Oligomeric compounds for the modulation HIF-1alpha expression

• US 20040096848A1
• Filed: 10/23/2003
• Published: 05/20/2004
• Est. Priority Date: 04/05/2002
• Status: Active Grant

• Current Assignee: Roche Innovation Center Copenhagen A/S (Roche Holding AG)
• Original Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC)
• Inventors: Thrue, Charlotte Albaek, Hog, Anja Molhart, Kristjansen, Paul E.G.
• Granted Patent: US 7,737,264 B2
• Time in Patent Office: Days
• US Class Current: 435/6.14
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• 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 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/334 : Modified C
• CPC Class Codes: C12N 2310/3341 : 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 : Modified T or U
• CPC Class Codes: C12N 2310/341 : Gapmers, i.e. of the type ===---===

Abstract

Oligonucleotides directed against the hypoxia-inducible factor-1α (HIF-1α) gene are provided for modulating the expression of HIF-1α. The compositions comprise oligonucleotides, particularly antisense oligonucleotides, targeted to nucleic acids encoding the HIF-1α. Methods of using these compounds for modulation of HIF-1α expression and for the treatment of diseases associated with the hypoxia-inducible factor-1α are provided. Examples of diseases are cancer and pre-eclampsia. The oligonucleotides may be composed of deoxyribonucleosides, a nucleic acid analogue, or Locked Nucleic Acid (LNA) or a combination thereof.

Claims

1. A compound of 8 to 50 nucleobases in length which modulates HIF-1α

and wherein said compound has a sequence comprising at least an 8-nucleobase portion of SEQ ID NO;

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.

3. The compound according to any one of claims 1-2, which is an antisense oligonucleotide.

4. The compound according to any one of claims 1-3, wherein at least one nucleotide comprises at least one Nucleic Acid Analogue.

5. The compound according to any one of claims 1-4, wherein at least one nucleotide comprises at least one Locked Nucleic Acid (LNA) unit.

6. The compound according to claims 1-5, wherein the LNA unit has the structure of the general formula in Scheme 2.

7. The compound according to claims 1 through 6, wherein at least one nucleotide comprises a LNA unit according any of the formulas in Scheme 3:

8. The compound according to claims 4 through 7, wherein the internucleoside linkage may be selected from the group consisting of —

O—

P(O)₂—

O—

, —

O—

P(O,S)—

O—

, —

O—

P(S)₂—

O—

, —

S—

P(O)₂—

O—

, —

S—

P(O,S)—

O—

, —

S—

P(S)₂—

O—

, —

O—

P(O)₂—

S—

—

O—

P(O,S)—

S—

, —

S—

P(O)₂—

S—

, —

O—

PO(R^H)—

O—

, O—

PO(OCH₃)—

O—

, —

O—

PO(NR^H)—

O—

, —

O—

PO(OCH₂CH₂S—

R)—

O—

, —

O—

PO(BH₃)—

O—

, —

O—

PO(NHR^H)—

O—

, —

O—

P(O)₂—

NR^H—

, —

NR^H—

P(O)₂—

O—

, —

NR^H—

CO—

O—

, where R^H is selected form hydrogen and C_1-4-alkyl.

9. The compound of claim 8, wherein the modified internucleoside linkage is a phosphorothioate linkage.

10. The compound according to claims 4 through 9, wherein the nucleobases is a modified nucleobases selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine.

11. The compound according to any one of claims 4-10, wherein the LNA unit is oxy-LNA, thio-LNA, amino-LNA in either the D-β

or L-α

configurations or combinations thereof.

12. The compound of any one of claims 1-11, wherein the antisense oligonucleotide is a chimeric oligonucleotide.

13. The compound of claim 12, wherein the antisense oligonucleotide is a gapmer, headmer or tailmer.

14. The compound according to any one of claims 1-13, wherein the antisense oligonucleotide is a 13, 14, 15, 16, 17, 18, 19, 20 or 21-mer.

15. The compound according to any one of claims 1-14, wherein the number of LNA units are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21.

16. The compound according to any one of claims 1-15, wherein at least four nucleotides are LNAs.

18. A composition comprising the compound of claims 1 through 17 and a pharmaceutically acceptable carrier or diluent.

19. A pharmaceutical composition comprising the compound of any one of claims 1-18, which further comprises a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the compound of any one of claims 1-18, which is employed in a pharmaceutically acceptable salt.

21. A pharmaceutical composition comprising the compound of any one of claims 1-18, which further comprises a conjugate or formulation.

22. A pharmaceutical composition comprising the compound of any one of claims 1-18, which constitutes a pro-drug.

23. A pharmaceutical composition comprising the compound of any one of claims 1-18, which further comprises other oligomeric compounds, chemotherapeutic compounds, antiinflamatory compounds and/or antiviral compounds.

24. A method of inhibiting the expression of HIF-1α

, in cells or tissues comprising contacting said cells or tissues with the compound according to any one of claims 1-23 so that expression of HIF-1α

is inhibited.

30. A method of modulating angiogenesis as well as red blood cell proliferation, cellular proliferation, iron metabolism, glucose and energy metabolism, pH regulation, tissue invasion, apoptosis, multi-drug resistance, cellular stress response or matrix metabolism comprising contacting a cell with the antisense compound of claim 1-18 so that the cell is modulated.

31. A method of inhibiting the proliferation of cells comprising contacting cells in vitro with an effective amount of the antisense compound of claim 1-18, so that proliferation of the cells is inhibited.

32. The method of claim 31 wherein said cells are cancer cells.

33. A method of modulating apoptosis in a cell comprising contacting a cell with the antisense compound of claim 1-18 so that apoptosis is modulated.

34. The method of claim 33 wherein said modulation of apoptosis is sensitization to an apoptotic stimulus.

35. The method of claim 33 or 34 wherein said apoptotic stimulus is a cytotoxic chemotherapeutic agent.

36. The method of claim 35 further comprising contacting said cells with a chemotherapeutic agent.

2. A compound of 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding HIF-1α

, wherein said compound specifically hybridises with a nucleic acid encoding HIF-1α and

inhibits the expression of HIF-1α and

wherein said compound has a sequence comprising at least an 8 nucleobase portion of SEQ ID NO;

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.

17. A compound of 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding HIF-1α

, wherein said compound specifically hybridises with a nucleic acid encoding HIF-1α and

inhibits the expression of HIF-1α and

wherein said compound has a sequence comprising at least an 8-nucleobase portion of SEQ ID NO;

2, 3, 12, 22, 23, 53, 54, 55, 56, 59, 60, 61, 89, 90, 93, 94, 95, 97, 100, 105, 106, 107, 108, 109, 110, 111, 112, 114 or 115.

25. A method of modulating expression of a gene involved in a disease comprising contacting the gene or RNA from the gene with an oligomeric compound wherein said compound has a sequence comprising at least an 8 nucleobase portion of SEQ ID NO:

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 whereby gene expression is modulated.

26. A method according to claim 25, wherein the compounds comprise one or more LNA units.

27. The method of claim 25 or 26, wherein the compound hybridizes with messenger RNA of the gene to inhibit expression thereof.

29. The method according to any one of claims 25-28 wherein the disease is a common cancer, as e.g. primary and metastatic breast, colorectal, prostate, pancreas, other GI-cancers, lung, cervical, ovarian, brain, head and neck, cervix, colon, liver, thyroid, kidney, testes, stomach, intestine, bowel, esophagus, spinal cord, sinuses, bladder or urinary tract tumors, as well as pre-eclampsia, inflammatory bowel disease and Alzheimers disease.

28. A method of treating a mammal suffering from or susceptible from an cancer disease, comprising:

administering to the mammal an therapeutically effective amount of an oligonucleotide targeted to HIF-1α

that comprises one or more LNA units.