ENA nucleic acid drugs modifying splicing in mRNA precursor

• US 7,902,160 B2
• Filed: 11/21/2003
• Issued: 03/08/2011
• Est. Priority Date: 11/25/2002
• Status: Active Grant

• Current Assignee: Daiichi Sankyo Company Limited
• Original Assignee: KNC Laboratories Co Ltd, Masafumi Matsuo, Nonprofit Organization Translational Research Organization Of Duchenne Muscular Dystrophy, Yasuhiro Takeshima
• Inventors: Matsuo, Masafumi, Koizumi, Makoto, Takeshima, Yasuhiro
• Primary Examiner(s): Schnizer; Richard
• Application Number:
  US10/536,258
• Publication Number:
  US 20070082861A1
• Time in Patent Office: 2,664 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Oligonucleotides having a nucleotide sequence complementary to nucleotide numbers such as 2571-2607, 2578-2592, 2571-2592, 2573-2592, 2578-2596, 2578-2601 or 2575-2592 of the dystrophin cDNA (Gene Bank accession No. NM_004006.1) and therapeutic agents for muscular dystrophy comprising such oligonucleotides.

Claims

1. A 10-20mer antisense oligonucleotide compound having the nucleotide sequence of SEQ ID NO:

14, SEQ ID NO;

15, SEQ ID NO;

16, or SEQ ID NO;

18, or a pharmacologically acceptable salt thereof.

2. The compound according to claim 1, which is represented by any one of the formulae selected from the group consisting of (AO33), (AO85), (AO88), (AO86), and (AO2), or a pharmacologically acceptable salt thereof:

HO-G^e2p-C^e2p-C^e2p-G^e2p-C^e2p-U^mp-G^mp-C^mp-C^mp-C^mp-A^e2p-A^e2p-T^e2p-G^e2p-C^e2p-CH₂CH₂OH (AO33) (SEQ ID NO;

     14)

HO-C^e2p-G^mp-C^e2p-T^e2p-G^mp-C^mp-C^e2p-C^e2p-A^mp-A^mp-T^e2p-G^mp-C^e2p-C^e2p-A^mp-A^mp-C^e2p-C^e2p-CH₂CH₂OH (AO85) (SEQ ID NO;

     15)

HO-C^e2s-G^ms-C^e2s-T^e2s-G^ms-C^ms-C^e2s-C^e2s-A^ms-A^ms-T^e2s-G^ms-C^e2s-C^e2s-A^ms-U^ms-C^e2s-C^e2s-CH₂CH₂OH (AO88) (SEQ ID NO;

     15)

HO-C^e2p-A^mp-G^mp-T^e2p-T^e2p-U^mp-G^mp-C^e2p-C^e2p-G^mp-C^e2p-T^e2p-G^mp-C^e2p-C^e2p-C^e2p-A^mp-A^mp-CH₂CH₂OH (AO86) (SEQ ID NO;

     16) and

HO-G^e2p-C^e2p-T^e2p-T^e2p-T^e2p-U^mp-C^mp-U^mp-U^mp-U^mp-U^mp-A^mp-G^mp-U^mp-U^mp-G^e2p-C^e2p-T^e2p-G^e2p-C^e2p-CH₂CH₂OH (AO2) (SEQ ID NO;

     18),

wherein A^e1p, G^e1p, C^e1p, T^e1p, A^e2p, G^e2p, C^e2p, T^e2p, A^mp, G^mp, C^mp, U^mp, A^e1s, G^e1s, C^e1s, T^e1s, A^e2s, G^e2s, C^e2s, T^e2s, A^ms, U^ms, C^ms, and U^ms are groups having the following structures, respectively;

3. The compound according to claim 1 or a pharmacologically acceptable salt thereof, having the nucleotide sequence of SEQ ID NO:

15.

4. The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein at least one of the sugars and/or the phosphates constituting the oligonucleotide is modified.

5. The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose and the modification of the sugar is modification of the hydroxyl group at position 2′

of D-ribofuranose.

6. The compound according to claim 5 or a pharmacologically acceptable salt thereof, wherein the modification of the sugar is 2′

-O-alkylation and/or 2′

-O,4′

-C-alkylenation of the D-ribofuranose.

7. The compound according to claim 4 or a pharmacologically acceptable salt thereof, wherein the modification of the phosphate is thioation of the phosphate group.

8. The antisense oligonucleotide compound according to claim 1 which is selected from the group consisting of compounds (v′

), (vi′

), (vii′

), (ix′

), or a pharmacologically acceptable salt thereof;

(v′

) a compound represented by formula (v′

);

HO-Bg-Bc-Bc-Bg-Bc-Bt-Bg-Bc-Bc-Bc-Ba-Ba-Bt-Bg-Bc-CH₂CH₂OH (v′

);

SEQ ID NO;

14

(vi′

) a compound represented by formula (vi′

);

HO-Bc-Bg-Bc-Bt-Bg-Bc-Bc-Bc-Ba-Ba-Bt-Bg-Bc-Bc-Ba-Bt-Bc-Bc-CH₂CH₂OH (vi′

);

SEQ ID NO;

15

(vii′

) a compound represented by formula (vii′

);

HO-Bc-Ba-Bg-Bt-Bt-Bt-Bg-Bc-Bc-Bg-Bc-B t-Bg-Bc-Bc-Bc-Ba-Ba-CH₂CH₂OH (vii′

);

SEQ ID NO;

16

(ix′

) a compound represented by formula (1x′

);

HO-Bg-Bc-Bt-Bt-Bt-Bt-Bc-Bt-Bt-Bt-Bt-Ba-Bg-Bt-Bt-Bg-Bc-Bt-Bg-Bc-CH₂CH₂OH (ix′

);

SEQ ID NO;

18

where Bg is a group represented by formula (G1) or (G2);

Ba is a group represented by formula (A1) or (A2);

Bc is a group represented by formula (C1) or (C2); and

Bt is a group represented by formula (U1) or (T2);

9. The antisense oligonucleotide compound according to claim 1 which is represented by any one of compounds selected from the group consisting of (I′

8), (I′

9), (I′

10), (I′

12), and (I′

13) or a pharmacologically acceptable salt thereof;

HO-Bg**-Bc**-Bc**-Bg**-Bc**-Bt*-Bg*-Bc*-Bc*-Bc*-Ba**-Ba **-Bt**-Bg**-Bc**-CH₂CH₂OH (I′

8);

SEQ ID NO;

14

HO-Bc**-Bg*-Bc**-Bt**-Bg*-Bc*-Bc**-Bc**-Ba*-Ba*-Bt**-Bg *-Bc**-Bc**-Ba*-Bt*-Bc**-Bc**-CH₂CH₂OH (I′

9);

SEQ ID NO;

15

HO-Bc**-Ba*-Bg*-Bt**-Bt**-Bt*-Bg*-Bc**-Bc**-Bg*-Bc**-Bt **-Bg*-Bc**-Bc**-Bc**-Ba*-Ba*-CH₂CH₂OH (I′

10);

SEQ ID NO;

16

HO-Bc**-Bg*-Bc**-Bt**-Bg*-Bc*-Bc**-Bc**-Ba*-Ba*-Bt**-Bg *-Bc**-Bc**-Ba*-Bt*-Bc**-Bc**-CH₂CH₂OH (I′

12);

SEQ ID NO;

15

HO-Bg**-Bc**-Bt**-Bt**-Bt**-Bt*-Bc*-Bt*-Bt*-Bt*-Bt*-Ba*-Bg*-Bt*-Bt*-Bg**-Bc**-Bt**-Bg**-Bc**-CH₂CH₂OH (I′

13);

SEQ ID NO;

18

where Bg* is a group represented by formula (G1^a);

Ba* is a group represented by formula (A1^a);

Bc* is a group represented by formula (C1^a);

Bt* is a group represented by formula (U1^a);

Bg** is a group represented by formula (G2);

Ba** is a group represented by formula (A2);

Bc** is a group represented by formula (C2); and

Bt** is a group represented by formula (T2);

10. The compound according to claim 9 which is represented by any one of formulae selected from the group consisting of (I′

8-a), (I′

9-a), (I′

10-a), (I′

12-a), and (I′

13-a) or a pharmacologically acceptable salt thereof;

11. The compound according to claim 8 where X in formulae (G1), (A1), (C1) and (U1) is a group represented by formula (X2) and X in formulae (G2), (A2), (C2) and (T2) is a group represented by formula (X1), or a pharmacologically acceptable salt thereof.

12. The compound according to claim 8 where X in all the formulae (G1), (A1), (C1), (U1), (G2), (A2), (C2) and (T2) is a group represented by formula (X2), or a pharmacologically acceptable salt thereof.

13. The compound according to claim 8 where Y in formulae (G1), (A1), (C1) and (U1) is a methoxy group and Z in formulae (G2), (A2), (C2) and (T2) is an ethylene group, or a pharmacologically acceptable salt thereof.

14. The compound according to claim 9 where X in formulas (G1^a), (A1^a), (C1^a) and (U1^a) is a group represented by formula (X2) and X in formulae (G2), (A2), (C2) and (T2) is a group represented by formula (X1), or a pharmacologically acceptable salt thereof.

15. The compound according to claim 9 where X in all the formulae (G1^a), (A1^a), (C1^a), (U1^a), (G2), (A2), (C2) and (T2) is a group represented by formula (X2), or a pharmacologically acceptable salt thereof.

16. The compound according to claim 9 where Y in formulae (G1), (A1), (C1) and (U1) is a methoxy group and Z in formulae (G2), (A2), (C2) and (T2) is an ethylene group, or a pharmacologically acceptable salt thereof.

17. The compound according to claim 10 where X in formulas (G1^a), (A1^a), (C1^a) and (U1^a) is a group represented by formula (X2) and X in formulae (G2), (A2), (C2) and (T2) is a group represented by formula (X1), or a pharmacologically acceptable salt thereof.

18. The compound according to claim 10 where X in all the formulae (G1^a), (A1^a), (C1^a), (U1^a), (G2), (A2), (C2) and (T2) is a group represented by formula (X2), or a pharmacologically acceptable salt thereof.

19. The compound according to claim 10 where Y in formulae (G1), (A 1), (C1) and (U1) is a methoxy group and Z in formulae (G2), (A2), (C2) and (T2) is an ethylene group, or a pharmacologically acceptable salt thereof.

20. A method of inducing a skipping of exon 45 or 46 of an RNA encoded by a dystrophin gene in a patient in need thereof, said method comprising administering by intravenous and/or intramuscular delivery to the patient an effective amount of a pharmaceutical composition comprising the compound of claim 1 or a pharmacologically acceptable salt thereof.

21. The method according to claim 20, wherein the total number of the amino acids enclosed in the open reading frame of the dystrophin gene in the patient will be a multiple of 3 when exon 45 of the dystrophin gene has been skipped.

22. The method according to claim 20, wherein said composition comprises a pharmacologically acceptable excipient or diluent.

23. The method according to claim 20, wherein said composition comprises 0.03-0.09 M of pharmacologically acceptable neutral salt.

24. The method according to claim 23, wherein said neutral salt is selected from the group consisting of sodium chloride, potassium chloride, and calcium chloride.

25. The method according to claim 20, wherein said composition comprises 0.002-0.05 M of pharmacologically acceptable buffer.

26. The method according to claim 25, wherein said pharmacologically acceptable buffer is selected from the group consisting of sodium citrate, sodium glycinate, sodium phosphate and tris(hydroxymethyl)aminomethane, or mixtures thereof.

27. The method according to claim 20, wherein said effective amount ranges from 0.1 to 100 mg/kg body weight per day.

28. The method according to claim 27, wherein said effective amount is administered in a single daily dosage.

29. The method according to claim 27, wherein said effective amount is administered in divided dosages.

30. The method according to claim 20, wherein said effective amount ranges from 1 to 50 mg/kg body weight per day.

31. The method according to claim 30, wherein said effective amount is administered in a single daily dosage.

32. The method according to claim 30, wherein said effective amount is administered in divided dosages.

33. The compound according to claim 1 or a pharmacologically acceptable salt thereof, having the nucleotide sequence of SEQ ID NO:

14.

34. The compound according to claim 1 or a pharmacologically acceptable salt thereof, having the nucleotide sequence of SEQ ID NO:

16.

35. The compound according to claim 1 or a pharmacologically acceptable salt thereof, having the nucleotide sequence of SEQ ID NO:

18.

36. The compound according to claim 9 or a pharmacologically acceptable salt thereof, wherein at least one of the sugars and/or the phosphates constituting the oligonucleotide is modified.

37. The compound according to claim 36 or a pharmacologically acceptable salt thereof, wherein the modification of the phosphate is thioation of the phosphate group.

38. The compound according to claim 9 or a pharmacologically acceptable salt thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose and the modification of the sugar is modification of the hydroxyl group at position 2′

of D-ribofuranose.

39. The compound according to claim 38 or a pharmacologically acceptable salt thereof, wherein the modification of the sugar is 2′

-O-alkylation and/or 2′

-O,4′

-C-alkylenation of the D-ribofuranose.

40. A method of inducing a skipping of exon 45 or 46 of an RNA encoded by a dystrophin gene in a patient in need thereof, said method comprising administering by intravenous and/or intramuscular delivery to the patient an effective amount of a pharmaceutical composition comprising the compound of claim 9 or a pharmacologically acceptable salt thereof.

41. The method according to claim 40, wherein the total number of the amino acids encoded in the open reading frame of the dystrophin gene in the patient will be a multiple of 3 when exon 45 of the dystrophin gene has been skipped.

42. The method according to claim 40, wherein said composition comprises a pharmacologically acceptable excipient or diluent.

43. The method according to claim 40, wherein said composition comprises 0.03-0.09 M of pharmacologically acceptable neutral salt.

44. The method according to claim 43, wherein said neutral salt is selected from the group consisting of sodium chloride, potassium chloride, and calcium chloride.

45. The method according to claim 40, wherein said composition comprises 0.002-0.05 M of pharmacologically acceptable buffer.

46. The method according to claim 45, wherein said pharmacologically acceptable buffer is selected from the group consisting of sodium citrate, sodium glycinate, sodium phosphate and tris(hydroxymethyl)aminomethane, or mixtures thereof.

47. The method according to claim 40, wherein said effective amount ranges from 0.1 to 100 mg/kg body weight per day.

48. The method according to claim 47, wherein said effective amount is administered in a single daily dosage.

49. The method according to claim 47, wherein said effective amount is administered in divided dosages.

50. The method according to claim 40, wherein said effective amount ranges from 1 to 50 mg/kg body weight per day.

51. The method according to claim 50, wherein said effective amount is administered in a single daily dosage.

52. The method according to claim 50, wherein said effective amount is administered in divided dosages.