Compounds and methods for modulating expression ApoB

• US 8,673,871 B2
• Filed: 05/07/2007
• Issued: 03/18/2014
• Est. Priority Date: 05/05/2006
• Status: Active Grant

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Bhanot, Sanjay, Geary, Richard S., McKay, Robert, Monia, Brett P., Seth, Punit P., Siwkowski, Andrew M., Swayze, Eric E., Wancewicz, Edward
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US12/299,764
• Publication Number:
  US 20090306180A1
• Time in Patent Office: 2,507 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 method of modulating expression of an apolipoprotein B (ApoB) by contacting a nucleic acid encoding ApoB with a short antisense compound 10 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, which are sugar-modified nucleotides that comprise a bridge between the 4′ and

the 2′

position of the sugar; and

wherein the short antisense compound is targeted to a nucleic acid encoding ApoB.

2. The method of claim 1, wherein each of said high-affinity modified nucleotides confers a T_m, of 1 to 4 degrees per nucleotide.

3. The method of claim 1, wherein the conformation of each of said sugar-modified nucleotides is, independently, β

-D or α

-L.

4. The method of claim 1, 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(R₁)—

;

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, COOJ₁, 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.

5. The method of claim 4, 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.

6. The method of claim 1, wherein at least one monomeric linkage is a modified monomeric linkage.

7. The method of claim 6, wherein the modified monomeric linkage is a phosphorothioate linkage.

8. The method of claim 1, wherein each monomeric linkage is a phosphorothioate internucleoside linkage.

9. The method of claim 1, wherein the short antisense compound comprises a motif selected from 1-12-1;

2-10-2;

1-10-1;

1-10-2;

3-8-3;

2-8-2;

1-8-1; and

3-6-3;

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.

10. The method of claim 9, wherein the motif is selected from 1-10-1;

2-10-2; and

1-9-2.

11. The method of claim 1, wherein the short antisense compound comprises 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.

12. The method of claim 1, wherein the short antisense compound comprises 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.

13. The method of claim 1, wherein the short antisense compound comprises 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.

14. The method of claim 1, wherein the ApoB nucleic acid is in a cell.

15. The method of claim 1, wherein the ApoB nucleic acid is in an animal.

16. The method of claim 15, wherein the animal is a human.

17. A method of inhibiting expression of ApoB RNA in an animal, comprising administering to said animal the short antisense compound 10 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, which are sugar-modified nucleotides that comprise a bridge between the 4′ and

the 2′

position of the sugar; and

wherein the short antisense compound is targeted to a nucleic acid encoding ApoB.

19. The method of claim 17, wherein said administering results in decreased total serum cholesterol, serum LDL, serum VLDL, serum HDL, serum triglycerides, serum apolipoprotein(a) and/or free fatty acids in the animal.

18. A method of treating a cardiovascular disorder in an animal, comprising administering to an animal in need of such therapy a short antisense compound 10 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, which are sugar-modified nucleotides that comprise a bridge between the 4′ and

the 2′

position of the sugar; and

wherein the short antisense compound is targeted to a nucleic acid encoding ApoB.