WO1995016032A1 - ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE - Google Patents
ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE Download PDFInfo
- Publication number
- WO1995016032A1 WO1995016032A1 PCT/EP1993/003461 EP9303461W WO9516032A1 WO 1995016032 A1 WO1995016032 A1 WO 1995016032A1 EP 9303461 W EP9303461 W EP 9303461W WO 9516032 A1 WO9516032 A1 WO 9516032A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pdgf
- bfgf
- nucleic acid
- seq
- dna
- Prior art date
Links
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 title claims abstract description 24
- 108010017843 platelet-derived growth factor A Proteins 0.000 title claims abstract description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 11
- 201000010099 disease Diseases 0.000 title claims abstract description 10
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 6
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 title claims abstract 8
- 102100037596 Platelet-derived growth factor subunit A Human genes 0.000 title claims abstract 8
- 230000000692 anti-sense effect Effects 0.000 title claims description 44
- 108020004707 nucleic acids Proteins 0.000 title claims description 43
- 102000039446 nucleic acids Human genes 0.000 title claims description 43
- 150000007523 nucleic acids Chemical class 0.000 title claims description 43
- 150000001875 compounds Chemical class 0.000 claims abstract description 16
- 208000023275 Autoimmune disease Diseases 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims description 23
- 125000003729 nucleotide group Chemical group 0.000 claims description 22
- 108091034117 Oligonucleotide Proteins 0.000 claims description 17
- 230000015572 biosynthetic process Effects 0.000 claims description 13
- 238000003786 synthesis reaction Methods 0.000 claims description 12
- 108020004999 messenger RNA Proteins 0.000 claims description 9
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 8
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 8
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 8
- 206010028980 Neoplasm Diseases 0.000 claims description 7
- -1 phosphite triester Chemical class 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 108020000948 Antisense Oligonucleotides Proteins 0.000 claims description 4
- 208000034038 Pathologic Neovascularization Diseases 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 claims description 3
- 230000033115 angiogenesis Effects 0.000 claims description 3
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical group OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 claims description 3
- 239000007790 solid phase Substances 0.000 claims description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 2
- 208000035475 disorder Diseases 0.000 claims 1
- 238000000034 method Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 230000001613 neoplastic effect Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 31
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 108010019674 Proto-Oncogene Proteins c-sis Proteins 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 229910019142 PO4 Inorganic materials 0.000 description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- 235000021317 phosphate Nutrition 0.000 description 8
- 239000003102 growth factor Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000011534 incubation Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 5
- 239000002336 ribonucleotide Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 102000004338 Transferrin Human genes 0.000 description 4
- 108090000901 Transferrin Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000004700 cellular uptake Effects 0.000 description 4
- 235000012000 cholesterol Nutrition 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 4
- 239000012581 transferrin Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000009826 neoplastic cell growth Effects 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- BCOSEZGCLGPUSL-UHFFFAOYSA-N 2,3,3-trichloroprop-2-enoyl chloride Chemical compound ClC(Cl)=C(Cl)C(Cl)=O BCOSEZGCLGPUSL-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Natural products CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- YRIZYWQGELRKNT-UHFFFAOYSA-N 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione Chemical compound ClN1C(=O)N(Cl)C(=O)N(Cl)C1=O YRIZYWQGELRKNT-UHFFFAOYSA-N 0.000 description 1
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 1
- JEPVUMTVFPQKQE-AAKCMJRZSA-N 2-[(1s,2s,3r,4s)-1,2,3,4,5-pentahydroxypentyl]-1,3-thiazolidine-4-carboxylic acid Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C1NC(C(O)=O)CS1 JEPVUMTVFPQKQE-AAKCMJRZSA-N 0.000 description 1
- 108091027075 5S-rRNA precursor Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108020004519 Antisense Oligoribonucleotides Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 101000957437 Homo sapiens Mitochondrial carnitine/acylcarnitine carrier protein Proteins 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- FSNCEEGOMTYXKY-JTQLQIEISA-N Lycoperodine 1 Natural products N1C2=CC=CC=C2C2=C1CN[C@H](C(=O)O)C2 FSNCEEGOMTYXKY-JTQLQIEISA-N 0.000 description 1
- 102100038738 Mitochondrial carnitine/acylcarnitine carrier protein Human genes 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000002825 antisense oligoribonucleotide Substances 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 229940106681 chloroacetic acid Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 125000000309 desoxyribosyl group Chemical class C1(C[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229960004132 diethyl ether Drugs 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 102000027411 intracellular receptors Human genes 0.000 description 1
- 108091008582 intracellular receptors Proteins 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005497 microtitration Methods 0.000 description 1
- 230000009707 neogenesis Effects 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Definitions
- the present invention is related to an antisense-nucleic acid or effective derivatives thereof hybridizing with an area of the messenger RNA (mRNA) or the DNA, encoding platelet derived growth factor-A (PDGF-A) or platelet derived growth factor-B (PDGF-B) or basic fibroblast growth factor (bFGF) , a pharmaceutical composition, comprising an antisense nucleic acid or effective derivatives thereof hybridizing with an area of the messenger RNA (mRNA) or the DNA, encoding PDGF-A or PDGF-B or bFGF as well as the use of said antisense nucleic acids and derivatives thereof for the manufacturing of a pharmacautieal composition for the treatment of neo ⁇ plasms, for the inhibtion of pathological angiogenesis or the treatment of rheumatoid arthritis and other autoimmune diseases .
- mRNA messenger RNA
- PDGF-A platelet derived growth factor-A
- PDGF-B platelet
- PDGF-A and PDGF-B as well as bFGF are growth factors, secreted by a variety of neoplastic cells including glioma cells, pancreas carcinoma cells, osteosarcoma cells, AIDS- related Kaposi's sarcoma cells, gastric carcinoma cells, lung carcinoma cells and melanoma cells and stimulate neoplastic cell growth in an autocrine manner. All three growth factors also stimulates neoangiogenesis in solid tumors. Furthermore, these factors may promote angiogenesis and pathological formation of tissue deposits in autoimmune diseases.
- the antisense nucleic acids described below could strongly inhibit tumor cell growth, capillary endo- thelial cell growth and fibroblast cell growth even when the purified growth factors were added to the culture. This suggests that these growth factors exert growth regulatory effects by mechanisms independent of exogenous binding of these factors to their known receptors on the cell surface.
- the intracellularly produced polypetides or fragments thereof may bind regulatory sites on the cellular DNA or to protein or to intra cellular receptor sites.
- the oligonucleotides may act as aptamers.
- antisense nucleic acids or effective derivatives thereof which hybridize with an area of the mRNA or DNA coding for PDGF-A or PDGF-B or bFGF can effectively treat the diseases addressed above.
- the antisense nucleic acid is able to hybridize with regions of the PDGF-A or PDGF-B or bFGF mRNAs . It is understood by the skilled person that fragments of the antisense nucleic acids and antisense nucleic acids containing these sequences work according to the invention so long as production of the PDGF-A or PDGF-B or bFGF polypeptides is reduced or in ⁇ hibited.
- the antisense-oligonucleotides are obtainable by solid phase synthesis using phosphite triester chemistry by growing the nucleotide chain in 3 '-5' direction in that the respective nucleotide is coupled to the first nucleotide which is covalently attached to the solid phase comprising the steps of cleaving 5'DMT protecting group of the previous nucleotide,
- oligodeoxy-ribonucleotides are given in figure 1 as well as the respective structures of antisense oligo-ribonucleotides are given in figure 2.
- the oligonucleotide chain is to be understood as a detail out of a longer nucleotide chain.
- B means an organic base such as adenine (A) , guanine (G) , cytosine (C) and thymine (T) which are coupled via N9(A,G) or N1(D,T) to the desoxyribose.
- A adenine
- G guanine
- C cytosine
- T thymine
- the sequence of the bases is the reverse complement of the genetic target sequence (mRNA-sequence) .
- the modifications used are
- B deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence
- p internucleotide phosphate
- n an oligodeoxy-ribonucleotide stretch of length 6 - 20 bases
- B deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence
- p internucleotide phosphate
- n an oligodeoxy-ribodinucleotide stretch o. length 4 - 12 dinucleotides
- RNA-oligonucleotides are the basis (adenine (A) , guanine (G) , cytosine (C) , uracil (U) ) coupled via N9 (A,G) or Nl (C,U) to the ribose.
- the sequence of the basis is the reverse complement of the genetic target sequence (mRNA-sequence) .
- the modifications in the oligo ⁇ nucleotide sequence used are as follows
- B ribonucleotide A, C, G or T depending on gene sequence
- p internucleotide phosphate
- n an oligo-ribonucleotide stretch of length 4 20 bases
- B ribonucleotide A, C, G or T depending on gene sequence
- p internucleotide phosphate
- n an oligo-ribodinucleotide stretch of length 4 - 12 dinucleotides
- the PDGF-A antisense-oligo- nucleotide has the sequence as disclosed in the sequence listing under Seq. ID No. 1 - 6, having a DNA- or RNA-type structure.
- the PDGF-B antisense-oligonuclotide has the sequence as disclosed in the sequence listing under Seq. ID No. 7 - 12, having a DNA- or RNA-type structure.
- the bFGF'antisense-oligonucleotide has the sequence as disclosed in the sequence listing under Seq. ID No. 13 - 27, having a DNA- or RNA-type structure.
- these oligonucleotides are phosphorothioate derivatives, having a DNA- or RNA-type structure.
- one single individual sequence as mentioned above works as an antisense nucleic acid or oligo ⁇ nucleotide structure according to the invention.
- one strand of nucleotides comprises more than one of the sequences as mentioned above directly covalently linked or with other nucleotides covalently linked in between.
- individual oligonucleotides are addressed .
- the randomized control sequence is disclosed in the sequence listing under Seq. ID No. 28.
- these oligo-nucleotides are phosphorothioate derivatives.
- the antisense-oligonucleotides are ad ⁇ vantageous since they are not as fast destroyed by endo- geneous factors when applied as this is valid for naturally occuring nucleotide sequences.
- the modification is a phosphorothioate modification.
- Oligodeoxy-nucleotides were synthesized by stepwise 5'- addition of protected nucleosides using phosphite triester chemistry.
- the nucleotide A was introduced as 5'dimethoxy- trityl-deoxyadenosine (N-benzoyl) -N,N' -diisopropyl-2-cyano- ethyl phosphoramidite (0.1 M) ;
- C was introduced by a 5'-
- Synthesis was performed on controlled pore glass particles of approximately 150 ⁇ m diameter (pore diameter 500 A) to which the most 3' nucleoside is covalently attached via a long-chain alkylamin linker (average loading 30 ⁇ mol/g solid support ) .
- the solid support was loaded into a cylindrical synthesis column, capped on both ends with filters which permit ade ⁇ quate flow of reagents but hold back the solid synthesis support. Reagents were delivered and withdrawn from the synthesis column using positive pressure of inert gas. The nucleotides were added to the growing oligonucleotide chain in 3'-> 5' direction. Each nucleotide was coupled using one round of the following synthesis cycle:
- the anisense-nucleic acid of the invention can be used as pharmaceutical composition or medicament.
- This medicament can be used for treating neoplasms in which the expression of platelet derived growth factor or basic fibroblast growth factor is of relevance for the pathogenicity. It can be used to reduce neoplastic cell growth in cells expressing these growth factors, and to inhibit pathological angiogenesis . Furthermore, it can be used to treat rheumatoid arthritis and other autoimmune diseases in which the production of PDGF-A, PDGF-B or bFGF is of pathogenetic relevance.
- PDGF-A and PDGF-B and bFGF specific antisense oligonucleotides were investigated. It was demonstrated that antisense oligodeoxynucleotides as well as phosphorothioate modified nucleic acids, comple ⁇ mentary to PDGF and bFGF mRNAs could specifically inhibit PDGF protein expression and bFGF protein expression re ⁇ spectively. Furthermore, they reduced cell proliferation in mammary carcinoma, glioma, pancreas carcinoma and melanoma cells with surprising efficiency.
- Cell lysates were diluted in 50 mM carbonate buffer at pH 9.0 and immobilized on immunon II plates (Dynatech Laborator ⁇ ies, Inc.) overnight. Antigen solution was removed and 200 ⁇ l/well phosphate buffered saline (PBS)/ l%/BSA/0.02% azide were added to block non-specific protein binding. Following incubation at room temperature for 2 h solution was removed. After washing with PBS plates were air dried for 3 h. Specific antibodies for PDGF-A, PDGF-B or bFGF (Oncogene or Santa Cruz Biotechnology Inc.) were added at 50 ⁇ l/well, diluted in blocking buffer.
- PBS phosphate buffered saline
- bFGF Oncogene or Santa Cruz Biotechnology Inc.
- TITLE OF INVENTION Antisense nucleic acid for the treatment of neoplasms and auto-immune diseases and diseases involving pathological angioneogenesis, in which expression of the growth factors bFGF, PDGF-A or PDGF-B..
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- ANTI-SENSE YES
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A compound which is capable of preventing and treating neoplastic diseases and/or autoimmune diseases in which expression of PDGF-A, PDGF-B and/or bFGF plays a pathogenic role.
Description
ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE.
The present invention is related to an antisense-nucleic acid or effective derivatives thereof hybridizing with an area of the messenger RNA (mRNA) or the DNA, encoding platelet derived growth factor-A (PDGF-A) or platelet derived growth factor-B (PDGF-B) or basic fibroblast growth factor (bFGF) , a pharmaceutical composition, comprising an antisense nucleic acid or effective derivatives thereof hybridizing with an area of the messenger RNA (mRNA) or the DNA, encoding PDGF-A or PDGF-B or bFGF as well as the use of said antisense nucleic acids and derivatives thereof for the manufacturing of a pharmacautieal composition for the treatment of neo¬ plasms, for the inhibtion of pathological angiogenesis or the treatment of rheumatoid arthritis and other autoimmune diseases .
PDGF-A and PDGF-B as well as bFGF are growth factors, secreted by a variety of neoplastic cells including glioma cells, pancreas carcinoma cells, osteosarcoma cells, AIDS- related Kaposi's sarcoma cells, gastric carcinoma cells, lung carcinoma cells and melanoma cells and stimulate neoplastic cell growth in an autocrine manner. All three growth factors also stimulates neoangiogenesis in solid tumors. Furthermore,
these factors may promote angiogenesis and pathological formation of tissue deposits in autoimmune diseases.
It is an object of the present invention to provide a com¬ pound for the treatment of neoplasms, autoimmune diseases and diseases involving pathological angioneogenesis . Surprisingly the antisense nucleic acids described below, could strongly inhibit tumor cell growth, capillary endo- thelial cell growth and fibroblast cell growth even when the purified growth factors were added to the culture. This suggests that these growth factors exert growth regulatory effects by mechanisms independent of exogenous binding of these factors to their known receptors on the cell surface. Thus, the intracellularly produced polypetides or fragments thereof may bind regulatory sites on the cellular DNA or to protein or to intra cellular receptor sites. Alternatively, the oligonucleotides may act as aptamers.
According to the invention antisense nucleic acids or effective derivatives thereof which hybridize with an area of the mRNA or DNA coding for PDGF-A or PDGF-B or bFGF can effectively treat the diseases addressed above. The antisense nucleic acid is able to hybridize with regions of the PDGF-A or PDGF-B or bFGF mRNAs . It is understood by the skilled person that fragments of the antisense nucleic acids and antisense nucleic acids containing these sequences work according to the invention so long as production of the PDGF-A or PDGF-B or bFGF polypeptides is reduced or in¬ hibited.
According to the invention the antisense-oligonucleotides are obtainable by solid phase synthesis using phosphite triester chemistry by growing the nucleotide chain in 3 '-5' direction in that the respective nucleotide is coupled to the first nucleotide which is covalently attached to the solid phase comprising the steps of
cleaving 5'DMT protecting group of the previous nucleotide,
adding the respective nucleotide for chain propagation,
modifying the phosphite group subsequently cap un- reacted 5'-hydroxyl groups and
cleaving the oligonucleotide from the solid support,
followed by working up the synthesis product.
The chemical structures of oligodeoxy-ribonucleotides are given in figure 1 as well as the respective structures of antisense oligo-ribonucleotides are given in figure 2. The oligonucleotide chain is to be understood as a detail out of a longer nucleotide chain.
In figure 1 lit. B means an organic base such as adenine (A) , guanine (G) , cytosine (C) and thymine (T) which are coupled via N9(A,G) or N1(D,T) to the desoxyribose. The sequence of the bases is the reverse complement of the genetic target sequence (mRNA-sequence) . The modifications used are
1. Oligodeoxy-ribonucleotides where all R are substituted by
1 . 1 R1 = 0
1 . 2 R1 = s
1 . 3 R1 = F
1 . 4 R1 = CH3 1 . 5 R1 = OEt
2. Oligodeoxy-ribonucleotides where R1 is varied at the internucleotide phosphates within one oligonucleotide
5' B-p-B-p-B-p- (B-p-) B-p-B-p-B-p-B 3
Rla Rla Rla Rlb Rla Rla Rla
where B = deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence p = internucleotide phosphate n = an oligodeoxy-ribonucleotide stretch of length 6 - 20 bases
2.1 Rla - S; Rlb = 0
2.2 Rla = CH ; Rlb = 0
2.3 Rla - S; Rlb = CH.
2.4 Rla = CH3; Rlb - S
3. Oligodeoxy-ribonucleotides where R is alternated at the internucleotide phosphates within one oligo¬ nucleotide
5' B-p-(B-p-B-p)n-B-p-B
Rla Rlb Rla Rlb
where B = deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence p = internucleotide phosphate n = an oligodeoxy-ribodinucleotide stretch o. length 4 - 12 dinucleotides
3.1 Rla = S; Rlb - 0
3.2 Rla = CH ; Rlb - 0
3.3 Rla - S; Rlb = CH3
4. Any of the compounds 1.1 - 1.5; 2.1 - 2.4; 3.1 - 3.3
2 coupled at R with the following compounds which are covalently coupled to increased cellular uptake
4.1 cholesterol
4.2 poly(L) lysine
4.3 transferrin
5. Any of the compounds 1.1 - 1.5; 2.1 - 2.4; 3.1 - 3.3 coupled at R with the following compounds which are covalently coupled to increase cellular uptake
5.1 cholesterol
5.2 poly(L) lysine
5.3 transferrin
In the case of the RNA-oligonucleotides (figure 2) are the basis (adenine (A) , guanine (G) , cytosine (C) , uracil (U) ) coupled via N9 (A,G) or Nl (C,U) to the ribose. The sequence of the basis is the reverse complement of the genetic target sequence (mRNA-sequence) . The modifications in the oligo¬ nucleotide sequence used are as follows
6. Oligo-ribonucleotides where all R are substituted by
6.1 R1 = 0
6.2 R1 = S
6.3 R1 = F
6.4 R1 = CH3 6.5 R1 = OEt
7. Oligo-ribonucleotides where R is varied at the inter¬ nucleotide phosphates within one oligonucleotide
B-p-B-p-B-p- (B-p-) B-p-B-p-B-p-B 3
Rla Rla Rla Rlb Rla Rla Rla
where B = ribonucleotide A, C, G or T depending
on gene sequence p = internucleotide phosphate n = an oligo-ribonucleotide stretch of length 4 20 bases
7.1 Rla = S; Rlb - 0
7.2 Rla = CH3; Rlb = 0
7.3 Rla = S; Rlb = CH
7.4 Rla = CH3; Rlb - S
Oligo-ribonucleotides where R is alternated at the internucleotide phosphates within one oligonucleotide
5' B-p- (B-p-B-p) -B-p-B 3'
Rla Rlb Rla Rlb
where B = ribonucleotide A, C, G or T depending on gene sequence p = internucleotide phosphate n = an oligo-ribodinucleotide stretch of length 4 - 12 dinucleotides
8.2 Rla = S; Rlb = 0
8.2 R1S = CH3; Rlb - 0
8.3 Rla = S; Rlb - CH3
9. Any of the compounds 6.1 - 6.5; 7.1 - 7.4; 8.1 - 8.3
2 coupled at R with the following compounds which are covalently coupled to increase cellular uptake
9.1 cholesterol
9.2 poly(L) lysine
9.3 transferrin
10. Any of the compounds 6.1 - 6.5; 7.1 - 7.4; 8.1 - 8.3 coupled at R the following compounds are covalently
coupled to increased cellular uptake
10.1 cholesterol
10.2 poly(L) lysine
10.3 transferrin
11. Any of the compounds 6.1 - 6.5; 7.1 - 7.4; 8.1 - 8.3; 9.1 - 9.3; 10.1 - 10.3 where all R are substituted by
4
11 . 1 R = 0
11 . 2 R4 = F
11 . 3 R4 = CH3
In a preferred embodiment the PDGF-A antisense-oligo- nucleotide has the sequence as disclosed in the sequence listing under Seq. ID No. 1 - 6, having a DNA- or RNA-type structure.
In a preferred embodiment the PDGF-B antisense-oligonuclotide has the sequence as disclosed in the sequence listing under Seq. ID No. 7 - 12, having a DNA- or RNA-type structure.
In a preferred embodiment the bFGF'antisense-oligonucleotide has the sequence as disclosed in the sequence listing under Seq. ID No. 13 - 27, having a DNA- or RNA-type structure.
In a preferred embodiment of these oligonucleotides they are phosphorothioate derivatives, having a DNA- or RNA-type structure.
It is possible that one single individual sequence as mentioned above works as an antisense nucleic acid or oligo¬ nucleotide structure according to the invention. However, it is also possible that one strand of nucleotides comprises more than one of the sequences as mentioned above directly covalently linked or with other nucleotides covalently linked in between. Preferably, individual oligonucleotides are
addressed .
The randomized control sequence is disclosed in the sequence listing under Seq. ID No. 28.
In a preferred embodiment of these oligo-nucleotides they are phosphorothioate derivatives.
Modifications of the antisense-oligonucleotides are ad¬ vantageous since they are not as fast destroyed by endo- geneous factors when applied as this is valid for naturally occuring nucleotide sequences. However, it is understood by the skilled person that also naturally occuring nucleotides having the disclosed sequence can be used according to the invention. In a very preferred embodiment the modification is a phosphorothioate modification.
The synthesis of the oligodeoxy-nucleotide of the invention is described as an example in a greater detail as follows.
Oligodeoxy-nucleotides were synthesized by stepwise 5'- addition of protected nucleosides using phosphite triester chemistry. The nucleotide A was introduced as 5'dimethoxy- trityl-deoxyadenosine (N-benzoyl) -N,N' -diisopropyl-2-cyano- ethyl phosphoramidite (0.1 M) ; C was introduced by a 5'-
4 dimethoxytrityl-deoxycytidine(N -benzoyl) -N,N' -diisopropyl-2- cyanoethyl phosphoramidite; G was introduced as 5' -dimethoxy-
Q trityl-deoxyguanosine (N -isobutyryl) -N,N' -diisopropyl-2- cyanoethyl phosphoramidite and the T was introduced as 5' - dimethodytrityl-deoxythymidine-N,N' -diisopropy1-2-cyanoethyl phosphoramidite. The nucleosides were preferably applied in 0.1 M concentration dissolved in acetonitrile.
Synthesis was performed on controlled pore glass particles of approximately 150 μm diameter (pore diameter 500 A) to which the most 3' nucleoside is covalently attached via a long-chain alkylamin linker (average loading 30 μmol/g solid
support ) .
The solid support was loaded into a cylindrical synthesis column, capped on both ends with filters which permit ade¬ quate flow of reagents but hold back the solid synthesis support. Reagents were delivered and withdrawn from the synthesis column using positive pressure of inert gas. The nucleotides were added to the growing oligonucleotide chain in 3'-> 5' direction. Each nucleotide was coupled using one round of the following synthesis cycle:
Cleave 5'DMT (dimethoxytrityl) protecting group of the previous nucleotide with 3-chloroacetic acid in dichloro- methane followed by washing the column with anhydrous aceton- itrile. Then simultaneously one of the bases in form of their protected derivative depending on the sequence was added plus tetrazole in acetonitrile. After reaction the reaction mixture has been withdrawn and the phosphite was oxidized with a mixture of sulfur (SoD) in carbon disulfid/pyridine/- triethylamine. After the oxidation reaction the mixture was withdrawn and the column was washed with acetonitrile . The unreacted 5'-hydroxyl groups were capped with simultaneous addition of 1-methylimidazole and acetic anhydryide/lutidine- /tetrahydrofuran. Thereafter, the synthesis column was washed with acetonitrile and the next cycle was started.
The work up procedure and purification of the synthesis products occured as follows.
After the addition of the last nucleotide the deoxynucleo- tides were cleaved from the solid support by incubation in ammonia solution. Exocyclic base protecting groups were removed by further incubation in ammonia. Then the ammonia was evaporated under vacuum. Full-length synthesis products still bearing the 5'DMT protecting group were separated from shorter failure contaminants using reverse phase high per¬ formance liquid chromatography on silica C.0 stationary
phase. Eluents from the product peak were collected, dried under vacuum and the 5' -DMT protecting group cleaved by incubation in acetic acid which was evaporated thereafter under vacuum. The synthesis products were solubilized in the deionized water and extracted three times with diethylether. Then the products were dried in vacuo. Another HPLC-AX chromatography was performed and the eluents from the product peak were dialysed against excess of Trisbuffer as well as a second dialysis against deionized water. The final products were lyophilized and stored dry.
The anisense-nucleic acid of the invention can be used as pharmaceutical composition or medicament. This medicament can be used for treating neoplasms in which the expression of platelet derived growth factor or basic fibroblast growth factor is of relevance for the pathogenicity. It can be used to reduce neoplastic cell growth in cells expressing these growth factors, and to inhibit pathological angiogenesis . Furthermore, it can be used to treat rheumatoid arthritis and other autoimmune diseases in which the production of PDGF-A, PDGF-B or bFGF is of pathogenetic relevance.
The effect of PDGF-A and PDGF-B and bFGF specific antisense oligonucleotides on neoplastic cell growth was investigated. It was demonstrated that antisense oligodeoxynucleotides as well as phosphorothioate modified nucleic acids, comple¬ mentary to PDGF and bFGF mRNAs could specifically inhibit PDGF protein expression and bFGF protein expression re¬ spectively. Furthermore, they reduced cell proliferation in mammary carcinoma, glioma, pancreas carcinoma and melanoma cells with surprising efficiency.
Surprisingly, cell extracts from tumor cells treated with the specific antisense oligodeoxynucleotides targeted against either of the three growth factors did not induce angio- neogenesis, while extracts from untreated cells were highly angiogenic.
The invention is further explained by the following non limiting example.
Example
Enzyme-linked immunosorbent assay (ELISA)
Cell lysates were diluted in 50 mM carbonate buffer at pH 9.0 and immobilized on immunon II plates (Dynatech Laborator¬ ies, Inc.) overnight. Antigen solution was removed and 200 μl/well phosphate buffered saline (PBS)/ l%/BSA/0.02% azide were added to block non-specific protein binding. Following incubation at room temperature for 2 h solution was removed. After washing with PBS plates were air dried for 3 h. Specific antibodies for PDGF-A, PDGF-B or bFGF (Oncogene or Santa Cruz Biotechnology Inc.) were added at 50 μl/well, diluted in blocking buffer. Following 1 h incubation at room temperature samples were removed and subsequently wells were washed four times with PBS/0.05% Tween 20. Then 50 μl of secondary antibody-phosphatase conjugate were added and removed after 1 h. Wells were washed with diethanolamine buffer (10 mM diethanolamine, 0.5 mM MgCl2, pH 9,5) . One tablet of Sigma 104 phosphatase substrate was dissolved in 5 ml diethanolamine buffer. 50 μl of the substrate solution were added per well. The reaction was stopped with 50 μl 0,1 M EDTA (pH 7.5) and plates were read on a microtitration plate reader.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Biognostik Gesellschaft fuer biomolekulare
Diagnostik mbH
(B) STREET: Carl-Giesecke-Str. 3
(C) CITY: Goettingen
(E) COUNTRY: Germany
(F) POSTAL CODE (ZIP) : 37079
(ii) TITLE OF INVENTION: Antisense nucleic acid for the treatment of neoplasms and auto-immune diseases and diseases involving pathological angioneogenesis, in which expression of the growth factors bFGF, PDGF-A or PDGF-B..
(iii) NUMBER OF SEQUENCES: 28
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCCAAGGTCC TCAT 14
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGAGTCTATC TCCA 14
(2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: IS base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCAAAGAATC CTCACT 16
(2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CACATGCTTA GTGG 14
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTCGTAAATG ACCG 14
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : AGGAATCTCG TAAATGAC 18
(2) INFORMATION FOR SEQ ID NO:7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : CAGCAGCGAT TCAT 14
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: IS base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGAGATCATC AAAGGA 16
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTCAGCAATG GTCA 14
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GATCTCGAAC ACCT 14
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CACAATCTCG ATCTTTCT 18
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCTTCTTAAA GATTGGCT 18
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CACATACCAA CTGG 14
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AGCTTGATGT GAGG 14
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GAAGTTGTAG CTTGATGT 18
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCTTGAAGTT GTAGCT 16
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTGCTTGAAG TTGTAG 16
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GACACAACTC CTCT 14
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TCCTTTGATA GACACAAC 18
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CTCCTTTGAT AGACAC 16
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GGTTAGCACA CACT 14
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GGTAACGGTT AGCA 14
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGTAACACAT TTAGAAGC 18
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CTCATCCGTA ACAC 14
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
( i) SEQUENCE DESCRIPTION: SEQ ID NO:25: CCGGTAAGTA TTGTAGTT 18
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGTGTATTTC CTTGAC 16
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ACATACCAAC TGGTGT 16
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GTCCCTATAC GAAC 14
Claims
1. A compound which is capable of treating or preventing neoplasms and/or autoimmune diseases and/or diseases involving pathological angiogenesis, in which ex¬ pression of PDGF-A, PDGF-B and/or bFGF plays a patho- genetic role.
2. The compound of claim 1 being an antisense nucleic acid or effective derivative thereof, said antisense nucleic acid hybridizing with an area of the messenger RNA
(mRNA) and/or DNA encoding bFGF, PDGF-A and/or PDGF-B.
3. The compound of claims 1 and/or 2 wherein the PDGF-A antisense oligonucleotide comprises the following sequences identified in the sequence listing under Seq ID No. 1 - 6, having a DNA- or RNA-type structure.
the PDGF-B antisense-oligonuclotide comprises the sequences identified in the sequence listing under Seq. ID No. 7 - 12, having a DNA- or RNA-type structure.
and the bFGF antisense-oligonucleotide comprises the sequences identified in the sequence listing under Seq. ID No. 13 - 27, having a DNA- or RNA-type structure.
4. Antisense oligonucleotides of the claims 2 and 3 wher¬ ein the oligonucleotides are modified oligonucleotides such as phosphorothioate derivatives.
5. Antisense nucleic acid or -oligonucleotides according to any one of the claims 1 to 4 obtainable by solid phase synthesis using phosphite triester chemistry by growing the nucleotide chain in 3' -5' direction in that the respective nucleotide is coupled to the first nucleotide which is covalently attached to the solid phase comprising the steps of cleaving 5'DMT protecting group of the previous nucleotide,
adding the respective nucleotide for chain pro¬ pagation,
modifying phosphite groups subsequently cap un- reacted 5'-hydroxyl groups and
cleaving the oligonucleotide from the solid support,
followed by working up the synthesis product.
A pharmaceutical composition comprising an effective amount of a compound of any one of the claims 1 to 5 for the prevention and treatment of neoplasms and/or autoimmune diseases and/or diseases involving patho¬ logical angiogenesis, in which expression of PDGF-A, PDGF-B and/or bFGF plays a pathogenetic role.
Use of a compound according to any one of the claims 1 to 5 for the preparation of a pharmaceutical com¬ position for the treatment and or prevention of neo¬ plasms and/or autoimmune diseases and/or diseases involving pathological angiogenesis related with the expression of PDGF-A, PDGF-B and/or bFGF.
Method of treating or preventing neoplasms and/or autoimmune diseases and/or diseases involving patho¬ logical angiogenesis by administering an effective amount of the compound according to any one of the claims 1 to 5 or a pharmaceutical composition of claim 6 to a patient suffering from disorders related with the expression of PDGF-A, PDGF-B and/or bFGF.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU56980/94A AU5698094A (en) | 1993-12-09 | 1993-12-09 | Antisense nucleic acid for the treatment of diseases in which expression of bfgf, pdgf-a or pdgf-b plays a pathogenic role |
| PCT/EP1993/003461 WO1995016032A1 (en) | 1993-12-09 | 1993-12-09 | ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP1993/003461 WO1995016032A1 (en) | 1993-12-09 | 1993-12-09 | ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1995016032A1 true WO1995016032A1 (en) | 1995-06-15 |
Family
ID=8165802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/003461 WO1995016032A1 (en) | 1993-12-09 | 1993-12-09 | ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU5698094A (en) |
| WO (1) | WO1995016032A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997044046A1 (en) * | 1996-05-17 | 1997-11-27 | Otsuka Pharmaceutical Co., Ltd. | Platelet-derived growth factor expression inhibitor |
| WO2005087269A1 (en) * | 2004-03-16 | 2005-09-22 | Dnavec Research Inc. | Method of inhibiting tumor proliferation |
| WO2005020972A3 (en) * | 2003-08-27 | 2005-11-17 | Eyetech Pharmaceuticals Inc | Combination therapy for the treatment of ocular neovascular disorders |
| WO2009033690A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Bfgf (119-126) for therapeutic applications |
| WO2009033687A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Spantide ii and bfgf (119-126) for therapeutic applications |
| US11273171B2 (en) | 2013-07-12 | 2022-03-15 | Iveric Bio, Inc. | Methods for treating or preventing ophthalmological conditions |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992017206A1 (en) * | 1991-03-28 | 1992-10-15 | The Victoria University Of Manchester | Wound healing |
-
1993
- 1993-12-09 WO PCT/EP1993/003461 patent/WO1995016032A1/en active Application Filing
- 1993-12-09 AU AU56980/94A patent/AU5698094A/en not_active Abandoned
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992017206A1 (en) * | 1991-03-28 | 1992-10-15 | The Victoria University Of Manchester | Wound healing |
Non-Patent Citations (6)
| Title |
|---|
| BEHL, C. ET AL.: "Autocrine growth regulation in neuroectodermal tumors as detected with oligodeoxynucleotide antisense molecules", NEUROSURGERY, vol. 33, no. 4, October 1993 (1993-10-01), pages 679 - 684 * |
| BEHL, C. ET AL.: "Autoinduction of platelet derived growth factor (PDGF) A-chain mRNA expression in a human melanoma cell line and growth inhibitory effects of PDGF A-chain mRNA-specific antisense molecules", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 193, no. 2, 15 June 1993 (1993-06-15), DULUTH, MINNESOTA US, pages 744 - 751 * |
| BETSHOLZ, C. ET AL.: "cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumor cell lines", NATURE, vol. 320, 24 April 1986 (1986-04-24), LONDON GB, pages 695 - 699 * |
| ECKSTEIN, F.: "Phosphorothioate analogues of nucleotides - tools for the investigation of biochemical processes", ANGEWANDTE CHEMIE. INTERNATIONAL EDITION, vol. 22, no. 6, June 1983 (1983-06-01), WEINHEIM DE, pages 423 - 439 * |
| ERICKSON, R. & IZANT, J.:'Gene regulation: biology of antisense RNA and DNA'; 1992, RAVEN PRESS, Ltd., NEW YORK pages 317-328, * |
| ITOH, H. ET AL.: "Antisense oligonucleotides complementary to PDGF mRNA attenuate angiotensin II-induced vascular hypertrophy", HYPERTENSION, vol. 16, no. 3, September 1990 (1990-09-01), pages 325 - 326 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997044046A1 (en) * | 1996-05-17 | 1997-11-27 | Otsuka Pharmaceutical Co., Ltd. | Platelet-derived growth factor expression inhibitor |
| WO2005020972A3 (en) * | 2003-08-27 | 2005-11-17 | Eyetech Pharmaceuticals Inc | Combination therapy for the treatment of ocular neovascular disorders |
| US7759472B2 (en) | 2003-08-27 | 2010-07-20 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
| EP2281885A1 (en) * | 2003-08-27 | 2011-02-09 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
| US8187597B2 (en) | 2003-08-27 | 2012-05-29 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
| US8206707B2 (en) | 2003-08-27 | 2012-06-26 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
| US8685397B2 (en) | 2003-08-27 | 2014-04-01 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
| WO2005087269A1 (en) * | 2004-03-16 | 2005-09-22 | Dnavec Research Inc. | Method of inhibiting tumor proliferation |
| WO2009033690A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Bfgf (119-126) for therapeutic applications |
| WO2009033687A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Spantide ii and bfgf (119-126) for therapeutic applications |
| US11273171B2 (en) | 2013-07-12 | 2022-03-15 | Iveric Bio, Inc. | Methods for treating or preventing ophthalmological conditions |
| US12016875B2 (en) | 2013-07-12 | 2024-06-25 | Iveric Bio, Inc. | Methods for treating or preventing ophthalmological conditions |
Also Published As
| Publication number | Publication date |
|---|---|
| AU5698094A (en) | 1995-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0736093B1 (en) | ANTISENSE NUCLEIC ACIDS FOR THE PREVENTION AND TREATMENT OF DISORDERS IN WHICH EXPRESSION OF c-erbB-2 PLAYS A ROLL | |
| US5780610A (en) | Reduction of nonspecific hybridization by using novel base-pairing schemes | |
| US5637459A (en) | Systematic evolution of ligands by exponential enrichment: chimeric selex | |
| AU694468B2 (en) | Solution phase nucleic acid sandwich assays having reduced background noise | |
| US5686242A (en) | Determination of oligonucleotides for therapeutics, diagnostics and research reagents | |
| Wyatt et al. | Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion. | |
| US6455689B1 (en) | Antisense-oligonucleotides for transforming growth factor-β (TGF-β) | |
| US5672472A (en) | Synthetic unrandomization of oligomer fragments | |
| EP0708829B1 (en) | A pharmaceutical composition comprising antisense-nucleic acid for prevention and/or treatment of neuronal injury, degeneration and cell death and for the treatment of neoplasms | |
| US5747253A (en) | Combinatorial oligomer immunoabsorbant screening assay for transcription factors and other biomolecule binding | |
| WO1996006950A9 (en) | Reduction of nonspecific hybridization by using novel base-pairing schemes | |
| EP0451221A1 (en) | CHIMER'S DNA RNS CATALYTIC SEQUENCES. | |
| EP0695354B1 (en) | Antisense-oligonucleotides for the treatment of immunosuppressive effects of transforming growth factor-beta1 (tgf-beta1) | |
| WO1995016032A1 (en) | ANTISENSE NUCLEIC ACID FOR THE TREATMENT OF DISEASES IN WHICH EXPRESSION OF bFGF, PDGF-A OR PDGF-B PLAYS A PATHOGENIC ROLE | |
| Gat et al. | Reading DNA differently | |
| WO1994021825A1 (en) | Combinatorial oligomer immunoabsorbant screening assay for transcription factors and other biomolecule binding | |
| WO1995006751A1 (en) | Methods for isolation of most abundant oligonucleotides from complex mixtures | |
| WO1995006751A9 (en) | Methods for isolation of most abundant oligonucleotides from complex mixtures | |
| AU1042000A (en) | Method for the exponential amplification of molecular matrices |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| NENP | Non-entry into the national phase |
Ref country code: CA |
|
| 122 | Ep: pct application non-entry in european phase |