Oligonucleotides comprising a C5-modified pyrimidine

• US 20050288244A1
• Filed: 04/29/2005
• Published: 12/29/2005
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Granted Patent: US 7,674,778 B2
• Time in Patent Office: Days
• US Class Current: 514/44.00A
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• 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 33/00 : Antiparasitic agents
• CPC Class Codes: A61P 33/04 : Amoebicides
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• 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: C07H 21/02 : with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 : General methods applicable to biologically active non-coding nucleic acids
• 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 15/1135 : against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3515 : Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 : chemically synthesised

Abstract

One aspect of the present invention relates to a double-stranded oligonucleotide comprising at least one ligand. In certain embodiments, a ligand is bound to only one of the two oligonucleotide strands comprising the double-stranded oligonucleotide. In certain embodiments, both of the oligonucleotide strands of the double-stranded oligonucleotide independently comprise a bound ligand. In certain embodiments, the oligonucleotide strands comprise at least one modified sugar moiety. In certain embodiments, a phosphate linkage in one or both of the strands of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. Another aspect of the present invention relates to a single-stranded oligonucleotide comprising at least one ligand. In certain embodiments, the oligonucleotide comprises at least one modified sugar moiety. In certain embodiments, a phosphate linkage of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. The ligand improves the pharmacokinetic properties of the oligonucleotide.

Claims

1. A single-stranded oligonucleotide represented by formula I:

2. The single-stranded oligonucleotide of claim 1, wherein n is 18, 19, 20, 21, or 22.

3. The single-stranded oligonucleotide of claim 1, wherein n is 20.

4. The single-stranded oligonucleotide of claim 1, wherein A⁵ occurs at least twice.

5. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence compound C, compound D, or a radical of cholesterol, 5β

-cholanic acid, progesterone, aldosterone, dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol, dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone, testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol, corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon, equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol, mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene, fuisidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol, dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone, hydrocortisone, parametasone, betametasone, cortisone, fluocinonide, fluorometholone, halcinonide, budesonide, or any one of them further substituted with one or more of hydroxyl, halogen, amino, alkylamino, alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

6. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence a radical of cholesterol or 5β

-cholanic acid.

7. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence:

10. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence a radical of cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, or chenodeoxycholic acid.

11. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence a radical of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, triacylglycerol, phosphoacylglycerol, sphingolipid, monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, or tetraterpene.

12. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence optionally substituted phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl, phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl, quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl, benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl, pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl, tetrazolyl, or furanyl.

13. The single-stranded oligonucleotide of claim 1, wherein A⁵ represents independently for each occurrence the radical of erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, or talose;

or a disaccharide or trisaccharide formed via a 1,4 glycoside linkage between any of them.

14. The single-stranded oligonucleotide of claim 1, wherein A⁵ is a hexose or pentose.

15. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

16. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

17. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

18. The single-stranded oligonucleotide of claim 1, wherein A⁵ is an optionally substituted cyclopentane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, or 1,1-difluorocyclopentane.

19. The single-stranded oligonucleotide of claim 1, wherein A⁵ is compound C.

20. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

21. The single-stranded oligonucleotide of claim 1, wherein A⁵ is compound D.

22. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

23. The single-stranded oligonucleotide of claim 1, wherein A⁵ is a radical of a polycyclic compound.

24. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

25. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

26. The single-stranded oligonucleotide of claim 1, wherein A⁵ is

27. A double-stranded oligonucleotide comprising a first strand and a second strand, wherein said first strand and said second strand are represented independently by formula II:

28. The double-stranded oligonucleotide of claim 27, wherein n is 18, 19, 20, 21, or 22.

29. The double-stranded oligonucleotide of claim 27, wherein n is 20.

30. The double-stranded oligonucleotide of claim 27, wherein n is 20, and said first strand and said second strand are hybridized so that there are two unhybridized nucleotides on said first strand and said second strand.

31. The double-stranded oligonucleotide of claim 27, wherein n is 20 for said first strand, and n is 22 for said second strand.

32. The double-stranded oligonucleotide of claim 27, wherein A⁵ occurs at least twice.

33. The double-stranded oligonucleotide of claim 27, wherein A⁵ occurs only in said first strand.

34. The double-stranded oligonucleotide of claim 27, wherein said first strand and said second strand each contain at least one occurrence of A⁵.

35. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence compound C, compound D, or a radical of cholesterol, 5β

-cholanic acid, progesterone, aldosterone, dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol, dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone, testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol, corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon, equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol, mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene, fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol, dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone, hydrocortisone, parametasone, betametasone, cortisone, fluocinonide, fluorometholone, halcinonide, budesonide, or any one of them further substituted with one or more of hydroxyl, halogen, amino, alkylamino, alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

36. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence a radical of cholesterol or 5β

-cholanic acid.

37. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence:

38. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence a radical of cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, or chenodeoxycholic acid.

39. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence a radical of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, triacylglycerol, phosphoacylglycerol, sphingolipid, monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, or tetraterpene.

40. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence optionally substituted phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl, phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl, quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl, benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl, pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl, tetrazolyl, or furanyl.

41. The double-stranded oligonucleotide of claim 27, wherein A⁵ represents independently for each occurrence the radical of erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, or talose;

or a disaccharide or trisaccharide formed via a 1,4 glycoside linkage between any of them.

42. The double-stranded oligonucleotide of claim 27, wherein A⁵ is a hexose or pentose.

43. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

44. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

45. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

46. The double-stranded oligonucleotide of claim 27, wherein A⁵ is an optionally substituted cyclopentane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, or 1,1-difluorocyclopentane.

47. The double-stranded oligonucleotide of claim 27, wherein A⁵ is compound C.

48. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

49. The double-stranded oligonucleotide of claim 27, wherein A⁵ is compound D.

50. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

51. The double-stranded oligonucleotide of claim 27, wherein A⁵ is a radical of a polycyclic compound.

52. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

53. The double-stranded oligonucleotide of claim 27, wherein A⁵ is

54. The double-stranded oligonucleotide of claim 27, wherein A⁵ is