Treatment

Abstract

To provide a method and a composition for delivering a nucleic acid to a cell or a cytosol of a target cell.SOLUTION: A method includes bringing a cell in contact with 1) a membrane-destabilizing polymer and 2) a nucleic acid complex. The nucleic acid complex includes an optionally selected linker and a target-directional ligand bonded to a nucleic acid.SELECTED DRAWING: None

Claims

1. A combination pharmaceutical for delivering nucleic acids to cells, comprising 1) a membrane destabilizing polymer and 2) a nucleic acid complex, The nucleic acid complex is given by formula (I): 【Chemistry 1】 [In the formula, R １ The targeting ligand has the following formula: 【Chemistry 2】 (In the formula, B １ teeth, 【Transformation 3】 A trivalent group selected from the group consisting of L １ , T １ , and T ２ It is covalently bonded to it; B ２ teeth, 【Chemistry 4】 is a trivalent group selected from the group consisting of, T １ , T ３ , and T ４ are covalently bonded to; B ３ teeth, 【Transformation 5】 A trivalent group selected from the group consisting of T ２ , T ５ , and T ６ It is covalently bonded to it; T １ and T ２ Each of these is either absent or a glycine linking group; T ３ , T ４ , T ５ , and T ６ Each of them is, 【Transformation 6】 (In the formula, n = 1, 2, or 3) Independently selected from the group consisting of; Each sugar is a monosaccharide; L １ A is a linking group, which independently is an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR Ｘ -, -NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S- may be optionally replaced, where R Ｘ is hydrogen or (C １ -C ６ ) is alkyl, and the hydrocarbon chain may be optionally substituted with one or more oxos (=O); Ring A is a 3- to 6-membered monocyclic heterocycloalkyl group that is either absent or contains one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; Each R Ａ is hydrogen, hydroxyl, -C １－２ Alkyl-OR Ｂ , and C １－４ Independently selected from the group consisting of alkyls; R Ｂ is hydrogen; n is 0, 1, 2, 3, or 4; L ２ A is a linking group, which independently is a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR Ｘ -, -NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S- may be optionally replaced, where R Ｘ is hydrogen or (C １ -C ６ ) is alkyl, and the hydrocarbon chain may be optionally substituted with one or more oxos (=O); R ２ [The nucleic acid is the nucleic acid, which is an oligonucleotide with a length of approximately 10 to approximately 100 nucleotides.] It is a compound or salt thereof, The aforementioned film-destabilizing polymer is given by formula (XX): 【Transformation 7】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M ２ is, (C ４ -C １８ ) is an alkyl-methacrylate residue; BMA is a butyl methacrylate residue; PAA is a propyl acrylic acid residue; DMAEMA is a dimethylaminoethyl methacrylate residue; m and n are molar ratios greater than 0, where m is greater than n and m + n = 1; q is a molar ratio between 0.2 and 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q + r + s = 1; v is between 1 and 25 kDa; w is between 1 and 25 kDa; The targeted portion is 【Transformation 8】 (In the formula, X is NR 3 and Y is selected from -(C=O)R 4, -SO 2R 5, and -(C=O)NR 6R 7, or X is -(C=O)- and Y is NR 8R 9, R3 is hydrogen or (C1-C4) alkyl, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and the (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. R10 is -OH, -NR8, R9, or -F. R11 is -OH, -NR8R9, -F, or a five-membered heterocycle, the five-membered heterocycle being optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. And; L is a connecting portion containing a polyethylene glycol (PEG) portion having 2 to 20 ethylene glycol units. The aforementioned combination drug is a polymer of the above.

2. The combination drug according to claim 1, wherein the nucleic acid complex and the membrane destabilizing polymer are administered separately.

3. The combination pharmaceutical according to claim 1, wherein the membrane destabilizing polymer is administered after the administration of the nucleic acid complex.

4. The combination pharmaceutical according to claim 1, wherein the nucleic acid complex and the membrane destabilizing polymer are included in a single composition and administered together.

5. A combination pharmaceutical formulation for administration by injection, according to any one of claims 1 to 4.

6. A combination pharmaceutical formulation for administration by subcutaneous injection, according to any one of claims 1 to 5.

7. The combination pharmaceutical according to any one of claims 1 to 6, wherein the cells are secretory cells, chondrocytes, epithelial cells, nerve cells, muscle cells, blood cells, endothelial cells, pericytes, fibroblasts, glial cells, or dendritic cells.

8. The combination pharmaceutical according to any one of claims 1 to 6, wherein the cells are cancer cells, immune cells, bacterial-infected cells, viral-infected cells, or cells having abnormal metabolic activity.

9. The combination pharmaceutical according to any one of claims 1 to 6, wherein the cells are hepatocytes.

10. The combination pharmaceutical according to any one of claims 1 to 6, wherein the targeted ligand specifically binds to a cell surface molecule selected from the group consisting of type 1 transferrin receptor, type 2 transferrin receptor, EGF receptor, HER2 / Neu, VEGF receptor, PDGF receptor, integrin, NGF receptor, CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, asialoglycoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), folate receptor, and sigma receptor.

11. The combination pharmaceutical according to any one of claims 1 to 10, wherein the nucleic acid is selected from the group consisting of low molecular weight interfering RNA (siRNA), dicer substrate dsRNA, low molecular weight hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplified RNA (sa-RNA), and combinations thereof.

12. The combination pharmaceutical according to any one of claims 1 to 11, wherein the nucleic acid is siRNA.

13. The aforementioned film-destabilizing polymer is defined by formula (XXI): 【Chemistry 9】 (In the formula, px is 11 or 12, and py is 4 or 5) A combination pharmaceutical according to any one of claims 1 to 12, wherein the polymer is...

14. The combination pharmaceutical according to any one of claims 1 to 13, wherein ring A is absent or is pyrrolidinyl.

15. Each R Ａ However, independently, hydroxy or C １－８ Alkyl, and the C １－８ The combination pharmaceutical according to any one of claims 1 to 14, wherein the alkyl group may be optionally substituted with a hydroxyl group.

16. The compound of formula (I) 【Chemistry 10】 (In the formula, Q is -L) １ -R １ And R' is C １－９ (It is alkyl.) A combination pharmaceutical according to any one of claims 1 to 14, selected from the group consisting of the following.

17. The compound of formula (I) 【Chemistry 11】 (In the formula, Q is -L) １ -R １ And R' is C １－９ (It is alkyl.) The combination pharmaceutical according to any one of claims 1 to 14 and 16.

18. L １ is -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO ２ )--via R １ A combination pharmaceutical according to any one of claims 1 to 17, which is connected to the

19. L １ but, 【Chemistry 12】 A combination pharmaceutical according to any one of claims 1 to 17, selected from the group consisting of the following.

20. L １ but, 【Chemistry 13】 A combination pharmaceutical according to any one of claims 1 to 17, selected from the group consisting of the following.

21. L ２ However, R via -O- ２ A combination pharmaceutical according to any one of claims 1 to 15, which is connected to the

22. L ２ However, -CH ２ O-, -CH ２ CH ２ O-, or -CH(OH)CH ２ A combination pharmaceutical according to any one of claims 1 to 15, wherein the compound is O-.

23. T １ and T ２ A combination pharmaceutical according to any one of claims 1 to 22, wherein neither of the above exists.

24. T １ and T ２ The combination pharmaceutical according to any one of claims 1 to 22, wherein at least one of the is glycine.

25. T １ and T ２ The combination pharmaceutical according to claim 24, wherein each of them is glycine.

26. B １ but, 【Chemistry 14】 The combination pharmaceutical according to any one of claims 1 to 25.

27. B ２ but, 【Chemistry 15】 A combination pharmaceutical according to any one of claims 1 to 26, selected from the group consisting of the following.

28. B ３ but, 【Chemistry 16】 A combination pharmaceutical according to any one of claims 1 to 27, selected from the group consisting of the following.

29. Each monosaccharide, 【Chemistry 17】 A combination pharmaceutical according to any one of claims 1 to 28, independently selected from the group consisting of the following.

30. Each monosaccharide, independently, [Chemistry 18] The combination pharmaceutical according to any one of claims 1 to 29.

31. The compound of formula (I) is given by the following formula: 【Chemistry 19】 A combination pharmaceutical according to any one of claims 1 to 13, wherein the compound is [the compound].

32. A composition, (a) Nucleic acid complexes which are compounds of formula (I) or salts thereof: 【Chemistry 20】 [In the formula, R １ The targeting ligand has the following formula: 【Chemistry 21】 (In the formula, B １ teeth, 【Chemistry 22】 A trivalent group selected from the group consisting of L １ , T １ , and T ２ It is covalently bonded to it; B ２ teeth, 【Chemistry 23】 A trivalent group selected from the group consisting of T １ , T ３ , and T ４ It is covalently bonded to it; B ３ teeth, 【Chemistry 24】 A trivalent group selected from the group consisting of T ２ , T ５ , and T ６ It is covalently bonded to it; T １ and T ２ Each of these is either absent or a glycine linking group; T ３ 、 T ４ 、 T ５ 、 and T ６ are each, 【Chemistry 25】 (In the formula, n = 1, 2, or 3) Independently selected from the group consisting of; Each sugar is a monosaccharide; L １ is a linking group, which is independently an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are —O—, —NR Ｘ —, —NR Ｘ —C(═O)—, —C(═O)—NR Ｘ —, or —S—, and may be optionally replaced thereby, where R Ｘ is hydrogen or (C １ —C ６ ) alkyl, and the hydrocarbon chain may be optionally substituted by one or more oxo (═O); Ring A is a 3- to 6-membered monocyclic heterocycloalkyl group that is either absent or contains one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; Each R Ａ is hydrogen, hydroxyl, -C １－２ Alkyl-OR Ｂ , and C １－４ Independently selected from the group consisting of alkyls; R Ｂ is hydrogen; n is 0, 1, 2, 3, or 4; L ２ A is a linking group, which independently is a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR Ｘ -, -NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S- may be optionally replaced, where R Ｘ is hydrogen or (C １ -C ６ ) is alkyl, and the hydrocarbon chain may be optionally substituted with one or more oxos (=O); R ２ [This is a nucleic acid, and the nucleic acid is an oligonucleotide with a length of approximately 10 to approximately 100 nucleotides.] (b) Film destabilizing polymer of formula (XX): 【Chemistry 26】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M ２ is, (C ４ -C １８ ) is an alkyl-methacrylate residue; BMA is a butyl methacrylate residue; PAA is a propyl acrylic acid residue; DMAEMA is a dimethylaminoethyl methacrylate residue; m and n are molar ratios greater than 0, where m is greater than n and m + n = 1; q is a molar ratio between 0.2 and 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q + r + s = 1; v is between 1 and 25 kDa; w is between 1 and 25 kDa; The targeted portion is 【Chemistry 27】 (In the formula, X is NR 3 and Y is selected from -(C=O)R 4, -SO 2R 5, and -(C=O)NR 6R 7, or X is -(C=O)- and Y is NR 8R 9, R3 is hydrogen or (C1-C4) alkyl, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and the (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. R10 is -OH, -NR8, R9, or -F. R11 is -OH, -NR8R9, -F, or a five-membered heterocycle, the five-membered heterocycle being optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. And; L is a connecting portion containing a polyethylene glycol (PEG) portion having 2 to 20 ethylene glycol units, and (c) Medicinally acceptable carriers The composition comprising the above.

33. The composition according to claim 32, formulated for administration by injection.

34. The composition according to claim 32 or 33, formulated for administration by subcutaneous injection.

35. A method for delivering nucleic acids to cells in vitro, the method comprising contacting the cells with 1) a membrane-destabilizing polymer and 2) a nucleic acid complex, The nucleic acid complex is given by formula (I): 【Chemistry 28】 [In the formula, R １ The targeting ligand has the following formula: 【Chemistry 29】 (In the formula, B １ teeth, 【Transformation 30】 A trivalent group selected from the group consisting of L １ , T １ , and T ２ It is covalently bonded to it; B ２ teeth, 【Chemistry 31】 A trivalent group selected from the group consisting of T １ , T ３ , and T ４ It is covalently bonded to it; B ３ teeth, 【Chemistry 32】 A trivalent group selected from the group consisting of T ２ , T ５ , and T ６ It is covalently bonded to it; T １ and T ２ Each of these is either absent or a glycine linking group; T ３ , T ４ , T ５ , and T ６ Each of them is, 【Transformation 33】 (In the formula, n = 1, 2, or 3) Independently selected from the group consisting of; Each sugar is a monosaccharide; L １ A is a linking group, which independently is an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR Ｘ -, -NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S- may be optionally replaced, where R Ｘ is hydrogen or (C １ -C ６ ) is alkyl, and the hydrocarbon chain may be optionally substituted with one or more oxos (=O); Ring A is a 3- to 6-membered monocyclic heterocycloalkyl group that is either absent or contains one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; Each R Ａ is hydrogen, hydroxyl, -C １－２ Alkyl-OR Ｂ , and C １－４ Independently selected from the group consisting of alkyls; R Ｂ is hydrogen; n is 0, 1, 2, 3, or 4; L ２ A is a linking group, which independently is a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR Ｘ -, -NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S- may be optionally replaced, where R Ｘ is hydrogen or (C １ -C ６ ) is alkyl, and the hydrocarbon chain may be optionally substituted with one or more oxos (=O); R ２ [The nucleic acid is the nucleic acid, which is an oligonucleotide with a length of approximately 10 to approximately 100 nucleotides.] It is a compound or salt thereof, The aforementioned film-destabilizing polymer is given by formula (XX): 【Transformation 34】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M ２ is, (C ４ -C １８ ) is an alkyl-methacrylate residue; BMA is a butyl methacrylate residue; PAA is a propyl acrylic acid residue; DMAEMA is a dimethylaminoethyl methacrylate residue; The method wherein m and n are each a molar ratio greater than 0, m is greater than n, and m + n = 1; the polymer is of the same type. q is a molar ratio between 0.2 and 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q + r + s = 1; v is between 1 and 25 kDa; w is between 1 and 25 kDa; The targeted portion is 【Chemistry 35】 (In the formula, X is NR 3 and Y is selected from -(C=O)R 4, -SO 2R 5, and -(C=O)NR 6R 7, or X is -(C=O)- and Y is NR 8R 9, R3 is hydrogen or (C1-C4) alkyl, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and the (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. R10 is -OH, -NR8, R9, or -F. R11 is -OH, -NR8R9, -F, or a five-membered heterocycle, the five-membered heterocycle being optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. And; L is a connecting portion containing a polyethylene glycol (PEG) portion having 2 to 20 ethylene glycol units.

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