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 PDF

Info

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
Application number
PCT/EP1993/003461
Other languages
French (fr)
Inventor
Georg Ferdinand Schlingensiepen
Wolfgang Brysch
Reimar Schlingensiepen
Karl-Hermann Schlingensiepen
Original Assignee
Biognostik Gesellschaft für Biomolekulare Diagnostik mbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Biognostik Gesellschaft für Biomolekulare Diagnostik mbH filed Critical Biognostik Gesellschaft für Biomolekulare Diagnostik mbH
Priority to AU56980/94A priority Critical patent/AU5698094A/en
Priority to PCT/EP1993/003461 priority patent/WO1995016032A1/en
Publication of WO1995016032A1 publication Critical patent/WO1995016032A1/en

Links

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

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

C l a i s
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.
PCT/EP1993/003461 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 WO1995016032A1 (en)

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)

* Cited by examiner, † Cited by third party
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
WO2009033687A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Spantide ii and bfgf (119-126) for therapeutic applications
WO2009033690A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag 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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992017206A1 (en) * 1991-03-28 1992-10-15 The Victoria University Of Manchester Wound healing

Patent Citations (1)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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 (11)

* Cited by examiner, † Cited by third party
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
WO2009033687A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Spantide ii and bfgf (119-126) for therapeutic applications
WO2009033690A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Bfgf (119-126) for therapeutic applications
US11273171B2 (en) 2013-07-12 2022-03-15 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
EP1097939B1 (en) Method for the synthesis of isoguanosine and of its 2'-derivatives
US5637459A (en) Systematic evolution of ligands by exponential enrichment: chimeric selex
AU694468B2 (en) Solution phase nucleic acid sandwich assays having reduced background noise
Crooke Therapeutic applications of oligonucleotides
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
EP0736093A1 (en) ANTISENSE NUCLEIC ACIDS FOR THE PREVENTION AND TREATMENT OF DISORDERS IN WHICH EXPRESSION OF c-erbB PLAYS A ROLE
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) Chimeric dna-rna 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
Kool Topological modification of oligonucleotides for potential inhibition of gene expression
Murakami Artificially Created Nucleic Acids and Peptides/Proteins in Chemical Biology

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