ENA nucleic acid drugs modifying splicing in mRNA precursor

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 -CH 2 CH 2 OH (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 -CH 2 CH 2 OH (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 -CH 2 CH 2 OH (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 -CH 2 CH 2 OH (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 -CH 2 CH 2 OH (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-CH 2 CH 2 OH (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-CH 2 CH 2 OH (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-CH 2 CH 2 OH (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-CH 2 CH 2 OH (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):

where X is individually and independently a group represented by formula (X1) or (X2):

Y is individually and independently a hydrogen atom, a hydroxyl group or an alkoxy group with 1-6 carbon atoms; and Z is individually and independently a single bond or an alkylene group with 1-5 carbon atoms.

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**-CH 2 CH 2 OH (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**-CH 2 CH 2 OH (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*-CH 2 CH 2 OH (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**-CH 2 CH 2 OH (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**-CH 2 CH 2 OH (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):

where X is individually and independently a group represented by formula (X1) or (X2); R 1 is individually and independently an alkyl group with 1-6 carbon atoms; and Z is individually and independently a single bond or an alkylene group with 1-5 carbon atoms:

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:

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); Bt** is a group represented by formula (T2); and in individual formulas, at least one of Bg*, Ba*, Bc*, Bt*, Bg**, Ba**, Bc** and Bt** has a group represented by formula (X2) as X and all of have a group represented by (X1) as X.

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.