CARBOXYLATED 2'-AMINO-LNA NUCLEOTIDES AND OLIGONUCLEOTIDES COMPRISING THE SAME

Abstract

The invention proves an oligonucleotide comprising one or more carboxylated 2-amino-LNA nucleotide units. The invention also provides a method of transfecting cells with the oligonucleotide, a method of treating a human or animal by therapy using the oligonucleotide, and a pharmaceutical composition comprising the oligonucleotide.

Claims

1. Carboxylated 2 amino LNA nucleotides having the general formula ##STR00017## wherein B is a nucleobase, L1 is an internucleoside linkage or absent and R is a carboxylated substituent comprising one or two carboxyl groups and wherein R is comprising from 6 to 40 carbon atoms and optionally from 1 to 3 heteroatoms selected from O, N, and S.

2. The carboxylated 2 amino LNA nucleotides of claim 1, wherein R has from 6 to 20, preferably from 8 to 16 carbon atoms.

3. The carboxylated 2 amino LNA nucleotides of claim 1 or 2, wherein the nucleobases are selected from the group of adenine, guanine, thymine, cytosine, uracil (resulting in uridine or pseudouridine), 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouracil, xanthine, hypoxanthine or 7-methylguanine and wherein further the internucleoside linkages are selected from phosphodiester, phosphorothioate or phosphotriester linkages.

4. Carboxylated 2 amino LNA nucleotide having one of the following structures (2) to (7), wherein Y is oxygen or absent thereby describing the occurrence of a methylene group at this position and ##STR00018## ##STR00019##

5. Oligonucleotide having between 3 and 100 nucleotides, wherein one or more of the carboxylated 2 amino LNA nucleotides of any of the claims 1 to 4 are present.

6. Oligonucleotide of claim 5 having between 10 and 30 nucleotides, further characterized in being essentially single stranded. Oligonucleotide of claim 5 or 6, wherein the nucleotides are linked by phosphodiester, phosphorothioate or phosphotriester linkages.

8. Oligonucleotides of any of the claims 5 to 7, further comprising one or more hydrophobic nucleotides.

9. Oligonucleotide of claim 8, wherein the one or more hydrophobic nucleotides are independently selected from the group of 2 amino LNA comprising a substituent RH and wherein RH is a hydrophobic moiety comprising from 8 to 40 carbon atoms and optionally from 1 to 3 heteroatoms selected from O, N, and S; 2-N-C.sub.3-C.sub.7-alkyl-amino-LNA nucleotides; 2-N-C.sub.3-C.sub.7-acyl-amino-LNA nucleotides; 2-O-C.sub.3-C.sub.26-alkyl-RNA nucleotides; 2-O-C.sub.6-C.sub.26-aryl-RNA nucleotides, 2-O-C.sub.1-C.sub.6-alkyloxy-C.sub.3-C.sub.26-alkyl-RNA nucleotides; or 2-O-C.sub.1-C.sub.6-alkyloxy-C.sub.6-C.sub.26-aryl-RNA nucleotides.

10. Oligonucleotide of claim 9, wherein the 2 amino LNA comprising a substituent RH are selected from the structures (11) to (16), wherein B and L1 are defined as in claim 1, Y is oxygen or absent thereby describing the occurrence of a methylene group at this position, and ##STR00020## ##STR00021##

11. Oligonucleotide of any of the claims 5 to 7, wherein less than 40%, preferably less than 30% of the total number of monomer units in the oligonucleotide are carboxylated 2 amino LNA nucleotides.

12. Oligonucleotide of the claims 8 to 11, wherein the combined portion of hydrophobic and carboxylated 2 amino LNA nucleotides does not exceed 40% of all nucleotides of the oligonucleotide, preferably said portion does not exceed 30% and the combined number of hydrophobic and carboxylated 2 amino LNA nucleotides is at least 2.

13. Oligonucleotide of any of the claims 5 to 12 having a sequence that is essentially identical or essentially complementary to a sequence found in a genome.

14. Oligonucleotide of claim 13, further characterized in that the carboxyalated 2 amino nucleotides and hydrophobic nucleotides are located close to the 5 and 3 ends of the oligonucleotide, preferably in between positions 1 to 5 from the 5 end of the oligonucleotide and/or positions t to (t-4) from its 3-terminal end, wherein t designates the number of the 3-terminal nucleotide counted from the 5-terminal nucleotide unit of the oligonucleotide.

15. An oligonucleotide comprising from 3 to 100 nucleotide units, comprising one or more carboxylated 2-amino LNA nucleotide units of the following general formula (1) ##STR00022## wherein B is a nucleobase, each of L1 is an internucleoside linkage, a bond to a neighboring nucleotide unit or a terminal moiety selected from the group consisting of OH, phosphate or a linker for the conjugation of the oligonucleotide to another molecule, and R is a carboxylated substituent comprising one or two carboxyl group(s) and wherein R comprises from 6 to 40 carbon atoms and optionally from 1 to 3 further heteroatoms selected from O, N, and S.

16. The oligonucleotide according to claim 15, wherein said oligonucleotide comprises from 5 to 50 nucleotide units and is double stranded or from 10 to 30 nucleotide units and is single stranded.

17. The oligonucleotide according to claim 15 or 16, wherein R has from 6 to 20, preferably from 8 to 16 carbon atoms.

18. The oligonucleotide according to any one of claims 15 to 17, wherein said carboxylated substituent is a carboxyhydrocarbyl group or a carboxyheterohydrocarbyl group, the heterohydrocarbyl moiety of which comprises from 1 to 3 heteroatoms selected from O, N, and S.

19. The oligonucleotide according to any one of claims 15 to 18, wherein said carboxylated substituent is a carboxyalkyl or a carboxyaryl substituent.

20. The oligonucleotide according to any one of claims 15 to 19, wherein the internucleoside linkage is selected from phosphodiester, phosphorothioate and/or phosphotriester linkages.

21. The oligonucleotide according to any one of claims 15 to 20, wherein less than 40%, preferably less than 30%, more preferably less than 20% of the total number of nucleotide units in the oligonucleotide are carboxylated 2-amino LNA nucleotide units of the general formulas (1).

22. The oligonucleotide according to any one of claims 15 to 20, wherein said oligonucleotide contains at most 8, preferably at most 6, more preferably at most 4, even more preferably at most 3 or even more preferably 2 of said carboxylated 2-amino LNA nucleotide units of the general formulas (1).

23. The oligonucleotide according to any one of claims 15 to 22, wherein said oligonucleotide has, from the 5-end to the 3-end, three segments: a 5-end segment of at least 2 nucleotide units, a central binding segment of at least 6 nucleotide units in length, and a 3-end segment of at least 2 nucleotide units in length, and wherein said oligonucleotide contains at least two, preferably two, carboxylated 2-amino-LNA nucleotide units in either the 5-end segment or the 3-end segment but none in the central segment; or contains at least one, preferably one, carboxylated 2-amino-LNA nucleotide unit in each of said end segments, but none in the central segment.

24. The oligonucleotide according to any one of claims 15 to 23, comprising 0, 1 or more hydrophobic nucleotide units that are independently selected from the group consisting of 2 amino LNA nucleotide moieties carrying on said 2-amino group a (hetero)hydrocarbyl substituent comprising from 8 to 40 carbon atoms and optionally from 1 to 3 heteroatoms selected from O, N, and S; 2-N-C.sub.3-C.sub.7-alkyl-amino-LNA nucleotide moieties; 2-N-C.sub.3-C.sub.7-alkyl-amido-LNA nucleotide moieties; 2-O-C.sub.3-C.sub.26-alkyl-RNA nucleotide moieties; 2-O-C.sub.6-C.sub.16-aryl-RNA nucleotide moieties, 2-O-C.sub.1-C.sub.6-alkyloxy-C.sub.3-C.sub.26-alkyl-RNA nucleotide moieties; and 2-O-C.sub.1-C.sub.6-alkyloxy-C.sub.6-C.sub.26-aryl-RNA nucleotide moieties.

25. The oligonucleotide according to claim 24, wherein less than 30%, preferably less than 20% of the total number of nucleotide monomer units in the oligonucleotide are hydrophobic nucleotide units as defined in claim 24.

26. The oligonucleotide according to claim 15, wherein the carboxylated 2-amino LNA nucleotide unit of formula (1) has, within the oligonucleotide, the following formula (1) or (1): ##STR00023##

27. A method of transfecting cells with the oligonucleotide as defined in any one of claims 1 to 26, comprising mixing said oligonucleotide with an aqueous buffer and treating said cells with the mixture obtained in the previous step.

28. A method of treating a human or animal by therapy, comprising administering the oligonucleotide as defined in any one of claims 1 to 26 in a suitable carrier to an animal or human.

29. A method of transfecting cells with the oligonucleotide as defined in any one of claims 1 to 26, comprising mixing said oligonucleotide with an aqueous buffer and treating said cells with the mixture obtained in the previous step.

30. A method of treating a human or animal by therapy, comprising administering the oligonucleotide as defined in any one of claims 1 to 26 in a suitable carrier to an animal or human.

31. A pharmaceutical composition comprising the oligonucleotide of any of claims 1 to 26 and a pharmaceutically acceptable diluent, carrier or adjuvant.

32. The oligonucleotide of any of claims 1 to 26 for use as a medicament.

Description

DESCRIPTION OF THE FIGURES

[0155] FIG. 1: Compounds obtained during the synthesis of the examples and used for the preparation of the modified oligonucleotides

[0156] FIG. 2: The dose response curves for oligonucleotides comprising one, two or three of the carboxylated 2 amido LNA modifications. FIG. 2 demonstrates the inhibition of cell growth using oligonucleotides targeting the Eg5 gene, whereas FIG. 2B shows the data obtained using control, non-targeting oligonucleotides comprising the exact same modifications.

EXAMPLES

[0157] The following examples illustrate, but not limit the invention:

Example 1Preparation of a Carboxyalkvlated 2 Amino LNA

[0158] 1 g of the 5 DMTO protected 2amino LNA thymidine 1 was brought in 40 mL dichloromethane and 0.4 mL acetic acid were added; the mixture was placed on ice. 498 mg of 16 oxo-hexadecanoic acid methyl ester were dissolved in 20 mL dichloromethane and given to the amino LNA. 275 mg sodium cyanoborohydride in 10 mL methanol were added dropwise to the solution. The ice was removed and the components were allowed to react overnight at room temperature. The carboxyalkylated 2 amino LNA was isolated using ethyl acetate:brine and chromatographed on silica using petrol ether:ethyl acetate as solvent. 1.2 g of 2 were obtained.

Example 2Phosphoamidate of the Carboxyalkvlated 2 Amino LNA

[0159] 0.95g of 2 were dissolved in 20 ml absolute dichloromethane and placed on ice. 585 mg of dry N,N-Diisopropylethylamine were added followed by 2 portions of 401 mg of 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (3-([Chloro(diisopropylamino)phosphanyl]oxy)-propanenitrile) in 5 mL dichloromethane each. Total reaction time was 24 h at room temperature. The organic phase was extracted two time with saturated sodium hydrogen carbonate and purified over silica using ethyl acetate as solvent. 800 mg of the phosphoamidate 3 were isolated.

Example 3Oligonucleotides Comprising Carboxyalkylated 2 Amino LNA

[0160] Gapmer antisense oligonucleotides were synthesized with the following sequences and modification patterns (noted in 5-3 direction):

TABLE-US-00003 TABLE 3 List of certain oligonucleotides targeting the Eg5 gene. Seq Nucle- ID otide T C C A A G T G C T A C T G T A DNA d d d d d d d d d d LNA b b b b b b special special c16 c16 special a16 a16 special a16 a16 Back- bone 1 s Tbs Cbs Cbs dAs dAs dGs dTs dGs dCs dTs dAs dCs Tbs Gbs Tbs dA 2 s Tbc16s Cbs Cbs dAs dAs dGs dTs dGs dCs dTs dAs dCs Tbs Gbs Tbc16s dA 3 s Tba16s Cbs Cbs dAs dAs dGs dTs dGs dCs dTs dAs dCs Tbs Gbs Tba16s dA 4 Tb Cb Cb dA dA dG dT dG dC dT dA dC Tb Gb Tb dA 5 Tbc16 Cb Cb dA dA dG dT dG dC dT dA dC Tb Gb Tbc16 dA 6 Tba16 Cb Cb dA dA dG dT dG dC dT dA dC Tb Gb Tba16 dA

TABLE-US-00004 TABLE 4 List of certain control oligonucleotides not targeting any human or mouse gene. Seq Nucle- ID otide T C C A A G T G C T A C T G T A G T A DNA d d d d d d d d d d LNA b b b b b b b b special special c16 c16 special a16 a16 special a16 a16 Back- bone 7 s Tbs Cbs Cbs Abs dAs dGs dTs dGs dCs dTs dAs dCs dTs dGs Tbs Abs Gbs Tbs dA 8 s Tbc16s Cbs Cbs Abs dAs dGs dTs dGs dCs dTs dAs dCs dTs dGs Tbs Abs Gbs Tbc16s dA 9 s Tba16s Cbs Cbs Abs dAs dGs dTs dGs dCs dTs dAs dCs dTs dGs Tbs Abs Gbs Tba16s dA 10 Tb Cb Cb Ab dA dG dT dG dC dT dA dC dT dG Tb Ab Gb Tb dA 11 Tbc16 Cb Cb Ab dA dG dT dG dC dT dA dC dT dG Tb Ab Gb Tbc16 dA 12 Tba16 Cb Cb Ab dA dG dT dG dC dT dA dC dT dG Tb Ab Gb Tba16 dA

[0161] Symbols

[0162] A,C,G,T denote nucleobases

[0163] d before nucleobase=DNA

[0164] nucleobase followed by b=LNA (a conventional 2,4-methoxyribose-LNA)

[0165] c16 denotes the carboxylated 2 amino LNA from example 1

[0166] a16 denotes a palmitoylated 2 amino LNA

[0167] s stands for phosphorothioate internucleoside linkage

[0168] where s is absent, the internucleoside linkage is a phosphodiester.

[0169] The oligonucleotides having the sequence ID1 to 12 were synthesized on an Expedite 8909 synthesized using the RNA coupling protocol of the supplier at a 1 mol scale. At the end of the synthesis cycle, oligonucleotides were cleaved from their support using 0.5M sodium hydroxide in methanol/water (5:1) for 30 min at room temperature. The material was further incubated for 10 hours at room temperature to afford deprotection and cleavage of the methyl ester from the carboxylated 2 amino LNA. The solution was desalted using NAP10 columns and lyophilized and the material was purified using anion exchange.

Example 4Preparation of Carboxyalkylated 2 Amido LNA

[0170] Hexadecanedioic acid monomethyl ester was prepared and activated as NHS ester. 25 g hexadecanedioic acid dimethyl ester were dissolved in 250 mL methanol and 25 g barium hydroxide octahydrate in 200 mL methanol were added dropwise. Upon completion 500 mL ice water, 200 mL 1N HCl were added and extracted using ethylacetate. The organic phase was reduced to a small volume and hexadecanedioic acid monomethyl ester was crystallized in the cold, filtered and dried. 1 g of the material was dissolved in 25 mL THF and 0.46 g N-Hydroxysuccinimide and 0.89 g N,N-Dicyclohexylcarbodiimide were added. The reaction mixture was stirred overnight, reduced under vacuum, heated under reflux upon addition of 50 mL hexane and 5 mL ethylacetate and filtered while hot. The filtrate was reduced under vaccum and the NHS ester of hexadecanedioic acid monomethyl ester precipitated in the cold.

[0171] The conjugate 4 of hexadecanedioic acid monomethyl ester with the 2 amido LNA thymidine 1 was prepared from equimolar components. 0.45 g 1 and 0.313 g of the NHS ester of hexadecanedioic acid monomethyl ester were dissolved in 10 mL THF. 0.11mL trimethylamine was added and the mixture was stirred at 40 C. for 2 days. The conjugate 4 was purified from the reaction mixture on silica using ethylacetate and methanol as solvents. Early eluting material was re-purified on silica to 90% purity. The C11 homologue 6 was prepared on the same route.

Example 5Phosphoramidates of the Carboxyalkylated 2 Amido LNA

[0172] 0.35 g of 4 or 6 were dissolved in 10 ml absolute dichloromethane and placed on ice. 212mg of dry N,N-diisopropylethylamine were added followed by 145 mg of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (3-([Chloro(diisopropylamino)phosphanyl]oxy)-propanenitrile) in 5 mL dichloromethane. Total reaction time was 24 h at room temperature. The organic phase was extracted two time with saturated sodium hydrogen carbonate and purified over silica using ethyl acetate as solvent. 300 mg of the phosphoramidate 5 were isolated using 4 as a precursor. 315 mg of the phosphoramidate 7 were isolated using 6 as a precursor.

Example 6A series of Oligonucleotides Comprising Carboxyalkylated 2 Amino LNA or 2 Amido LNA

[0173] Gapmer antisense oligonucleotides targeting the Eg5 gene or respective mismatch controls were synthesized. The parent sequence was taken from Koller et al. (2006) in Cancer Res 66 (4): 2059-2066.

[0174] The C16 carboxyalkylated LNA amine 3 or the C16 carboxyalkylated LNA Amide 5 were inserted into the sequence as indicated in the table below. In addition, a C16 alkylated LNA Amide 8 was used, synthesis described in I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768. The sequences and modification patterns (noted in 5-3 direction) are as follows:

TABLE-US-00005 TABLE 5 List of certain oligonucleotides targeting the Eg5 gene. Eg5 on-target T C C A A G T G C T A C T G T A G T A DNA d d d d d d d d d d d LNA b b b b b b b b Group SeqID Modification Parent 16/38 Amide C 47 C 5 17/35 CC 5 5 17/37 CCC 5 5 5 Amine C 51 C-C 3 3 53 C-CC 3 3 3 Amine C/ 59 A-C 8 3 Amide A 55 A-CC 8 3 3 mixed 61 A-AC 8 8 3 63 C-AC 3 8 3

[0175] Symbols A,C,G,T in the first line denote the nucleobases, numbers 3,5 or 8 denote the modified 2amino or 2 amido LNA compounds used for the synthesis of the oligonucleotide. Within the modification patterns C denotes a carboxyalkylated 2 amino LNA or a carboxyalkylated 2 amido LNA and A denotes an alkylated 2 amino LNA.

[0176] For comparison, a mutated, non-targeting sequences was used. The C16 carboxyalkylated LNA amine 3 or the C16 carboxyalkylated LNA Amide 5 were inserted into the non-targeting sequence as indicated in the table below. In addition, a C16 alkylated LNA Amide 8 was used, synthesis described in I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768. The sequences and modification patterns (noted in 5-3 direction) are as follows:

TABLE-US-00006 TABLE 6 List of certain control oligonucleotides not targeting any human or mouse gene. Mismatch Controls T A C G C C T T C T A T T C T C A T G Seq ID DNA d d d d d d d d d d d LNA b b b b b b b b Group Seq ID Modification Control 16/39 Amide C 46 C 5 17/36 CC 5 5 17/38 CCC 5 5 5 Amine C 52 C-C 3 3 54 C-CC 3 3 3 Amine C/ 60 A-C 8 3 Amide A 56 A-CC 8 3 3 mixed 62 A-AC 8 8 3 64 C-AC 3 8 3

[0177] Symbols A,C,G,T in the first line denote the nucleobases, numbers 3,5 or 8 denote the modified 2amino or 2 amido LNA compounds used for the synthesis of the oligonucleotide. Within the modification patterns C denotes a carboxyalkylated 2 amino LNA or a carboxyalkylated 2 amido LNA and A denotes an alkylated 2 amino LNA.

Example 7Transfection of Cells

[0178] HeLa cells were grown in RPMI plus 10% FCS using 96 well microplates. Cells were seeded at a density of 3000 cells per well in 100 l medium, grown over night and transfected with the oligonucleotides as follows: oligonucleotides were dissolved at 100 M in 20 mM NaCl. Serial dilutions of this concentrated stock were prepared using Opti-MEM as diluent and transfection complexes were formed using lipofectamine 3000. 10 l of said complexes were added to the cells to arrive at final concentration between 10 pM and 250 nM. The cells were incubated at 37 C., 5% CO2 and 100% humidity for 72 hours and cell viability was determined at the end of this period using a Cell Titer Blue cell vailbility assay (Promega, USA). A low cell viability can result from a specific inhibition of the Eg5 gene and resulting cell cycle arrest, it can also be caused by unspecific toxicity of the compounds or of the transfectant. To differentiate between the two, parallel experiments were performed using the Eg5 on-target compounds and their respective mismatch controls.

Example 8Efficacy and Specificity of Oligonucleotides Comprising a Carboxylated 2 Amido LNA

[0179] Oligonucleotides comprising carboxylated 2 amido LNA have a much improved efficacy and specificity. See FIG. 2. When compared to the parent compound 16/38, the oligonucleotide carrying a single C16 carboxylated 2 amido LNA (SeqID 47) was about 10 fold more potent. Introduction of a second and third modification in Seq ID 17/35 and 17/37 gave oligonucleotides having 20 to 70fold better potency. The modified oligonucleotides also feature an improved specificity, as demonstrated in table 7.

TABLE-US-00007 TABLE 7 Potency and specificity of oligonucleotides comprising carboxyalkylated 2 amido LNA. EC50 Seq EC50 Group Modification SeqID in nM ID in nM specificity Parent 16/38 7.6 16/39 23 3 Amide C C 47 0.85 46 11 13 CC 17/35 0.1 17/36 11 115 CCC 17/37 0.3 17/38 18 60

Example 9Efficacy and Specificity of Oligonucleotides Comprising a Carboxylated 2 Amino LNA

[0180] Oligonucleotides comprising carboxylated 2 amino LNA feature improved efficacy and specificity. When compared to the parent compound 16/38, the oligonucleotide carrying a double C16 carboxylated 2 amino LNA (SeqID 51) was about 10 fold more potent. Introduction of a third modification in Seq ID 53 gave oligonucleotides having a potency similar to the parent compound, but much improved specificity.

TABLE-US-00008 TABLE 8 Potency and specificity of oligonucleotides comprising carboxyalkylated 2 amino LNA. EC50 Seq EC50 Group Modification SeqID in nM ID in nM specificity Parent 16/38 1.5 16/39 23 5 Amine C CC 51 0.2 52 25 151 CCC 53 7.5 54 470 62

Example 10Efficacy and Specificity of Oligonucleotides Comprising a Carboxylated 2 amino LNA in Combination with Alkylated 2 Amido LNA

[0181] This example compares the potency and specificity of oligonucleotides comprising carboxylated 2 amino LNA in combination with alkylated 2 amido LNA, specifically the palmitoylated 2 amido LNA described in I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768.

[0182] For oligonucleotides comprising two modified position such as Seg ID 51 or 59, a replacement of a carboxylated with an alkylated 2 LNA resulted in somewhat reduced potency, but a stronger loss of specificity.

[0183] For oligonucleotides comprising three modified position such as Seg ID 53 or the group of 55, 63, 61 a replacement of a single carboxylated with an alkylated 2 LNA resulted in a slightly reduced potency and specificity. A second replacement rendered the compound inactive.

TABLE-US-00009 TABLE 9 Potency and specificity of oligonucleotides comprising carboxyalkylated 2 amino LNA and mixed modifications of carboxyalkylated 2 amino LNA and alkylated 2 amido LNA. Modification SeqID EC50 in nM Seq ID EC50 in nM specificity CC 51 0.3 52 10.6 35 A-C 59 1.1 60 3.8 4 CCC 53 1.3 54 89 70 A-CC 55 1.7 56 20.2 12 C-AC 63 2.7 64 28 10 A-AC 61 >250 62 >250