Compounds and methods for modulating expression of PCSK9

• US 8,143,230 B2
• Filed: 05/07/2007
• Issued: 03/27/2012
• Est. Priority Date: 05/05/2006
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Siwkowski, Andrew M., Swayze, Eric, Wancewicz, Edward, Bhanot, Sanjay, Geary, Richard S., McKay, Robert, Monia, Brett P., Seth, Punit P.
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US12/299,572
• Publication Number:
  US 20090306005A1
• Time in Patent Office: 1,786 Days
• Field of Search: None
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• 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 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/46 for decreasing, blocking or antagonising the activity of glucocorticosteroids
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• 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 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/341 : Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/3515 : Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12Y 203/0102 : Diacylglycerol O-acyltransferase (2.3.1.20)

Abstract

The present disclosure describes short antisense compounds, including such compounds comprising chemically-modified high-affinity monomers 8-16 monomers in length. Certain such short antisense compound are useful for the reduction of target nucleic acids and/or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing.

Claims

1. A short antisense compound 8 to 14 monomers in length, comprising a 2′

-deoxyribonucleotide gap region flanked on each side by at least one wing, wherein each wing independently comprises 1 to 3 high-affinity modified monomers and wherein the short antisense compound is targeted to a nucleotide encoding PCSK9.

2. The short antisense compound of claim 1, wherein said high-affinity modified monomers are sugar-modified nucleotides.

3. The short antisense compound of claim 2, wherein at least one of the sugar-modified nucleotides comprises a bridge between the 4′

and the 2′

position of the sugar.

4. The short antisense compound of claim 2, wherein each of said high-affinity modified nucleotides confers a Δ

T_m of 1 to 4 degrees per nucleotide.

5. The short antisense compound of claim 2, wherein each of said sugar-modified nucleotides comprises a 2′

-substituent group that is other than H or OH.

6. The short antisense compound of claim 5, wherein at least one of said sugar-modified nucleotides is a 4′

to 2′

bridged bicyclic nucleotide.

7. The short antisense compound of claim 5, wherein each of the 2′

-substituent groups is, independently, alkoxy, substituted alkoxy, or halogen.

8. The short antisense compound of claim 7, wherein each of the 2′

-substituent groups is OCH₂CH₂OCH₃.

9. The short antisense compound claim 3, wherein the conformation of each of said sugar-modified nucleotides is, independently, β

-D or α

-L.

10. The short antisense compound claim 3, wherein each of said bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —

[C(R₁)(R₂)]_n—

, —

C(R₁)═

C(R₂)—

, —

C(R₁)═

N—

, —

C(═

NR₁)—

, —

C(═

O)—

, —

C(═

S)—

, —

O—

, —

Si(R₁)₂—

, —

S(═

O)_x— and

—

N(Ri)—

;

wherein

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ_I, acyl (C(═

O)—

H), substituted acyl, CN, sulfonyl (S(═

O)₂-J₁), or sulfoxyl (S(═

O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═

O)—

H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

11. The short antisense compound of claim 10, wherein each of said bridges is, independently, 4′

-CH₂-2′

, 4′

-(CH₂)₂-2′

, 4′

-CH₂—

O-2′

, 4′

-(CH₂)₂—

O-2′

, 4′

-CH₂—

O—

N(R₁)-2′ and

4′

-CH₂—

N(R₁)—

O-2′

- wherein each R₁ is, independently, H, a protecting group or C₁-C₁₂ alkyl.

12. The short antisense compound of claim 1, wherein each of the high-affinity modified monomer is independently selected from bicyclic nucleotides or other 2′

-modified nucleotides.

13. The short antisense compound of claim 12, wherein the 2′

-modified nucleotides are selected from halogen, allyl, amino, azido, thio, O-allyl, O—

C₁-C₁₀ alkyl, —

OCF₃, O—

(CH₂)₂—

O—

CH₃, 2′

-O(CH₂)₂SCH₃, O—

(CH₂)₂—

O—

N(R_m)(R_n) or O—

CH₂—

C(═

O)—

N(R_m)(R_n), where each R_m and R_n is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

14. The short antisense compound of claim 13, wherein the 2′

-modified nucleotide is a 2′

-OCH₂CH₂OCH₃ nucleotide.

15. The short antisense compound of claim 1, wherein at least one monomeric linkage is a modified monomeric linkage.

16. The antisense compound of claim 15, wherein the modified monomeric linkage is a phosphorothioate linkage.

17. The short antisense compound of claim 1, wherein each monomeric linkage is a phosphorothioate internucleoside linkage.

18. The short antisense compound of claim 1, that is 8-13 monomers in length.

19. The short antisense compound of claim 18 that is 8-10 monomers in length.

20. The short antisense compound of claim 18 that is 11-13 monomers in length.

21. The short antisense compound of claim 1 that is 11-12 monomers in length.

22. The short antisense compound of claim 18 that is 9-10 monomers in length.

23. The short antisense compound of claim 18 that is 9-13 monomers in length.

24. The short antisense compound of claim 18 that is 10-13 monomers in length.

25. The short antisense compound of claim 18 that is 9-12 monomers in length.

26. The short antisense compound of claim 18 that is 10-12 monomers in length.

27. The short antisense compound of claim 18 that is 9-11 monomers in length.

28. The short antisense compound of claim 18 that is 10-11 monomers in length.

29. The short antisense compound of claim 18 that is 8 monomers in length.

30. The short antisense compound of claim 18 that is 9 monomers in length.

31. The short antisense compound of claim 18 that is 10 monomers in length.

32. The short antisense compound of claim 18 that is 11 monomers in length.

33. The short antisense compound of claim 18 that is 12 monomers in length.

34. The short antisense compound of claim 18 that is 13 monomers in length.

35. The short antisense compound of claim 1 that is 14 monomers in length.

36. The short antisense compound of claim 1, having a motif selected from 1-12-1;

2-10-2;

1-10-1;

1-10-2;

1-9-1;

1-9-2;

2-9-1;

2-8-1;

2-7-1;

2-7-2;

2-6-3;

2-7-2;

2-6-2;

3-8-3;

2-8-2;

1-8-1;

3-6-3; and

1-6-1 wherein, the first number represents the number of monomers in the 5′

-wing, the second number represents the number of monomers in the gap, and the third number represents the number of monomers in the 3′

wing.

37. The short antisense compound of claim 36 wherein the motif is selected from 1-10-1;

2-10-2; and

1-9-2.

38. The short antisense compound of claim 1 having a motif selected from 1-1-10-2, 1-1-8-2, 1-1-6-3, and 1-2-8-2, wherein the first number represents the number of monomers in a first 5′

wing, the second number represents the number of monomers in a second 5′

wing, the third number represents the number of monomers in the gap, and the fourth number represents the number of monomers in the 3′

wing.

39. The short antisense compound of claim 1 having a motif selected from 2-10-1-1, 2-8-1-1, 3-6-1-1, and 2-8-2-1, wherein the first number represents the number of monomers in the 5′

wing, the second number represents the number of monomers in the gap, the third number represents the number of monomers in a first 3′

wing, and the fourth number represents the number of monomers in a second 3′

wing.

40. The short antisense compound of claim 1 having a motif selected from 1-1-8-1-1;

2-1-6-1-1; and

1-2-8-2-1, wherein the first number represents the number of monomers in a first 5′

wing, the second number represents the number of monomers in a second 5′

wing, the third number represents the number of monomers in the gap, the fourth number represents the number of monomers in a first 3′

wing and the fifth number represents the number of monomers in a second 3′

wing.

41. A method of inhibiting PCSK9 expression comprising contacting a nucleic acid encoding PCSK9 with the short antisense compound of claim 1.

42. The method of claim 41 wherein the PCSK9 nucleic acid is in a cell.

43. The method of claim 42, wherein the PCSK9 nucleic acid is in an animal, and wherein the method further comprises administering the short antisense compound of claim 1 to the animal.

44. The method of claim 43, wherein the animal is a human.

45. The method of claim 43, wherein said contacting decreases total serum cholesterol, serum LDL, serum VLDL, serum HDL, serum triglycerides, serum apolipoprotein(s) and/or free fatty acids in the animal.

46. The method of claim 43, wherein inhibiting PCSK9 expression treats a cardiovascular disorder in the animal.