Peptide oligonucleotide conjugates

• US 9,161,948 B2
• Filed: 11/17/2011
• Issued: 10/20/2015
• Est. Priority Date: 05/05/2011
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Hanson, Gunnar J.
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US13/299,310
• Publication Number:
  US 20120289457A1
• Time in Patent Office: 1,433 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/455, 435/6.1, 435/375, 435/458, 514/1, 514/2, 514/44, 514/3.1, 514/3.7, 514/17.7, 514/20.9, 530/300, 530/322, 536/23.1, 536/24.5
• US Class Current: 1/1
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• 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 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Oligonucleotide analogues conjugated to carrier peptides are provided. The disclosed compounds are useful for the treatment of various diseases, for example diseases where inhibition of protein expression or correction of aberrant mRNA splice products produces beneficial therapeutic effects.

Claims

1. A conjugate comprising:

(a) a carrier peptide comprising amino acid subunits, the carrier peptide comprising a glycine (G) amino acid subunit at the carboxy terminus of the carrier peptide;

(b) a nucleic acid analogue comprising a substantially uncharged backbone and a targeting base sequence for sequence-specific binding to a target nucleic acid; and

(c) a covalent attachment between the nucleic acid analog and the carrier peptide, the covalent attachment comprising the carboxy-terminal glycine and an optional linker group;

wherein;

two or more of the amino acid subunits are positively charged amino acids, no more than seven contiguous amino acid subunits are arginine, and the covalent attachment between the nucleic acid analog and the carrier peptide is not 6-aminohexanoic acid or β

-alanine.

2. The conjugate of claim 1, wherein the carrier peptide comprises from 4 to 40 amino acid subunits.

3. The conjugate of claim 1, wherein the carrier peptide comprises from 6 to 20 amino acid subunits.

4. The conjugate of claim 1, wherein the positively charged amino acids are histidine (H), lysine (K), arginine (R) or combinations thereof.

5. The conjugate of claim 1, wherein at least one of the positively charged amino acids is arginine.

6. The conjugate of claim 1, wherein each of the amino acid subunits, except the carboxy terminal glycine, are positively charged amino acids.

7. The conjugate of claim 1, wherein each of the positively charged amino acids is arginine.

8. The conjugate of claim 1, wherein at least one of the positively charged amino acids is an arginine analog, the arginine analog being a cationic α

amino acid comprising a side chain of the structure R^aN═

C(NH₂)R^b, where R^a is H or R^c;

R^b is R^c, NH₂, NHR, or N(R^c)₂, where R^c is lower alkyl or lower alkenyl and optionally comprises oxygen or nitrogen or R^a and R^b may together form a ring; and

wherein the side chain is linked to the amino acid via R^a or R^b.

9. The conjugate of claim 1, wherein at least 20% of the amino acid subunits are positively charged amino acids.

10. The conjugate of claim 1, wherein at least 50% of the amino acid subunits are positively charged amino acids.

11. The conjugate of claim 1, wherein at least 80% of the amino acid subunits are positively charged amino acids.

12. The conjugate of claim 1, wherein the carrier peptide comprises the sequence (R^d)_m, wherein R^d is independently, at each occurrence, a positively charged amino acid and m is an integer ranging from 2 to 12.

13. The conjugate of claim 12, wherein R^d is independently, at each occurrence, arginine, histidine, or lysine.

14. The conjugate of claim 12, wherein each R^d is arginine.

15. The conjugate of claim 1, wherein each amino acid subunit, except the carboxy terminal glycine, is arginine.

16. The conjugate of claim 1, wherein the carrier peptide comprises at least one hydrophobic amino acid, the hydrophobic amino acid comprising a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain wherein the alkyl, alkenyl and alkynyl side chain includes at most one heteroatom for every six carbon atoms.

17. The conjugate of claim 16, wherein the carrier peptide comprises two or more hydrophobic amino acids.

18. The conjugate of claim 16, wherein the carrier peptide comprises two or more contiguous hydrophobic amino acids.

19. The conjugate of claim 16, wherein at least one of the hydrophobic amino acids is phenylalanine.

20. The conjugate of claim 16, wherein each of the hydrophobic amino acids are phenylalanine.

21. The conjugate of claim 1, wherein the carrier peptide comprises at least one neutral amino acid having the following structure (Y^b):

—

NH—

(CHR^e)_n—

C(O)—

 

 

(Y^b)

wherein n is 2 to 7 and each R^e is independently, at each occurrence, hydrogen or methyl.

22. The conjugate of claim 21, wherein the carrier peptide comprises the sequence [(R^dY^bR^d)_x(R^dR^dY^b)_y]_z, wherein R^d is independently, at each occurrence, a positively charged amino acid and x and y are independently, at each occurrence, 0 or 1, provided that x +y is 1 or 2 and z is an integer ranging from 1 to 6.

23. The conjugate of claim 22, wherein R^d is independently, at each occurrence, arginine, histidine or lysine.

24. The conjugate of claim 22, wherein each R^d is arginine.

25. The conjugate of claim 22, wherein at least one Y^b is 6aminohexanoic acid or β

-alanine.

26. The conjugate of claim 1, wherein the carrier peptide comprises the sequence ILFQY (SEQ ID NO:

576).

27. The conjugate of claim 1, wherein the carrier peptide comprises the sequence ILFQ (SEQ ID NO:

577), IWFQ (SEQ ID NO;

     578) or ILIQ (SEQ ID NO;

     579).

28. The conjugate of claim 1, wherein the carrier peptide comprises the sequence PPMWS (SEQ ID NO:

580), PPMWT (SEQ ID NO;

     581), PPMFS (SEQ ID NO;

     582) or PPMYS (SEQ ID NO;

     583).

29. The conjugate of claim 1, wherein, in addition to the covalent linkage, the carrier peptide comprises at least one of alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.

30. The conjugate of claim 1, wherein the carrier peptide comprises an acetyl, benzoyl or stearoyl moiety at the amino terminus of the carrier peptide.

31. The conjugate of claim 1, wherein each of the amino acid subunits are natural amino acids.

32. The conjugate of claim 1, wherein the uncharged backbone comprises a sequence of morpholino ring structures joined by intersubunit linkages, the intersubunit linkages joining a 3′

-end of one morpholino ring structure to a 5′

-end of an adjacent morpholino ring structure, wherein each morpholino ring structure is bound to a base-pairing moiety, such that the oligomer can bind in a sequence-specific manner to the target nucleic acid, wherein the intersubunit linkages have the following general structure (I);

33. The conjugate of claim 32, wherein at least one of the morpholino ring structures has the following structure (i):

34. The conjugate of claim 32, wherein at least one of the intersubunit linkages is linkage (A).

35. The conjugate of claim 32, wherein each of the intersubunit linkages is linkage (A).

36. The conjugate of claim 32, wherein each linkage (A) has the same structure at each occurrence.

37. The conjugate of claim 32, wherein for at least one occurrence of linkage (A), X is —

N(CH₃)₂.

38. The conjugate of claim 32, wherein X is —

N(CH₃)₂ at each occurrence of linkage (A).

39. The conjugate of claim 32, wherein each linkage is linkage (A) and X is —

N(CH₃)₂.

40. The conjugate of claim 32, wherein for at least one occurrence of linkage (A), X has the following structure:

41. The conjugate of claim 32, wherein for at least one occurrence of linkage (B), X has the following structure:

42. The conjugate of claim 32, wherein for at least one occurrence of linkage (B), X has the following structure:

43. The conjugate of claim 32, wherein W and Y are each O at each occurrence.

44. The conjugate of claim 32, wherein at least 5% of the intersubunit linkages are linkage (B).

45. The conjugate of claim 32, wherein each linkage (B) has the same structure at each occurrence.

46. The conjugate of claim 32, wherein the conjugate has the following structure (XVII):

47. The conjugate of claim 46, wherein R¹⁸ is the carrier peptide.

48. The conjugate of claim 46, wherein R¹⁹ is the carrier peptide.

49. The conjugate of claim 46, wherein at least one of R¹⁸ or R¹⁹ is R²⁰.

50. The conjugate of claim 46, wherein R¹⁹ is —

C(═

O)OR²¹.

51. The conjugate of claim 50, wherein R¹⁹ is

52. The conjugate of claim 46, wherein L¹ comprises phosphorodiamidate and piperazine bonds.

53. The conjugate of claim 52, wherein L¹ has the following structure (XXIX):

54. The conjugate of claim 53, wherein R²⁴ is absent.

55. The conjugate of claim 46, wherein R¹⁸ is the carrier peptide, L² is a direct bond, and the carrier peptide forms a bond between the carboxy terminus of the carrier peptide and the 3′

terminal nitrogen of the nucleic acid analogue.

56. A composition comprising the conjugate of claim 1 and a pharmaceutically acceptable vehicle.

57. A method of treating a disease in a subject, the method comprising administering a therapeutically effective amount of the conjugate of claim 1 to the subject.

58. The method of claim 57, wherein the disease is a viral infection, a neuromuscular disease, a bacterial infection, inflammation or polycystic kidney disease.

59. The method of claim 58, wherein the viral infection is influenza.

60. The method of claim 58, wherein the neuromuscular disease is Duchenne muscular dystrophy.

61. The conjugate of claim 1, wherein the covalent attachment includes the optional linker.

62. The conjugate of claim 1, wherein the glycine amino acid subunit is covalently linked to the nucleic acid analogue at the 5′

end of the nucleic acid analogue.

63. The conjugate of claim 1, wherein the glycine amino acid subunit is covalently linked to the nucleic acid analogue at the 3′

end of the nucleic acid analogue.

64. The conjugate of claim 46, wherein the carrier peptide is selected from SEQ ID NOS:

60, 69, 70, 89-121, 125, 130-160, 162-257, 276, 277, 281-288, 293-297, 300, 302-412, 419-552, and 554-566.

65. The conjugate of claim 46, wherein the carrier peptide is selected from SEQ ID NOS:

130, 157-160, 251, 256, 386-388, and 540.

66. The conjugate of claim 46, wherein the carrier peptide is SEQ ID NO:

159.

67. The conjugate of claim 1, wherein the carrier peptide is of a formula selected from:

68. The conjugate of claim 67, wherein the carrier peptide is of the formula:

69. The conjugate of claim 1, wherein the conjugate is selected from

70. The conjugate of claim 69, wherein the carrier peptide is selected from SEQ ID NOS:

130, 157-160, 251, 256, 386-388, and 540.

71. The conjugate of claim 69, wherein the carrier peptide is SEQ ID NO:

159.

72. The conjugate of claim 1, wherein the conjugate is selected from:

73. The conjugate of claim 72, wherein the carrier peptide is selected from SEQ ID NOS:

130, 157-160, 251, 256, 386-388, and 540.

74. The conjugate of claim 72, wherein the carrier is SEQ ID NO:

159.

75. The conjugate of claim 72, wherein each Pi is independently selected from adenine, cytosine, guanine, uracil, thymine, and inosine.

76. A compound selected from:

77. The compound of claim 76, wherein each Pi is independently selected from adenine, cytosine, guanine, uracil, thymine, and inosine.

78. The compound of claim 76, wherein the compound is:

79. The compound of claim 78, wherein R is H.

80. The compound of claim 78, wherein R is acetyl.

81. The compound of claim 78, wherein each Pi is independently selected from adenine, cytosine, guanine, uracil, thymine, and inosine.