Potent LNA oligonucleotides for the inhibition of HIF-1a expression

• US 20060252721A1
• Filed: 11/09/2005
• Published: 11/09/2006
• Est. Priority Date: 11/09/2004
• Status: Active Grant

• Current Assignee: Roche Innovation Center Copenhagen A/S (Roche Holding AG)
• Original Assignee: Santaris Pharma A/S (Roche Holding AG)
• Inventors: Thrue, Charlotte A., Rasmussen, Frank W., Westergaard, Majken, Hansen, Henrik F.
• Granted Patent: US 7,589,190 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/08 Antiseborrheics
• CPC Class Codes: A61P 17/12 Keratolytics, e.g. wart or anti-corn preparations
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/113 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 when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/345 : having at least two different backbone modifications
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications

Abstract

The present disclosure relates to an LNA oligonucleotide consisting of a sequence selected from the group consisting of 5′-(T_x)G_xG_xc_sa_sa_sg_sc_sa_st_sc_sc_sT_xG_xT-3′ and 5′-(G_x)T_xT_xa_sc_st_sg_sc_sc_st_st_sc_sT_xT_xA-3′, wherein capital letters designate a beta-D-oxy-LNA nucleotide analogue, small letters designate a 2-deoxynucleotide, underline designates either a beta-D-oxy-LNA nucleotide analogue or a 2-deoxynucleotide, subscript “s” designates a phosphorothioate link between neighbouring nucleotides/LNA nucleotide analogues, and subscript “x” designates either a phosphorothioate link or a phosphorodiester link between neighbouring nucleotides/LNA nucleotide analogues, and wherein the sequence is optionally extended by up to five 2-deoxynucleotide units. The LNA oligonucleotides are useful for modulating the expression of hypoxia-inducible factor-1a (HIF-1a), e.g. in the treatment of cancer diseases, inhibiting angiogenesis, inducing apoptosis, preventing cellular proliferation, or treating an angiogenic disease, e.g. diabetic retinopathy, macular degeneration (ARMD), psoriasis, rheumatoid arthritis and other inflammatory diseases.

Claims

1. An LNA oligonucleotide consisting of a sequence selected from the group consisting of

2. The LNA oligonucleotide according to claim 1, wherein the nucleotide unit in the bracket, (T_x) or (G_x), respectively, is present.

3. The LNA oligonucleotide according to claim 1, where one or both of the underlined nucleotide units designate a beta-D-oxy-LNA nucleotide analogue.

4. The LNA oligonucleotide according to claim 1, wherein the LNA oligonucleotide consists of 15, 16, 17, 18, 19 or 20 nucleotide units selected from 2-deoxynucleotides and beta-D-oxy-LNA nucleotide analogues.

5. The LNA oligonucleotide according to claim 4, wherein the LNA oligonucleotide consists of 16 nucleotide units selected from 2-deoxynucleotides and beta-D-oxy-LNA nucleotide analogues.

6. The LNA oligonucleotide according to claim 1, wherein the sequence is extended by one 2-deoxynucleotide unit at the 3′

-end.

8. The LNA oligonucleotide according to claim 1, which is selected from the group consisting of

9. The LNA oligonucleotide according to claim 8, which is

10. The LNA oligonucleotide according to claim 1, which is selected from the group consisting of

11. The LNA oligonucleotide according to claim 10, which is

12. A conjugate comprising an LNA oligonucleotide according to claim 1 and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said LNA oligonucleotide.

13. A pharmaceutical composition comprising an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12, and a pharmaceutically acceptable diluent, carrier or adjuvant.

14. The pharmaceutical composition according to claim 13, which comprises an aqueous carrier, said carrier comprising a buffer for keeping the pH in the range of 4.0-8.5 and having an ionic strength of 20-2000 mM.

15. The pharmaceutical composition according to claim 13, which is adapted for intraocular administration.

16. The pharmaceutical composition according to claim 13 further comprising at least one chemotherapeutic agent.

17. A method for treating cancer, said method comprising administering an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13 to a patient in need thereof.

18. The method according to claim 17, wherein said cancer is in the form of a solid tumor.

19. The method according to claim 17, wherein the cancer is selected from the group consisting of multiple myeloma, renal cancer, cervical cancer, colon cancer, brain cancer, and breast cancer.

20. A method for treating a disease selected from the group consisting of artherosclerosis, psoriasis, diabetic retinopathy, macular degeneration, rheumatoid arthritis, asthma, inflammatory bowel disease, warts, allergic dermatitis, inflammation, and skin inflammation, said method comprising administering an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13 to a patient in need thereof.

21. The method according to claim 20, wherein the disease is selected from inflammatory bowel disease, psoriasis and rheumatoid arthritis.

22. A method of treating a mammal suffering from or susceptible from a disease caused by abnormal angiogenesis, comprising administering to the mammal a therapeutically effective amount of an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12.

23. A method of inhibiting angiogenesis comprising the administration of an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13.

24. A method of inducing cellular apoptosis comprising the administration of an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13.

25. A method of preventing cellular proliferation comprising the administration of an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13.

26. A method of treating an angiogenic disease comprising the administration of an LNA oligonucleotide as defined in claim 1 or a conjugate as defined in claim 12 or a pharmaceutical composition as defined in claim 13, such that angiogenesis associated with the angiogenic disease is inhibited.

27. The method according to claim 26, wherein the angiogenic disease is selected from the group consisting of diabetic retinopathy, macular degeneration, and inflammatory diseases.

28. The method according to claim 27, wherein the angiogenic disease is an inflammatory disease selected from inflammatory bowel disease, psoriasis and rheumatoid arthritis.

29. The method according to claim 26, wherein the angiogenic disease is macular degeneration and diabetic retinopathy.

30. A kit comprising

(a) a first component containing an LNA oligonucleotide as defined in claims 1 or a conjugate as defined in claim 12 in solid form, and

(b) a second component containing saline or a buffer solution adapted for reconstitution of said LNA oligonucleotides.

7. The LNA oligonucleotide according to claim, wherein all nucleotide units in the sequence are linked by a phosphorothioate group.