CpG Oligonucleotide Analogs Containing Hydrophobic T Analogs with Enhanced Immunostimulatory Activity

• US 20140163213A1
• Filed: 10/04/2013
• Published: 06/12/2014
• Est. Priority Date: 09/27/2006
• Status: Active Grant

• Current Assignee: Pfizer Inc.
• Original Assignee: Coley Pharmaceutical GmbH (Pfizer Inc.)
• Inventors: Debelak, Harald, Uhlmann, Eugen, Jurk, Marion
• Granted Patent: US 9,382,545 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 31/22 for herpes viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• 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
• CPC Class Codes: C12N 15/117 Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/17 Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/332 Abasic residue
• CPC Class Codes: C12N 2310/335 : Modified T or U

Abstract

The invention relates to oligonucleotides including at least one lipophilic substituted nucleotide analog and a pyrimidine-purine dinucleotide. The invention also relates to pharmaceutical compositions and methods of use thereof.

Claims

1. An oligonucleotide comprising:

the sequence R₁YZR₂ wherein R₁ and R₂ are selected from the group consisting of;

a lipophilic substituted nucleotide analog (L), a nucleotide, and a linkage, wherein at least one of R₁ and R₂ is a lipophilic substituted nucleotide analog (L), wherein Y is a pyrimidine nucleotide and wherein Z is a purine, a pyrimidine, or an abasic residue.

2. The oligonucleotide of claim 1, wherein L comprises a 5- or 6-membered ring nucleobase analog.

3. The oligonucleotide of claim 1, wherein L comprises a group of formula I

4. The oligonucleotide of claim 3, wherein n is 1.

5. The oligonucleotide of claim 3, wherein n is 0.

6. The oligonucleotide of claim 3, wherein all of the atoms A, B, X, D, E, F are carbon (C).

7. The oligonucleotide of claim 3, wherein one of the atoms A, B, X, D, E, F is nitrogen (N).

8. The oligonucleotide of claim 3, wherein two of the atoms A, B, X, D, E, F are nitrogen (N).

9. The oligonucleotide of claim 3, wherein three of the atoms A, B, X, D, E, F are nitrogen (N).

10. The oligonucleotide of claim 3, wherein at least one of the atoms A, B, X, D, E, F is substituted by a member of the group consisting of:

F, Cl, Br, I, alkyl, alkenyl, alkinyl, halogenated alkyl, halogenated alkenyl, cycloalkyl, O-alkyl, O-alkenyl, —

NH-alkyl, —

N(alkyl)₂;

—

S-alkyl, —

SO-alkyl, —

SO₂-alkyl, nitro, cyano, carboxylester, phenyl, thiophenyl, benzyl, oxo, thio, hydroxy, mercapto and imino, wherein at least one substituent is not oxo, thio, hydroxy, mercapto, imino, amino or methyl.

11. The oligonucleotide of claim 3, wherein one of the two atoms A or E is substituted by F, Cl, Br, I, C₂-C₆-alkyl, alkenyl, alkinyl, halogenated alkyl, halogenated alkenyl, cycloalkyl, O-alkyl, O-alkenyl, —

NH-alkyl, —

N(alkyl)₂;

—

S-alkyl, —

SO-alkyl, —

SO₂-alkyl, nitro, cyano, carboxylester, phenyl, thiophenyl, benzyl, or methyl, provided that if methyl then A, B, X, D, E, and F are all C.

12. The oligonucleotide of claim 3, wherein the group of formula I comprises a substituted pyrimidine, a substituted uracil, a substituted toluene, a substituted imidazole, a substituted pyrazole, or a substituted triazole.

17. The oligonucleotide of claim 3, wherein the group of formula I is 5-chloro-uracil, 5-bromo-uracil, 5-iodo-uracil, 5-ethyl-uracil, 5-propyl-uracil, 5-propinyl-uracil, (E)-5-(2-bromovinyl)-uracil, or 2.4-difluoro-toluene.

21. The oligonucleotide of claim 3, wherein the group of formula I is fused with a 3-to-6-membered aromatic or aliphatic ring system.

22. The oligonucleotide of claim 3, wherein the group of formula I is linked to a 5- to 6-membered sugar moiety.

23. The oligonucleotide of claim 22, wherein the 5- to 6-membered sugar moiety is a pentose or hexose.

24. The oligonucleotide of claim 23, wherein the pentose is a furanose and hexose is a pyranose, which can optionally be substituted by F, amino, alkoxy, alkoxy-ethoxy, aminopropyl, alkenyl, alkinyl, or a O2,C4-alkylene bridge.

25. The oligonucleotide of claim 23, wherein the furanose is ribose or deoxyribose.

31. The oligonucleotide of claim 1, comprising:

two 5′

R₁NR₁YZ 3′

motifs.

32. The oligonucleotide of claim 1, wherein Y comprises a pyrimidine selected from the group consisting of:

cytosine, 5-methyl-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-halogeno-cytosine, 2-thio-cytosine, 4-thio-cytosine, N3-methyl-cytosine, N4-alkyl-cytosine and a 6-substituted cytosine.

33. The oligonucleotide of claim 1, wherein Z is a purine nucleotide.

34. The oligonucleotide of claim 1, wherein Z comprises a purine selected from the group consisting of:

guanine, 7-deaza-guanine, hypoxanthine, 7-deaza-hypoxanthine, 2-amino-purine, 4-thio-purine, 2.6-diamino-purine, 8-oxo-7.8-dihydroguanine, 7-thia-8-oxo-7.8-dihydroguanine, 7-allyl-8-oxo-7,8-dihydroguanine, 7-deaza-8-aza-guanine, 8-aza-guanine and N1-methyl-guanine or purine.

35. The oligonucleotide of claim 1, wherein Z is a pyrimidine nucleotide.

36. The oligonucleotide of claim 1, wherein Z is T.

38. The oligonucleotide of claim 1, wherein the oligonucleotide is 3 nucleotides in length.

39. The oligonucleotide of claim 1, wherein the oligonucleotide is 3-6 nucleotides in length.

40. The oligonucleotide of claim 1, wherein the oligonucleotide is 3-100 nucleotides in length.

41. The oligonucleotide of claim 1, wherein the oligonucleotide is 7-100 nucleotides in length.

42. The oligonucleotide of claim 41, wherein the oligonucleotide is T-rich.

43. The oligonucleotide of claim 42, wherein the oligonucleotide includes at least 80% T.

44. The oligonucleotide of claim 1, comprising at least one palindromic sequence.

45. The oligonucleotide of claim 1, comprising two palindromic sequences.

46. The oligonucleotide of claim 44, comprising one to four unmethylated CG dinucleotides.

47. The oligonucleotide of claim 1, comprising at least one (G)_m sequence, wherein m is 4 to 10.

48. The oligonucleotide of claim 1, comprising at least one methylated CG dinucleotide.

49. The oligonucleotide of claim 1, wherein all CG dinucleotides are unmethylated.

50. The oligonucleotide of claim 1, comprising additionally a non-nucleotidic modification.

51. The oligonucleotide of claim 50, wherein the non-nucleotidic modification is selected from the group consisting of:

C₆-C₄₈-polyethyleneglycol, C₃-C₂₀-alkane-diol, C₃-C₁₈-alkylamino linker and C₃-C₁₈-alkylthiol linker.

52. The oligonucleotide of claim 50, wherein the non-nucleotidic modification is selected from the group consisting of:

cholesterol, bile acid, saturated or unsaturated fatty acid, and folate.

53. The oligonucleotide of claim 50, wherein the non-nucleotidic modification is a hexadecyl-glycerol or dihexadecyl-glycerol group.

54. The oligonucleotide of claim 50, wherein the non-nucleotidic modification is an octadecyl-glycerol or dioctadecyl-glycerol group.

55. The oligonucleotide of claim 50, wherein the non-nucleotidic modification is a vitamin E group.

56. The oligonucleotide of claim 1, further comprising a non-nucleotidic brancher moiety.

57. The oligonucleotide of claim 1, further comprising a nucleotidic brancher moiety.

58. The oligonucleotide of claim 1, further comprising a brancher moiety, wherein the oligonucleotides has at least two 5′

-ends.

59. The oligonucleotide of claim 1, wherein at least two nucleotides of the oligonucleotide have a stabilized linkage.

60. The oligonucleotide of claim 59, wherein the stabilized linkage is phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphonothioate boranophosphonate, phosphoramidate, or a dephospho linkage, either as enantiomeric mixture or as enantiomeric pure S- or R-configuration.

61. The oligonucleotide of claim 1, wherein the YZ of R₁YZR₂ has a phosphodiester linkage.

62. The oligonucleotide of claim 1, wherein the YZ of R₁YZR₂ has a phosphorothioate linkage.

63. The oligonucleotide of claim 1, wherein the R₁Y of R₁YZR₂ has a phosphorothioate linkage.

64. The oligonucleotide of claim 1, wherein the ZR₂ of R₁YZR₂ has a phosphorothioate linkage.

65. The oligonucleotide of claim 61, wherein all other nucleotides have a phosphorothioate linkage.

66. The oligonucleotide of claim 1, wherein the oligonucleotide is free of a microcarrier.

67. The oligonucleotide of claim 66, wherein the oligonucleotide is free of a lipid carrier.

68. The oligonucleotide of claim 1, wherein the oligonucleotide is an A class oligonucleotide.

69. The oligonucleotide of claim 1, wherein the oligonucleotide is a B class oligonucleotide.

70. The oligonucleotide of claim 69, wherein the B class oligonucleotide has the sequence 5′

TCN₁TX₁X₂CGX₃X₄ 3′

wherein X₁ is G or A X₂ is T, G, or A, X₃ is T or C and X₄ is T or C and N is any nucleotide and N₁ and N₂ are nucleic acid sequences composed of from about 0-25 N'"'"'s each.

71. The oligonucleotide of claim 1, wherein the oligonucleotide is a C class oligonucleotide.

72. The oligonucleotide of claim 1, wherein the oligonucleotide is a P class oligonucleotide.

73. The oligonucleotide of claim 1, wherein the oligonucleotide is a T class oligonucleotide.

74. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one 3′

-3′

linkage.

75. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one 5′

-5′

linkage.

13-16. -16. (canceled)

18-20. -20. (canceled)

26-30. -30. (canceled)

37. (canceled)

76-94. -94. (canceled)