Poly-substituted-phenyl-oligoribo nucleotides having enhanced stability and membrane permeability and methods of use

• US 5,858,988 A
• Filed: 02/22/1996
• Issued: 01/12/1999
• Est. Priority Date: 02/24/1993
• Status: Expired due to Fees

• Current Assignee: DNP Pharmaceuticals, Inc.
• Original Assignee: Jui H. Wang
• Inventors: Wang, Jui H.
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/604,871
• Time in Patent Office: 1,055 Days
• Field of Search: 435/6, 435/375, 435/325, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.5, 536/25.1, 514/44, 935/33, 935/34
• US Class Current: 514/44.00A
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

In accordance with the present invention, antisense oligonucleotides are provided with enhanced membrane permeability and stability. This is accomplished in accordance with the invention through conjugating oligoribonucleotides with a hydrophobic carrier agent at the 2'"'"'-O position of the oligonucleotides. The hydrophobic carrier agent comprises a compound of the following general structure: ##STR1## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphothionate, or phosphothioate.

In a preferred embodiment, R¹ and R³ are NO₂. In such embodiment, it will be appreciated that when R², R⁴, and R⁵ are H, the compound is DNP and when R⁴ is F, the compound is FDNP.

Claims

1. An antisense oligoribonucleotide conjugated at the 2'"'"'-O position with a substituted phenyl group to produce a derivatized compound of the following general structure:

##STR18## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphorothioate, or phosphothioate.

2. The oligoribonucleotide of claim 1, wherein R¹ and R³ are NO₂ as follows:

##STR19##

3. The oligoribonucleotide of claim 2, wherein R², R⁴, and R⁵ are H.

4. The oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

5. The oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

6. The oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

7. The oligoribonucleotide of claim 2, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

8. The oligoribonucleotide of claim 2, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

9. The oligoribonucleotide of claim 2, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

10. The oligoribonucleotide of claim 3, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

11. The oligoribonucleotide of claim 3, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

12. The oligoribonucleotide of claim 3, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

13. The antisense oligoribonucleotide of claim 1, wherein at least one of the 5'"'"'-OH and 3'"'"'-OH ends of the oligoribonucleotide is conjugated with a substituted phenyl group at a 2'"'"'-O ribose position to produce a derivatized compound of the following general structure:

##STR20## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphorothioate, or phosphothioate.

14. The oligoribonucleotide of claim 13, wherein R¹ and R³ are NO₂.

15. The oligoribonucleotide of claim 14, wherein R², R⁴, and R⁵ are H.

57. The oligonucleotide of any of claims 1-56, wherein the oligonucleotide is an antisense oligonucleotide.

16. An antisense oligoribonucleotide derivatized at a plurality of 2'"'"'-O positions with a hydrophobic group selected from the group consisting of a 2,4-dinitrophenyl group and a 3-fluoro-4,6-dinitrophenyl group.

17. The oligoribonucleotide of claim 16, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

18. The oligoribonucleotide of claim 16, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

19. The oligoribonucleotide of claim 16, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

20. A method of enhancing membrane permeability and stability of an oligoribonucleotide, comprising:

providing an oligoribonucleotide having a plurality of 2'"'"'-O positions;

conjugating a substituted phenyl group at said plurality of 2'"'"'-O ribose positions to produce a derivatized compound of the following general structure;

##STR21## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphorothioate, or phosphorothioate.

21. The method claim 20, wherein R¹ and R³ are NO₂ as follows:

##STR22##

22. The method claim 21, wherein R², R⁴, and R⁵ are H.

23. The method of claim 20, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

24. The method of claim 20, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

25. The method of claim 20, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

26. The method of claim 21, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

27. The method of claim 21, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

28. The method of claim 21, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

29. The method of claim 22, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

30. The method of claim 22, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

31. The method of claim 22, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

32. The method of claim 20, wherein at least one of the 5'"'"'-OH and 3'"'"'-OH ends of the oligoribonucleotide is conjugated with a substituted phenyl group at a 2'"'"'-O ribose position to produce a derivatized compound of the following general structure:

##STR23## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphorothioate, or phosphorothioate.

33. The method of claim 32, wherein R¹ and R³ are NO₂.

34. The method of claim 33, wherein R², R⁴, and R⁵ are H.

35. In an antisense therapeutic method, comprising administering an antisense oligonucleotide comprising a plurality of 2'"'"'-O positions in a manner designed inhibit gene expression, the improvement comprising:

derivatizing the antisense oligonucleotide at a plurality of the 2'"'"'-O positions with a substituted phenyl group to produce a derivatized compound of the following structure;

##STR24## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphothionate, or phosphorothioate.

36. In the improvement of claim 35, wherein R¹ and R³ are NO₂ as follows:

##STR25##

37. In the improvement of claim 36, wherein R², R⁴, and R⁵ are H.

38. In the improvement of claim 35, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

39. In the improvement of claim 35, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

40. In the improvement of claim 35, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

41. In the improvement of claim 36, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

42. In the improvement of claim 36, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

43. In the improvement of claim 36, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

44. In the improvement of claim 37, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

45. In the improvement of claim 37, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

46. In the improvement of claim 37, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

47. In the improvement of claim 35, wherein at least one of the 5'"'"'-OH and 3'"'"'-OH ends of the oligoribonucleotide is conjugated to a substituted phenyl group at a 2'"'"'-O ribose position to produce a derivatized with a compound of the following general structure:

##STR26## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphothionate, or phosphorothioate.

48. In the improvement of claim 47, wherein R¹ and R³ are NO₂.

49. In the improvement of claim 48, wherein R², R⁴, and R⁵ are H.

50. In a method for administering an oligoribonucleotide, wherein the oligoribonucleotide comprises a plurality of 2'"'"'-O positions in a manner designed to inhibit gene expression the improvement comprising:

derivatizing the O at a plurality of the 2'"'"'-O positions with a moiety selected from the group consisting of a 2,4-dinitrophenyl moiety and a 3-fluoro-4,6-dinitrophenyl moiety.

51. In the improvement of claim 50, wherein the oligoribonucleotide has a length of between 10 and 40 nucleotides.

52. In the improvement of claim 50, wherein the oligoribonucleotide has a length of between 12 and 30 nucleotides.

53. In the improvement of claim 50, wherein the oligoribonucleotide has a length of between 15 and 25 nucleotides.

54. In the improvement of claim 50, wherein at least one of the 5'"'"'-OH and 3'"'"'-OH ends of the oligoribonucleotide is conjugated with a substituted phenyl group at a 2'"'"'-O ribose position to produce a derivatized compound of the following general structure:

##STR27## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphorothioate, or phosphorothioate.

55. In the improvement of claim 50, wherein R¹ and R³ are NO₂.

56. In the improvement of claim 50, wherein R², R⁴, and R⁵ are H.

58. A method of derivatizing an oligoribonucleotide, comprising:

providing an oligoribonucleotide having a plurality of 2'"'"'-O positions;

conjugating a substituted phenyl group at a plurality of 2'"'"'-O positions to produce a derivatized compound of the following general structure;

##STR28## wherein R¹, R², R³, R⁴, and R⁵ are independently H, NO₂, halide, linear or branched alkyl, linear or branched acyl, linear or branched alkylene, linear or branched O-alkyl, linear or branched amido, linear or branched S-alkyl, mono or disubstituted amine, linear or branched thioamido, phosphothionate, or phosphorothioate.

59. The method of claim 58, wherein R¹ and R³ are NO₂.

60. The method of claim 58, wherein R², R⁴, and R⁵ are H.

61. A method of derivatizing an oligoribonucleotide, comprising:

providing an oligoribonucleotide having a plurality of 2'"'"'-O positions;

reacting said oligoribonucleotide with a Sanger-type reagent at a plurality of 2'"'"'-O positions to produce a derivatized oligoribonucleotide of the following general structure;

##STR29##

62. The method of claim 61, wherein R¹ and R³ are NO₂.

63. The method of claim 61, wherein R², R⁴, and R⁵ are H.