１２３５）抗体において、Ｋ５６２細胞の影よりも、イマチニブ耐性細胞の影の方が濃い影であった。 The result of immunoblotting is shown in FIG. FIG. 6 shows photographs of immunoblotting results of K562 cells and imatinib resistant cells for each antibody. Luminescence appears as a black shadow on the photo. In the anti-phospho-Met (Tyr1234 / 1235) antibody, the shadow of imatinib resistant cells was darker than the shadow of K562 cells.
また抗ｐｈｏｓｐｈｏ−Ｍｅｔ（Ｔｙｒ１３４９）抗体においては、Ｋ５６２細胞は、薄く確認できる程度であったが、イマチニブ耐性細胞は、明らかにＫ５６２細胞の場合より濃い影であった。つまり、チロシンキナーゼドメイン内にあるＴｙｒ１２３４／１２３５もＣ末端にあるＴｙｒ１３４９でもイマチニブ耐性細胞のイムノブロッティングの結果はＫ５６２細胞の場合より、濃い影であった。 In the anti-phospho-Met (Tyr1349) antibody, K562 cells were thin enough to be confirmed, but imatinib resistant cells were clearly darker than the K562 cells. In other words, the results of immunoblotting of imatinib-resistant cells were darker than those of K562 cells in both Tyr1234 / 1235 in the tyrosine kinase domain and Tyr1349 in the C-terminal.
抗ｍｅｔ抗体では、Ｋ５６２細胞、イマチニブ耐性細胞共に明確に黒い影であった。ただし、イマチニブ耐性細胞の方が黒い影が小さかった。抗β−ａｃｔｉｎ抗体は、Ｋ５６２細胞およびイマチニブ耐性細胞共に明確な黒い影が観察された。 With anti-met antibody, both K562 cells and imatinib resistant cells were clearly black shadows. However, imatinib-resistant cells had a smaller black shadow. With the anti-β-actin antibody, a clear black shadow was observed in both K562 cells and imatinib resistant cells.
以上のことより、Ｋ５６２細胞におけるリン酸化Ｍｅｔの発現と比較し、イマチニブ耐性細胞ではリン酸化Ｍｅｔが著しく増加していることが明らかになった。これは、イマチニブ耐性細胞では、ｍｅｔによるシグナル伝達が活性化していることを示唆するものである。なお、β−ａｃｔｉｎはＫ５６２細胞もイマチニブ耐性細胞も同程度有していると観察されたので、このイムノブロッティングの結果は正しいと判断できる。 From the above, it was revealed that phosphorylated Met was significantly increased in imatinib-resistant cells compared to the expression of phosphorylated Met in K562 cells. This suggests that signal transduction by met is activated in imatinib resistant cells. Since β-actin was observed to have the same degree in both K562 cells and imatinib resistant cells, it can be judged that the result of this immunoblotting is correct.
（実施例７）＜Ｍｅｔ阻害剤によるイマチニブ耐性克服効果の検討＞
イマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、イマチニブを最終濃度が５μＭになるように添加した。また、それとは別にイマチニブと併用してＰＨＡ−６６５７５２（Ｍｅｔ阻害剤）を最終濃度が１、２、２．５、３μＭになるように添加した。
Example 7 <Examination of Imatinib Resistance Overcoming Effect by Met Inhibitor>
Imatinib-resistant cells (Imatinib-resistant K562) were seeded in 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 5 μM. Separately, PHA-6655752 (Met inhibitor) was added in combination with imatinib so that the final concentration was 1, 2, 2.5, or 3 μM.
Ｃｏｎｔｒｏｌとして薬剤を溶解した溶媒を添加したものを同様に培養した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌに対する細胞生存率として評価した。 What added the solvent which melt | dissolved the chemical | medical agent as Control was cultured similarly. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control.
Ｍｅｔ阻害剤併用による結果を図７に示す。横軸はイマチニブおよびＰＨＡ−６６５７５２の量の違いを表し、縦軸は細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はｃｏｎｔｒｏｌ（Ｋ５６２細胞）を表し、黒棒はイマチニブ耐性細胞を表す。イマチニブ、ＰＨＡ−６６５７５２が単独で添加された場合は、ＰＨＡ−６６５７５２を３μＭ添加したときも、細胞生存率は約７０％あった。 The results of the Met inhibitor combination are shown in FIG. The horizontal axis represents the difference in the amount of imatinib and PHA-666552, and the vertical axis represents the cell viability (Cell viability:%). The white bar represents control (K562 cells), and the black bar represents imatinib resistant cells. When imatinib and PHA-666552 were added alone, the cell viability was about 70% even when 3 μM PHA-665752 was added.
一方、イマチニブ５μＭとＰＨＡ−６６５７５２を組み合わせて使用すると、ＰＨＡ−６６５７５２の添加量にしたがって、細胞生存率は減少し、ＰＨＡ−６６５７５２ ３μＭの添加で細胞生存率は０％になった。 On the other hand, when imatinib 5 μM and PHA-665752 were used in combination, the cell viability decreased according to the amount of PHA-665752 added, and the cell viability became 0% with the addition of PHA-665752 3 μM.
このことより、イマチニブ耐性細胞においてＭｅｔ阻害剤の濃度依存的にイマチニブ耐性が克服されることが認められた。これらの結果から、Ｍｅｔがイマチニブ耐性に関与することが明らかとなった。 From this, it was recognized that imatinib resistance was overcome in imatinib-resistant cells depending on the concentration of the Met inhibitor. From these results, it was revealed that Met is involved in imatinib resistance.
（実施例８）＜Ｍｅｔ下流シグナル伝達因子におけるイマチニブ耐性因子の検討＞
イマチニブ耐性株において活性化が確認されたＭｅｔの下流シグナルのＥＲＫ１／２、Ａｋｔ、ＳＴＡＴ３の活性動態について、イムノブロッティングで検討を行った。
(Example 8) <Examination of imatinib resistance factor in Met downstream signaling factor>
The activity kinetics of ERK1 / 2, Akt, and STAT3 of downstream signals of Met whose activation was confirmed in imatinib resistant strains were examined by immunoblotting.
Ｋ５６２細胞、イマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を１５０ｃｍ２フラスコに播種した後、７２時間培養したものから細胞溶解液にてをタンパク質を抽出し、サンプルとした。また、タンパク定量はＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ （Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ； Ｗａｌｔｈａｍ， ＭＡ， ＵＳＡ）を用いて行った。 After seeding K562 cells and imatinib-resistant cells (Imatinib-resistant K562) in a 150 cm 2 flask, the protein was extracted from the cells cultured for 72 hours, and used as a sample. Protein quantification was performed using BCA Protein Assay (Thermo Fisher Scientific; Waltham, MA, USA).
各サンプルをＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜に転写し、抗ｐｈｏｓｐｈｏ−ＥＲＫ１／２抗体、抗ＥＲＫ１／２抗体、抗ｐｈｏｓｐｈｏ−Ａｋｔ抗体、抗Ａｋｔ抗体、抗ｐｈｏｓｐｈｏ− ＳＴＡＴ３抗体、及び抗ＳＴＡＴ３抗体（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ； Ｂｅｖｅｒｌｙ， ＭＡ， ＵＳＡ）を用いてＥＲＫ１／２、Ａｋｔ、ＳＴＡＴ３のリン酸化を検討した。 Each sample was transferred to a PVDF membrane after SDS-PAGE, and anti-phospho-ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-STAT3 antibody, and anti-STAT3 antibody (Cell The phosphorylation of ERK1 / 2, Akt, and STAT3 was examined using Signaling Technology (Beverly, MA, USA).
イムノブロッティングの結果を図８に示す。図８では、Ｋ５６２細胞とイマチニブ耐性細胞のイムノブロッティングの結果をそれぞれの抗体毎に並べて示した。図８によれば、抗ＥＲＫ１／２抗体、抗Ａｋｔ抗体、および抗ＳＴＡＴ３抗体では、Ｋ５６２細胞とイマチニブ耐性細胞は、同程度の黒い影が映っている。一方、抗リン酸化ＥＲＫ１／２抗体、抗リン酸化Ａｋｔ抗体、および抗リン酸化ＳＴＡＴ３抗体ではＫ５６２細胞よりもイマチニブ耐性細胞の黒い影の方が明らかに濃い。 The result of immunoblotting is shown in FIG. In FIG. 8, the results of immunoblotting of K562 cells and imatinib resistant cells are shown side by side for each antibody. According to FIG. 8, with the anti-ERK1 / 2 antibody, the anti-Akt antibody, and the anti-STAT3 antibody, K562 cells and imatinib resistant cells have the same level of black shadow. On the other hand, in the anti-phosphorylated ERK1 / 2 antibody, the anti-phosphorylated Akt antibody, and the anti-phosphorylated STAT3 antibody, the dark shadow of imatinib-resistant cells is clearly darker than K562 cells.
さらに詳説すると、抗ＥＲＫ１／２抗体においては、上下に２つの影があるのが確認できる。上側の影がＥＲＫ１で、下側の影はＥＲＫ２に相当する。Ｋ５６２細胞もイマチニブ耐性細胞もＥＲＫ１／２の両方を有していることを示している。 More specifically, it can be confirmed that the anti-ERK1 / 2 antibody has two shadows at the top and bottom. The upper shadow corresponds to ERK1, and the lower shadow corresponds to ERK2. It shows that both K562 and imatinib resistant cells have both ERK1 / 2.
一方、抗リン酸化ＥＲＫ１／２抗体の場合は、Ｋ５６２細胞では、リン酸化ＥＲＫ２の影は薄く、リン酸化ＥＲＫ１は殆ど影がない。これに比較し、イマチニブ耐性細胞ではリン酸化ＥＲＫ１は薄いながら影を確認でき、リン酸化ＥＲＫ２は明らかに濃い影がある。 On the other hand, in the case of the anti-phosphorylated ERK1 / 2 antibody, phosphorylated ERK2 has a light shadow and phosphorylated ERK1 has almost no shadow in K562 cells. Compared to this, in imatinib-resistant cells, the phosphorylated ERK1 has a thin shadow, but phosphorylated ERK2 has a clearly dark shadow.
以上のことから、Ｋ５６２細胞におけるリン酸化ＥＲＫ１／２、リン酸化Ａｋｔ、リン酸化ＳＴＡＴ３の発現と比較し、イマチニブ耐性細胞ではリン酸化ＥＲＫ１／２、リン酸化Ａｋｔ、リン酸化ＳＴＡＴ３が著しく増加していることが明らかになった。 Based on the above, phosphorylated ERK1 / 2, phosphorylated Akt, and phosphorylated STAT3 are significantly increased in imatinib-resistant cells compared to the expression of phosphorylated ERK1 / 2, phosphorylated Akt, and phosphorylated STAT3 in K562 cells. It became clear.
このことは、イマチニブ耐性細胞では、イマチニブが主として阻害するＢｃｒ−Ａｂｌ活性によるシグナル経路だけでなく、ＥＲＫ−ＭＡＲＫ経路、ＰＩ３Ｋ−Ａｋｔ経路といったシグナル経路も活性化していることを示唆する。 This suggests that imatinib-resistant cells activate not only signal pathways due to Bcr-Abl activity that is mainly inhibited by imatinib but also signal pathways such as ERK-MARK pathway and PI3K-Akt pathway.
（実施例９）＜ＭＥＫ阻害剤、ＰＩ３Ｋ阻害剤、ＪＡＫ２阻害剤によるイマチニブ耐性克服効果の検討＞
イマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を９６ｗｅｌｌプレートに播種し、２４時間前培養した。その後、イマチニブを最終濃度が５μＭになるように添加した。また、それとは別にイマチニブと併用してＵ０１２６（ＭＥＫ阻害剤）を最終濃度が０．１、１、１０μＭ、ＬＹ２９４００２（ＰＩ３Ｋ阻害剤）を最終濃度が０．１、１、１０μＭ、ＡＧ４９０（ＪＡＫ２阻害剤）を最終濃度が１、５、１０μＭになるように添加した。
(Example 9) <Examination of effect of overcoming imatinib resistance by MEK inhibitor, PI3K inhibitor, and JAK2 inhibitor>
Imatinib-resistant cells (Imatinib-resistant K562) were seeded in 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 5 μM. Separately, in combination with imatinib, U0126 (MEK inhibitor) has a final concentration of 0.1, 1, 10 μM, and LY294002 (PI3K inhibitor) has a final concentration of 0.1, 1, 10 μM, AG490 (JAK2 inhibition). Agent) was added to a final concentration of 1, 5, 10 μM.
Ｃｏｎｔｒｏｌとして薬剤を溶解した溶媒を添加したものを同様に培養した。７２時間培養した後の細胞数の変化をトリパンブルー色素排除染色試験法により算定し、この測定値をｃｏｎｔｒｏｌに対する細胞生存率として評価した。 What added the solvent which melt | dissolved the chemical | medical agent as Control was cultured similarly. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control.
各種阻害剤併用による結果を図９〜図１１に示す。図９〜図１１のグラフにおいて、横軸はイマチニブおよびＵ０１２６（図９）、イマチニブとＬＹ２９４００２（図１０）、イマチニブとＡＧ４９０（図１１）の量の違いを表し、縦軸はいずれのグラフにおいても細胞生存率（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ：％）を示す。また白棒はｃｏｎｔｒｏｌ（Ｋ５６２細胞）を表し、黒棒はイマチニブ耐性細胞を表す。 The results of the combined use of various inhibitors are shown in FIGS. 9 to 11, the horizontal axis represents imatinib and U0126 (FIG. 9), imatinib and LY294002 (FIG. 10), imatinib and AG490 (FIG. 11), and the vertical axis represents any graph. The cell viability (Cell viability:%) is shown. The white bar represents control (K562 cells), and the black bar represents imatinib resistant cells.
図９を参照して、イマチニブとＵ０１２６（ＭＥＫ阻害剤）が単独で添加された場合は、Ｕ０１２６を１０μＭ添加したときも、細胞生存率は約７０％あった。一方、イマチニブ５μＭとＵ０１２６を組み合わせて使用すると、Ｕ０１２６の添加量にしたがって、細胞生存率は明らかに減少した。なお、イマチニブ５μＭとＵ０１２６を１０μＭ併用したときは、細胞生存率は約３５％まで低下した。 Referring to FIG. 9, when imatinib and U0126 (MEK inhibitor) were added alone, the cell viability was about 70% even when 10 μM of U0126 was added. On the other hand, when imatinib 5 μM and U0126 were used in combination, the cell viability was clearly reduced according to the amount of U0126 added. When imatinib 5 μM and U0126 were combined 10 μM, the cell viability decreased to about 35%.
図１０を参照して、イマチニブとＬＹ２９４００２（ＰＩ３Ｋ阻害剤）の組み合わせの場合は、それぞれを単独で用いた時も、これらを併用したときも、細胞生存率に大きな変化はなかった。なお、イマチニブ５μＭとＬＹ２９４００２を１０μＭ併用したときも細胞生存率は約７５％程度あった。 Referring to FIG. 10, in the case of a combination of imatinib and LY294002 (PI3K inhibitor), there was no significant change in cell viability when used alone or in combination. The cell viability was about 75% when imatinib 5 μM and LY294002 were combined 10 μM.
図１１を参照して、イマチニブとＡＧ４９０（ＪＡＫ２阻害剤）の組み合わせの場合は、それぞれを単独で用いた時も、これらを併用したときも、細胞生存率に大きな変化はなかった。なお、イマチニブ５μＭとＬＹ２９４００２を１０μＭ併用したときも細胞生存率は約９０％程度あった。 Referring to FIG. 11, in the case of a combination of imatinib and AG490 (JAK2 inhibitor), there was no significant change in cell viability when used alone or in combination. The cell viability was about 90% when imatinib 5 μM and LY294002 were combined 10 μM.
以上のことから、イマチニブ耐性細胞において５μＭのイマチニブと、１０μＭのＵ０１２６（ＭＥＫ阻害剤）併用時にイマチニブ耐性が克服されることが認められた。これらの結果から、ＭＥＫ／ＥＲＫ経路がイマチニブ耐性に関与することが明らかとなった。 From the above, it was recognized that imatinib resistance was overcome when imatinib-resistant cells were combined with 5 μM imatinib and 10 μM U0126 (MEK inhibitor). From these results, it became clear that the MEK / ERK pathway is involved in imatinib resistance.
（実施例１０）＜Ｍｅｔ阻害剤によるＥＲＫ抑制効果の検討＞
イマチニブ耐性細胞においてリン酸化Ｍｅｔの増加が耐性獲得に関与していることが示唆された（実施例６、図６参照）ため、Ｍｅｔ阻害剤添加時におけるＭｅｔおよび下流シグナルの発現変化についてイムノブロッティングにて検討した。
(Example 10) <Examination of ERK inhibitory effect by Met inhibitor>
It was suggested that the increase in phosphorylated Met in imatinib-resistant cells is involved in the acquisition of resistance (see Example 6 and FIG. 6). Therefore, the expression changes of Met and downstream signals when Met inhibitor was added were immunoblotted. And examined.
Ｋ５６２細胞、イマチニブ耐性細胞（Ｉｍａｔｉｎｉｂ−ｒｅｓｉｓｔａｎｔ Ｋ５６２）を１５０ｃｍ２フラスコに播種し、３７℃、５％ ＣＯ２の条件下で４８時間培養したものおよびイマチニブ耐性細胞を１５０ｃｍ２フラスコに播種し、２４時間前培養後、イマチニブ耐性細胞にＰＨＡ−６６５７５２を最終濃度が１、２、２．５μＭになるように添加し３７℃、５％ ＣＯ２の条件下で２４時間培養した。この培養液から細胞溶解液にてタンパク質を抽出し、サンプルとした。 K562 cells, imatinib-resistant cells (Imatinib-resistant K562) were seeded in 150 cm 2 flasks, cultured at 37 ° C. under 5% CO 2 for 48 hours, and imatinib resistant cells were seeded in 150 cm 2 flasks for 24 hours. After pre-culture, PHA-665752 was added to imatinib-resistant cells to a final concentration of 1, 2 , and 2.5 μM, and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2 . Protein was extracted from this culture solution with a cell lysate to prepare a sample.
また、タンパク定量はＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙを用いて行った。各サンプルをＳＤＳ−ＰＡＧＥ後、ＰＶＤＦ膜に転写し、抗ｐｈｏｓｐｈｏ−Ｍｅｔ （Ｔｙｒ１２３４／１２３５）抗体、抗ｐｈｏｓｐｈｏ−Ｍｅｔ （Ｔｙｒ１３４９）抗体、抗Ｍｅｔ抗体、抗ｐｈｏｓｐｈｏ−ＥＲＫ１／２抗体、及び抗ＥＲＫ１／２抗体を用いてＭｅｔ、ＥＲＫ１／２のリン酸化を検討した。なお、各細胞サンプルに対して抗β−ａｃｔｉｎ抗体を作用させ、イムノブロッティングの妥当性を確認した。 Protein quantification was performed using BCA Protein Assay. Each sample was transferred to a PVDF membrane after SDS-PAGE, anti-phospho-Met (Tyr1234 / 1235) antibody, anti-phospho-Met (Tyr1349) antibody, anti-Met antibody, anti-phospho-ERK1 / 2 antibody, and anti-ERK1 / Two antibodies were used to examine phosphorylation of Met and ERK1 / 2. In addition, anti-β-actin antibody was allowed to act on each cell sample, and the validity of immunoblotting was confirmed.
イムノブロッティングの結果を図１０に示す。写真横方向には、Ｋ５６２細胞の場合と、イマチニブ耐性細胞の場合を並べて示し、イマチニブ耐性細胞では、ＰＨＡ−６６５７５２の添加量（μＭ）を数字で示した。写真縦方向には、各抗体毎の結果（写真）を示した。 The result of immunoblotting is shown in FIG. In the horizontal direction of the photograph, the case of K562 cells and the case of imatinib resistant cells are shown side by side. In the case of imatinib resistant cells, the addition amount (μM) of PHA-665752 is indicated by a number. In the photo vertical direction, the results (photos) for each antibody are shown.
抗リン酸化Ｍｅｔ（Ｔｙｒ１２３４／１２３５）抗体の写真では、イマチニブ耐性細胞におけるＰＨＡ−６６５７５２が０μＭの時に最も黒い影が濃かった。イマチニブ耐性細胞では、ＰＨＡ−６６５７５２の増加にしたがって、黒い影は薄くなった。Ｋ５６２細胞の黒い影は、イマチニブ耐性細胞にＰＨＡ−６６５７５２を２μＭ添加した場合と同じ程度の濃さであった。 In the photo of anti-phosphorylated Met (Tyr1234 / 1235) antibody, the darkest shadow was darkest when PHA-665752 in imatinib resistant cells was 0 μM. In imatinib resistant cells, the black shadow faded with increasing PHA-666552. The black shadow of K562 cells was as thick as when 2 μM of PHA-666552 was added to imatinib resistant cells.
抗リン酸化Ｍｅｔ（Ｔｙｒ１３４９）抗体の写真でもほぼ同様の結果であった。ただし、抗リン酸化Ｍｅｔ（Ｔｙｒ１３４９）抗体は、抗リン酸化Ｍｅｔ（Ｔｙｒ１２３４／１２３５）抗体と比べて全体的に影の濃さは薄かった。 The result of the anti-phosphorylated Met (Tyr1349) antibody was almost the same. However, the anti-phosphorylated Met (Tyr1349) antibody was generally less shaded than the anti-phosphorylated Met (Tyr1234 / 1235) antibody.
抗Ｍｅｔ抗体では、イマチニブ耐性細胞はＰＨＡ−６６５７５２の添加量に係らず、ほぼ同じ濃さの影であった。Ｋ５６２細胞の写真は、イマチニブ耐性細胞の影より一回り大きな影であった。 With anti-Met antibody, imatinib resistant cells were shaded with almost the same density regardless of the amount of PHA-665752 added. The photo of K562 cells was a shadow that was slightly larger than the shadow of imatinib resistant cells.
抗リン酸化ＥＲＫ１／２抗体では、Ｋ５６２細胞の写真では、ＥＲＫ２にわずかな影が映っただけであった。濃さも薄かった。イマチニブ耐性細胞では、Ｋ５６２よりも明確に黒い大きな影がＥＲＫ１、ＥＲＫ２のいずれにもあった。また、ＰＨＡ−６６５７５２の添加にしたがい、黒い影は薄くなった。 With anti-phosphorylated ERK1 / 2 antibody, only a slight shadow was seen in ERK2 in the photo of K562 cells. The thickness was also thin. In imatinib resistant cells, both ERK1 and ERK2 had a clearly darker shadow than K562. In addition, the black shadow became lighter with the addition of PHA-665752.
抗ＥＲＫ１／２抗体では、Ｋ５６２細胞の影がイマチニブ耐性細胞の影より一回り小さな影であったが、濃さは充分黒かった。イマチニブ耐性細胞では、ＰＨＡ−６６５７５２の添加量に係らず、大きく濃い陰影が観察された。 With the anti-ERK1 / 2 antibody, the shadow of the K562 cell was slightly smaller than the shadow of imatinib resistant cells, but the darkness was sufficiently black. In imatinib resistant cells, a large and dark shadow was observed regardless of the amount of PHA-665752 added.
β−ａｃｔｉｎでは、Ｋ５６２細胞もイマチニブ耐性細胞もほぼ変化がなく、いずれも黒い大きな影が観察された。 In β-actin, both K562 cells and imatinib resistant cells were almost unchanged, and a large black shadow was observed in both.
Ｋ５６２細胞ではリン酸化ＥＲＫ１のバンドが観察されなかった。一方、イマニチブ耐性細胞ではリン酸化ＥＲＫ１のバンドが確認され、リン酸化ＥＲＫ２のバンドもＫ５６２細胞と比較し強くなっていた。つまり、Ｋ５６２細胞では、ＭＥＫ／ＥＲＫ経路は活性ではないのに対し、イマチニブ耐性細胞は、ＭＥＫ／ＥＲＫ経路が活性になっていたことを示した。 No phosphorylated ERK1 band was observed in K562 cells. On the other hand, the phosphorylated ERK1 band was confirmed in imatinib resistant cells, and the phosphorylated ERK2 band was also stronger compared to K562 cells. That is, the MEK / ERK pathway was not active in K562 cells, whereas the imatinib resistant cells showed that the MEK / ERK pathway was active.
このイマチニブ耐性細胞に、Ｍｅｔ阻害剤（ＰＨＡ−６６５７５２）を添加していくと、リン酸化ＥＲＫ１のバンドが無くなり、リン酸化ＥＲＫ２のバンドも薄くなり、Ｋ５６２細胞の状態に近づいていた。 When a Met inhibitor (PHA-665752) was added to this imatinib-resistant cell, the phosphorylated ERK1 band disappeared and the phosphorylated ERK2 band became thinner, approaching the state of K562 cells.
以上のことから、Ｍｅｔ阻害剤ＰＨＡ−６６５７５２により、リン酸化Ｍｅｔが阻害されていることを確認した。実施例９で、ＭＥＫ／ＥＲＫ経路がイマチニブ耐性に関与することが明らかであった。したがって、ＭＥＫ／ＥＲＫ経路の阻害よりイマチニブ耐性が克服されることが明らかとなった。 From the above, it was confirmed that phosphorylated Met was inhibited by the Met inhibitor PHA-665752. In Example 9, it was clear that the MEK / ERK pathway is involved in imatinib resistance. Thus, it was revealed that imatinib resistance was overcome by inhibition of the MEK / ERK pathway.
本発明に係る慢性骨髄性白血病の治療用組成物は、慢性骨髄性白血病に劇的に効果があるとされたＢｃｒ−Ａｂｌチロシンキナーゼ阻害剤に対して、耐性を有する原因はＭｅｔ／ＭＥＫ／ＥＲＫ経路が原因であることを明らかにし、この経路の阻害剤とＢｃｒ−Ａｂｌチロシンキナーゼ阻害剤と併用することで、耐性を克服するものである。したがって、イマチニブよりよりＢｃｒ−Ａｂｌとの結合性が高いとされるダサチニブやニロチニブに対が耐性を獲得した患者に対しても、耐性克服することができる。 The composition for the treatment of chronic myelogenous leukemia according to the present invention is resistant to Bcr-Abl tyrosine kinase inhibitor, which is considered to be dramatically effective for chronic myelogenous leukemia. It is clarified that the pathway is the cause, and the resistance is overcome by using an inhibitor of this pathway in combination with a Bcr-Abl tyrosine kinase inhibitor. Therefore, resistance can be overcome even for patients who have acquired resistance to dasatinib or nilotinib, which has higher binding to Bcr-Abl than imatinib.
Claims (4)
Ｍｅｔチロシンキナーゼを阻害する第２医薬組成物で構成されることを特徴とする慢性骨髄性白血病の治療用組成物。 A first pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase;
A composition for treating chronic myeloid leukemia, comprising a second pharmaceutical composition that inhibits Met tyrosine kinase.
前記第２医薬組成物が、カボザンチニブ、クリゾチニブ、フォアチニブ、ＢＭＳ７７７６０７、ゴルバチニブ、ＭＫ−２４６１、ＭＧＣＤ−２６５、ＭＫ−８０３３、アムバチニブ、ＴＡＳ−１１５、Ｓ４９０７６、ＢＭＳ−７５４８０７、ＢＭＳ−７９４８３３、ＬＹ２８０１６５３、ＣＫＩ２７／ＲＧ７３０４／ＲＯ５１２６７６６／ＣＨ５１２６７６６、ＢＩ８４７３２５、ＰＨＡ−６６５７５２、ＥＭＤ１２１４０６３、ＪＮＪ−３８８７７６０５、ＰＦ−４２１７９０３、ＳＧＸ５２３、ＩＮＣＢ−０２８０６０、チバンチニブ、ＳＡＲ１２５８４４、ボリチニブ、ＡＭＧ−４５８、ＮＶＰ−ＢＶＵ９７２、ＳＵ１１２７４、ＡＭＧ２０８、ＡＭＧ３３７から選ばれる少なくとも１つの医薬組成物であることを特徴とする請求項１に記載された慢性骨髄性白血病の治療用組成物。 The first pharmaceutical composition is at least one pharmaceutical composition selected from imatinib, nilotinib, dasatinib, ponatinib, bafetinib;
Said second pharmaceutical composition is cabozantinib, crizotinib, foatinib, BMS777607, golvatinib, MK-2461, MGCD-265, MK-8033, ambatinib, TAS-115, S49076, BMS-7504807, BMS-779453, LY2801653, LY2801653 RG7304 / RO5126766 / CH5126766, BI847325, PHA-666552, EMD1214063, JNJ-38877605, PF-4217903, SGX523, INCB-028060, Tivantinib, SAR125844, Boritinib, AMG-458A, NVP-BVU972G, S 2. At least one pharmaceutical composition The therapeutic composition of the listed chronic myelogenous leukemia.
Ｍｅｔ／ＭＥＫ／ＥＲＫのシグナル経路のいずれかのキナーゼを阻害する第３医薬組成物で構成されることを特徴とする慢性骨髄性白血病の治療用組成物。 A first pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase;
A composition for treating chronic myelogenous leukemia, characterized by comprising a third pharmaceutical composition that inhibits any kinase in the signal pathway of Met / MEK / ERK.
前記第３医薬組成物は、セルメチニブ、レファメチニブ、ピマセルチブ、ＭＥＫ１６２／ＡＲＲＹ−１６２、ＡＺＤ８３３０／ＡＲＲＹ−４２４７０４、コビメチニブ、ＧＤＣ−０６２３／ＲＧ７４２１／ＸＬ５１８、ＣＩＦ／ＲＧ７１６７／ＲＯ４９８７６５５、Ｅ６２０１、ＴＡＫ−７３３、ＰＤ−０３２５９０１、ＣＩ−１０４０／ＰＤ１８４３５２、ＡＳ７０３０２６、ＭＥＫ ｉｎｈｉｂｉｔｏｒ、ＰＤ３１８０８８、ＰＤ９８０５９、ＳＬ３２７、トラメチニブ、Ｕ０１２６、ＡＳ７０３９８８／ＭＳＣ２０１５１０３Ｂ、ＷＸ−５５４、ＲＲＹ−３００、ＢＶＤ−５２３、ＳＣＨ７７２９８４、ＶＴＸ１１ｅ、ＡＥＺＳ−１３１／ＡＥＺＳ−１３４、ＦＲ１８０２０４、ＭＫ−８３５３／ＳＣＨ９０００３５３から選ばれる少なくとも１つの医薬組成物であることを特徴とする請求項３に記載された慢性骨髄性白血病の治療用組成物。 The first pharmaceutical composition is at least one pharmaceutical composition selected from imatinib, nilotinib, dasatinib, ponatinib, bafetinib;
The third pharmaceutical composition is selmetinib, refametinib, pimaceltib, MEK162 / ARRY-162, AZD8330 / ARRY-424704, kobimetinib, GDC-0623 / RG7421, CIF / RG7167 / RO497655, E6201, TAK-7. 0325901, CI-1040 / PD184352, AS703026, MEK inhibitor, PD318088, PD98059, SL327, Trametinib, U0126, AS70388 / MSC2015103B, WX-554, RRY-300, BVD-523, SCH772984, SCH772984, SCH772944 , FR180204, MK-8353 / SCH9000353 The composition for treating chronic myeloid leukemia according to claim 3, wherein the composition is at least one pharmaceutical composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014202529A JP6267619B2 (en) | 2014-09-30 | 2014-09-30 | Composition for the treatment of chronic myeloid leukemia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014202529A JP6267619B2 (en) | 2014-09-30 | 2014-09-30 | Composition for the treatment of chronic myeloid leukemia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2016069348A true JP2016069348A (en) | 2016-05-09 |
| JP6267619B2 JP6267619B2 (en) | 2018-01-24 |
Family
ID=55866043
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014202529A Active JP6267619B2 (en) | 2014-09-30 | 2014-09-30 | Composition for the treatment of chronic myeloid leukemia |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6267619B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017199678A1 (en) * | 2016-05-18 | 2017-11-23 | 富士フイルム株式会社 | Imaging device and method |
| EP3808350A4 (en) * | 2018-06-18 | 2022-03-23 | Industry-Academic Cooperation Foundation, Yonsei University | Composition comprising flt3 inhibitor as effective ingredient for inhibiting drug resistance in chronic myelogenous leukemia |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007513967A (en) * | 2003-12-11 | 2007-05-31 | セラヴァンス， インコーポレーテッド | Compositions for use in the treatment of cell proliferative diseases driven by mutant receptor tyrosine kinases |
| JP2011520908A (en) * | 2008-05-14 | 2011-07-21 | アムジエン・インコーポレーテツド | Combinations of VEGF (R) inhibitors and hepatocyte growth factor (C-MET) inhibitors for the treatment of cancer |
-
2014
- 2014-09-30 JP JP2014202529A patent/JP6267619B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007513967A (en) * | 2003-12-11 | 2007-05-31 | セラヴァンス， インコーポレーテッド | Compositions for use in the treatment of cell proliferative diseases driven by mutant receptor tyrosine kinases |
| JP2011520908A (en) * | 2008-05-14 | 2011-07-21 | アムジエン・インコーポレーテツド | Combinations of VEGF (R) inhibitors and hepatocyte growth factor (C-MET) inhibitors for the treatment of cancer |
Non-Patent Citations (6)
| Title |
|---|
| "PS-4-29 R115777/Imatinib併用によるImatinib耐性細胞増殖抑制効果", 臨床血液, vol. 44, no. 8, JPN6016032951, 2003, pages p.869(369) * |
| BIOTHERAPY, vol. 25, no. 1, JPN6016032955, 2011, pages 589 - 596 * |
| CANCER CELL, vol. Vol.20, JPN6016032958, 2011, pages 715 - 727 * |
| NEOPLASIA, vol. 14, no. 7, JPN6016048745, 2012, pages 572 - 584 * |
| PHARMA MEDICA, vol. 31, no. 11, JPN6016032954, 2013, pages 43 - 48 * |
| 日本臨床, vol. 29, no. 11, JPN6017026103, 1971, pages p.304(2686)-309(2691) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017199678A1 (en) * | 2016-05-18 | 2017-11-23 | 富士フイルム株式会社 | Imaging device and method |
| JP2017205053A (en) * | 2016-05-18 | 2017-11-24 | 富士フイルム株式会社 | Imaging apparatus and method |
| US10878571B2 (en) | 2016-05-18 | 2020-12-29 | Fujifilm Corporation | Imaging apparatus and imaging method for imaging biological samples in a time-series order |
| EP3808350A4 (en) * | 2018-06-18 | 2022-03-23 | Industry-Academic Cooperation Foundation, Yonsei University | Composition comprising flt3 inhibitor as effective ingredient for inhibiting drug resistance in chronic myelogenous leukemia |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6267619B2 (en) | 2018-01-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | The clinical development of MEK inhibitors | |
| RU2704811C2 (en) | Treating cancer with combination of radiation therapy, cerium oxide nanoparticles and chemotherapeutic agent | |
| JP2022520079A (en) | Pharmaceutical combination containing TNO155 and KRASG12C inhibitor | |
| CN105392499B (en) | The combination treatment for including TOR kinase inhibitors and cytidine analog for treating cancer | |
| JP2018528206A (en) | MDM2 inhibitors and combinations thereof | |
| CN104703623A (en) | Compounds and methods for kinase modulation, and indications therefor | |
| CN110072528B (en) | Pharmaceutical composition for treating tumor | |
| JPWO2017200016A1 (en) | Lung cancer therapeutic agent that has acquired EGFR-TKI resistance | |
| CN104519885A (en) | Treatment of cancer with TOR kinase inhibitors | |
| US20190134034A1 (en) | Method of Treating Liver Cancer | |
| TW201526894A (en) | Treatment of cancer with dihydropyrazino-pyrazines | |
| US20140155372A1 (en) | Combinations of akt and mek inhibitor compounds, and methods of use | |
| WO2022111714A1 (en) | Combination therapy for treating pik3ca-mutated cancer | |
| SG188207A1 (en) | Combination anti - cancer therapy | |
| CN109152758A (en) | The composition and method of the cancer including glioma are treated using Eflornithine and the like and derivative | |
| CN107206090A (en) | Ah 's not purposes of the moral in colorectal cancer | |
| CN105611928A (en) | Pim kinase inhibitor combinations | |
| CN109069511A (en) | BRAF inhibitor is used to treat the purposes of dermoreaction | |
| TW201244716A (en) | Combination of a phosphatidylinositol-3-kinase (PI3K) inhibitor and a mTOR inhibitor | |
| WO2022271964A1 (en) | Erk1/2 and shp2 inhibitors combination therapy | |
| JP6267619B2 (en) | Composition for the treatment of chronic myeloid leukemia | |
| Gainor et al. | A phase II study of the multikinase inhibitor ponatinib in patients with advanced, RET-rearranged NSCLC | |
| TW201711679A (en) | Use of ureidomustine in cancer treatment | |
| US20180015075A1 (en) | Methods and compositions for treatment of venous malformation | |
| JP2022501390A (en) | Compounds and their uses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20160830 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20161028 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20161220 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20170718 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20171018 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20171025 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20171212 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6267619 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |