Oligomeric compounds for the modulation HIF-1α expression

• US 7,737,264 B2
• Filed: 04/04/2003
• Issued: 06/15/2010
• Est. Priority Date: 04/05/2002
• Status: Expired due to Fees

• Current Assignee: Roche Innovation Center Copenhagen A/S (Roche Holding AG)
• Original Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC), Santaris Pharma A/S (Roche Holding AG)
• Inventors: Kristjansen, Paul E. G., Thrue, Charlotte Albæk, Høg, Anja Mølhart
• Primary Examiner(s): Chong; Kimberly
• Application Number:
  US10/407,807
• Publication Number:
  US 20040096848A1
• Time in Patent Office: 2,629 Days
• Field of Search: 514/44, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/24.5
• US Class Current: 536/24.5
• 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 16 to 50 nucleotides or nucleotide analogues, wherein adjacent nucleotides or nucleotide analogues are covalently linked by an internucleotide linkage, which is capable of modulating HIF-1α

as encoded by a nucleic acid molecule having the sequence of SEQ ID NO. 1 and wherein a region of said compound has a sequence that is identically present in SEQ ID NO;

55.

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

4. The compound according to claim 3, wherein the antisense oligonucleotide comprises at least one nucleotide analogue.

5. The compound according to claim 4, wherein the nucleotide analogue is a Locked Nucleic Acid (LNA) unit.

6. The compound according to claim 5, wherein the LNA unit has the structure:

7. The compound according to claim 5, wherein the LNA unit has a structure of any of the following formulas:

8. The compound according to claim 6 or 7, wherein the internucleotide linkage is 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—

HR^H—

, —

NR^H—

P(O)₂—

O—

, and —

NR^H—

CO—

O—

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

9. The compound of claim 8, wherein at least one internucleotide linkage is a phosphorothioate linkage.

10. The compound according to claim 6 or 7, wherein at least one nucleobase is a modified nucleobase independently selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

11. The compound according to claim 5, wherein each of the LNA units is independently selected from β

-D-oxy-LNA, β

-D-thio-LNA, β

-D-amino-LNA, α

-L-oxy-LNA, α

-L- thio-LNA and α

-L-amino-LNA.

12. The compound of claim 3, wherein the antisense oligonucleotide is a chimeric oligonucleotide.

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

14. The compound according to claim 3, wherein the antisense oligonucleotide is a 16, 17, 18, 19, 20or 21-mer.

15. The compound according to claim 5, which comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 LNA units.

16. The compound according to claim 15, which comprises at least four LNA units.

17. A composition comprising the compound of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.

18. A pharmaceutical composition comprising the compound of claim 1 or 2 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the compound of claim 1 or 2 in the form of a pharmaceutically acceptable salt.

20. A pharmaceutical composition comprising the compound of claim 1 or 2, which is in the form of a conjugate or is in a formulation.

21. A pharmaceutical composition comprising the compound of claim 1 or 2, which is in the form of a pro-drug.

22. A pharmaceutical composition comprising the compound of claim 1 or 2, which further comprises other oligomeric compounds, chemotherapeutic compounds, antiinflamatory compounds and/or antiviral compounds.

35. The compound according to any of claims 1-2, which contains at least one nucleotide analogue.

2. A compound of 16 to 50 nucleotides or nucleotide analogues, wherein adjacent nucleotides or nucleotide analogues are covalently linked by an internucleotide linkage, targeted to a nucleic acid molecule encoding HIF-1α

(SEQ ID NO.

     1), wherein said compound hybridizes with said nucleic acid molecule and is capable of inhibiting the expression of HIF-1α

in a cell and wherein a region of said compound has a sequence that is identically present in SEQ ID NO;

55.

23. A compound represented by the formula:

T_sG_sG_sC_sa_sa_sg_sc_sa_st_sc_sc_sT_sG_sT_sA (SEQ ID NO.

     55) wherein uppercase letters denote oxy-LNA nucleotides, lowercase letters denote deoxynucleotides and the subscript “

s”

denotes a phosphorothioate linkage.

25. A composition comprising the compound of claim 23 or 24 and a pharmaceutically acceptable carrier, diluent or salt.

26. A pharmaceutical composition comprising the compound of claim 23 or 24, which is in the form of a conjugate or is in a formulation.

27. A pharmaceutical composition comprising the compound of claim 23 or 24 which is in the form of a pro-drug.

28. A pharmaceutical composition comprising the compound of claim 23 or 24 which further comprises other oligomeric compounds, chemotherapeutic compounds, antiinflamatory compounds and/or antiviral compounds.

24. A compound selected from T_sG_sG_sC_sa_sa_sg_sc_sa_st_sc_sc_sT_sG_sT_sa (SEQ ID NO. 55) and t_sG_sG_sC_sa_sa_sg_sc_sa_st_sc_sc_sT_sG_sT_sA (SEQ ID NO. 55) wherein uppercase letters denote oxy-LNA nucleotides, lowercase letters denote deoxynucleotides and the subscript “

s”

denotes a phosphorothioate linkage.

29. A compound of 16 to 50 nucleotides or nucleotide analogues, wherein adjacent nucleotides are covalently linked by an internucleotide linkage, which is capable of modulating HIF-1α

as encoded by the nucleic acid molecule having the sequence of SEQ ID NO. 1, wherein said compound has a sequence that is complementary to the sequence of a target site located at nucleotides 1177 to 1192 of said nucleic acid molecule, and wherein said compound comprises at least one nucleotide analogue that is an LNA unit.

30. The compound of claim 29, wherein the compound is a chimeric antisense oligonucleotide having a melting temperature of at least 45°

C. when measured in a solution comprising 100mM NaCl, 0.1mM EDTA, 10mM phosphate buffer (pH of 7.0), 1.5 μ

M of said compound, and 1.5 μ

M of the nucleic acid molecule having the sequence represented by SEQ ID NO. 1.

31. A composition comprising the compound of claim 29 and a pharmaceutically acceptable carrier, diluent or salt.

32. A pharmaceutical composition comprising the compound of claim 31 which is in the form of a conjugate or is in a formulation.

33. A pharmaceutical composition comprising the compound of claim 31 which is in the form of a pro-drug.

34. A pharmaceutical composition comprising the compound of claim 31 which further comprises another oligomeric compound, chemotherapeutic compound, anti-inflammatory compound or an antiviral compound.