Oligonucleotides and related compounds

• US 7,846,436 B2
• Filed: 01/30/2004
• Issued: 12/07/2010
• Est. Priority Date: 11/28/2003
• Status: Active Grant

• Current Assignee: Chemgenes Corporation
• Original Assignee: Chemgenes Corporation, Kwok-Hung Sit
• Inventors: Bajpai, Satya K., Sit, Kwok-Hung, Srivastava, Suresh C.
• Primary Examiner(s): Fetterolf; Brandon J
• Application Number:
  US10/768,996
• Publication Number:
  US 20050159375A1
• Time in Patent Office: 2,503 Days
• Field of Search: 424/133.1
• US Class Current: 424/133.1
• 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: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present invention relates generally to oligonucleotides and more specifically to oligonucleotides which have a sequence including at least two CpG dinucleotides and a prodrug of an antimetabolite. The prodrug can be part of a CpG dinucleotide or may be attached elsewhere on the oligonucleotide.

Claims

1. A synthetic oligonucleotide for preferentially killing cancerous cells over noncancerous cells comprising at least two CpG moieties and a covalently linked nucleoside antimetabolite.

3. The oligonucleotide of claim 1 or 2, wherein two of said at least two CpG moieties are separated by a number of nucleotides selected from the group of numbers 2, 5, and 9.

4. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is 5′

to said at least two CpG moieties.

5. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is 3′

to said at least two CpG moieties.

6. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is 3′

to at least one of said at least two CpG moieties and 5′

to at least a second of said at least two CpG moieties.

7. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is linked by a 3′

-3′

linkage.

8. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is linked by a 5′

-5′

linkage.

9. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is linked by a 3′

-5′

linkage.

10. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is linked by a 5′

-3′

linkage.

11. The oligonucleotide of claim 1 or 2, wherein said nucleoside antimetabolite is linked at a position that is selected from the following positions:

10 nucleotides upstream from one of the said at least two CpG moieties, 9 nucleotides upstream from one of the said at least two CpG moieties, 8 nucleotides upstream from one of the said at least two CpG moieties, 7 nucleotides upstream from one of the said at least two CpG moieties, 6 nucleotides upstream from one of the said at least two CpG moieties, 5 nucleotides upstream from one of the said at least two CpG moieties, 4 nucleotides upstream from one of the said at least two CpG moieties, 3 nucleotides upstream from one of the said at least two CpG moieties, 2 nucleotides upstream from one of the said at least two CpG moieties, 1 nucleotides upstream from one of the said at least two CpG moieties, 10 nucleotides downstream from CpG moieties, 9 nucleotides downstream from one of the said at least two CpG moieties, 8 nucleotides downstream from one of the said at least two CpG moieties, 7 nucleotides downstream from one of the said at least two CpG moieties, 6 nucleotides downstream from one of the said at least two CpG moieties, 5 nucleotides downstream from one of the said at least two CpG moieties, 4 nucleotides downstream from one of the said at least two CpG moieties, 3 nucleotides downstream from one of the said at least two CpG moieties, 2 nucleotides downstream from one of the said at least two CpG moieties, and 1 nucleotides downstream from one of the said at least two CpG moieties.

12. The oligonucleotide of claim 1 or 2, wherein the nucleoside antimetabolite is linked by a linker having the formula

13. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one nucleotide having a ribose sugar moiety.

14. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one nucleotide having a 2′

-deoxyribose sugar moiety.

15. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one 2′

-halogen nucleotide.

16. The oligonucleotide of claim 1 or 2, comprising at least one 2′

-N-alkyl nucleotide, wherein the alkyl has between 1 and 6 carbon atoms.

17. The oligonucleotide of claim 1 or 2, comprising at least one 2′

-O-alkyl nucleotide, 2′

-n-alkyl nucleotide, or 2′

-O-halogen nucleotide, wherein an alkyl has between 1 and 6 carbon atoms.

18. The oligonucleotide of claim 17, wherein each alkyl is methyl.

19. The oligonucleotide of claim 1 or 2, comprising a plurality of nucleotides connected by covalent internucleotide linkages selected from the group consisting of phosphodiester linkage, C1-C6 alkoxy phosphotriester linkage, phosphorothioate linkage and phosphoramidate linkage.

20. A pharmaceutical composition comprising the oligonucleotide of any of claims 1 or 2-19.

21. A pharmaceutical composition of claim 20 further comprising a pharmaceutically acceptable carrier.

22. The oligonucleotide of claim 21 wherein said pharmaceutically acceptable carrier is lipofectin.

2. A synthetic oligonucleotide for preferentially killing cancerous cells over noncancerous cells comprising at least two CpG moieties and a covalently linked nucleoside antimetabolite, wherein, the antimetabolite is selected from the group consisting of 2′

-deoxy-3′

-thiacytidine, 3′

-azido-3′

-deoxythymidine, 2′

,3′

-dideoxycytidine, 2′

,3′

-didehydro-3′

-deoxythymidine, 2′

,3′

-dideoxyinosine, 5-fluoro-2′

-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′

-deoxycytidine, 6-mercaptopurineriboside, 2-chlorodeoxyadenosine, pentostatin and 2′

-deoxy, 2′

,2′

-difluorocytidine.

23. A synthetic oligonucleotide for preferentially killing cancerous cells over noncancerous cells comprising a motif represented by one of the group of formulas 5′

-PCGXCG-3′ and

5′

-CGXCGP-3′

, wherein P is a nucleoside antimetabolite and X represents between 0 and 50 nucleotides.

25. The oligonucleotide of claim of 23 or 24, where X is selected from the group consisting of 2, 5, and 9 nucleotides.

26. The oligonucleotide of claim 23 or 24, wherein X has at least one nucleotide and the nucleoside antimetabolite is covalently linked to one of the nucleotides by a 3′

-3′

linkage.

27. The oligonucleotide of claim 23 or 24, wherein X has at least one nucleotide and the nucleoside antimetabolite is covalently linked to one of the nucleotides by a 5′

-5′

linkage.

28. The oligonucleotide of claim 23 or 24, wherein X has at least one nucleotide and the nucleoside antimetabolite is covalently linked to one of the nucleotides by a 3′

-5′

linkage.

29. The oligonucleotide of claim 23 or 24, wherein X has at least one nucleotide and the nucleoside antimetabolite is covalently linked to one of the nucleotides by a 5′

-3′

linkage.

30. The oligonucleotide of claim 23 or 24, comprising at least one nucleotide having a ribose sugar moiety.

31. The oligonucleotide of claim 23 or 24, comprising at least one nucleotide having a 2′

-deoxyribose sugar moiety.

32. The oligonucleotide of claim 23 or 24, comprising at least one 2′

-O-alkyl nucleotide, 2′

-N-Alkyl nucleotide, or 2′

-O-halogen nucleotide, wherein an alkyl has between 1 and 6 carbon atoms.

33. The oligonucleotide of claim 23 or 24, comprising a plurality of nucleotides connected by covalent internucleoside linkages, wherein the linkages are selected from the group consisting of phosphodiester linkage, C1-C6 alkoxy phosphotriester linkage, phosphorothioate linkage and phosphoramidate linkage.

34. The oligonucleotide of claim 23 or 24, wherein X has at least one nucleotide and the nucleoside antimetabolite is attached to at least one of the multiple nucleotides by a linker having the formula

35. A pharmaceutical composition comprising the oligonucleotide of any of claims 23-34.

36. A pharmaceutical composition of claim 35 further comprising a pharmaceutically acceptable carrier.

37. The oligonucleotide of claim 36 wherein said pharmaceutically acceptable carrier is lipofectin.

24. A synthetic oligonucleotide for preferentially killing cancerous cells over noncancerous cells comprising a motif represented by one of the group of formulas 5′

-PCGXCG-3′ and

5′

-CGXCGP-3′

, and wherein X represents between 0 and 50 nucleotides and P is a nucleoside antimetabolite selected from the group consisting of 2′

-deoxy-3′

-thiacytidine, 3′

-azido-3′

-deoxythymidine, 2′

,3′

-dideoxycytidine, 2′

,3′

-didehydro-3′

-deoxythymidine, 2′

,3′

-dideoxyinosine, 5-fluoro-2′

-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′

-deoxycytidine, 6-mercaptopurineriboside, 2-chlorodeoxyadenosine, pentostatin and 2′

-deoxy, 2′

-,2′

-difluorocytidine.

38. A method of synthesizing an oligonucleotide product for preferentially killing cancerous cells over non-cancerous cells comprising the steps of:

(a) Selecting a oligonucleotide comprising at least two CpG moieties; and

(b) Covalently linking a nucleoside antimetabolite to said oligonucleotide comprising at least two CpG moieties.

40. The method of claim 38 or 39, wherein said oligonucleotide comprising at least two CpG moieties comprises between 2 and 50 nucleotides.

39. A method of synthesizing an oligonucleotide product for preferentially killing cancerous cells over non-cancerous cells comprising the steps of:

(a) Selecting a oligonucleotide comprising at least two CpG moieties; and

(b) Covalently linking a nucleoside antimetabolite to said oligonucleotide comprising at least two CpG moieties,

wherein, said antimetabolite is selected from the group consisting of 2′

-deoxy-3′

-thiacytidine, 3′

-azido-3′

-deoxythymidine, 2′

,3′

-dideoxycytidine, 2′

,3′

-didehydro-3′

-deoxythymidine, 2′

,3′

-dideoxyinosine, 5-fluoro-2′

-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′

-deoxycytidine, 6-mercaptopurineriboside, 2-chlorodeoxyadenosine, pentostatin and 2′

-deoxy, 2′

,2′

-difluorocytidine.