Permeability enhancers for negatively charged polynucleotides

• US 5,583,020 A
• Filed: 11/19/1993
• Issued: 12/10/1996
• Est. Priority Date: 11/24/1992
• Status: Expired due to Fees

• Current Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Sullivan, Sean
• Primary Examiner(s): Stone, Jacqueline M.
• Assistant Examiner(s): Crouch, Deborah
• Application Number:
  US08/155,474
• Time in Patent Office: 1,117 Days
• Field of Search: 514/44, 435/172.3, 536/22.1, 536/23.1, 536/24.5, 530/350, 560/1, 564/230, 564/284, 564/436, 564/509
• US Class Current: 435/458
• CPC Class Codes: A61K 47/541 Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
• CPC Class Codes: A61K 9/127 Liposomes
• 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: C08G 63/912 derived from hydroxycarboxylic acids

Abstract

This invention features permeability enhancer molecules, and methods, to increase membrane permeability of negatively charged polymers thereby facilitating cellular uptake of such polymers.

Claims

1. A method for enhancing permeability of a negatively charged polynucleotide, comprising the step of contacting said polynucleotide with a permeability enhancer molecule comprising a cationic group which carries between 0 and 10% of a positive charge at pH 7.4, and carries a greater than 50% positive charge at a pH between 5.0 and 6.0 covalently joined to an anionic group which is greater than 50% unprotonated at pH 7.4, and no greater than 10% unprotonated at pH 5.0 to 6.0, wherein said negatively charged polynucleotide is ion-paired to said cationic group.

3. The method of claim 1 or 2, wherein said enhancer molecule is provided in an amount of at least one enhancer molecule per negative charge on said polynucleotide.

4. The method of claim 1, wherein said contacting is at acidic pH so that said enhancer molecule has a net positive charge and ion-pairs with said polynucleotide.

6. The method of claim 4 wherein formation of the ion-pair increases the intrinsic hydrophobicity of said polynucleotide allowing said polynucleotide to diffuse across a cell membrane.

7. The method of claim 6, wherein when said polynucleotide is present in a cytoplasm having a pH of about 7.4, said cationic group is deprotonated resulting in a negative charge, and said anionic group is deprotonated yielding no charge, wherein said enhancer molecule dissociates from said polynucleotide and remains in solution.

8. The method of claim 3, wherein when said polynucleotide is present in a cytoplasm having a pH of about 7.0, said cationic group is deprotonated resulting in a negative charge, and said anionic group is deprotonated yielding no charge, wherein said enhancer molecule dissociates from said polynucleotide and remains in solution.

9. The method of claim 1 or 2, wherein said permeability enhancer molecule is provided co-encapsulated within a liposome with said polynucleotide.

10. The method of claim 1, wherein said cationic group is an aminobenzyl group or an imidazole group having a pK_a between 4.0 and 5.0.

11. The method of claim 1 or 2, wherein said anionic group is a carboxylate group having a pK_a between 4.5 and 5.5.

12. The method of claim 1 or 2, wherein said enhancer molecule further comprises a linker between said cationic and anionic groups.

13. The method of claim 12, wherein said linker is a hydrocarbon chain or a simple or complex aromatic hydrocarbon.

15. The method of claims 1, 2, 10 or 14, wherein said enhancer molecule is a polynucleotide comprising at least two monomeric units.

2. A method for enhancing permeability of a negatively charged polynucleotide, comprising the step of contacting said polynucleotide with a permeability enhancer molecule comprising a cationic group which carries at least a 90% positive charge at both pH 7.4 and 5.0 covalently joined to an anionic group which is greater than 50% unprotonated at pH 7.4, and no greater than 10% unprotonated at pH 5.0 to 6.0, wherein said negatively charged polynucleotide is ion-paired to said cationic group.

5. The method of claim 2 or 4, wherein said polynucleotide is RNA or DNA and said enhancer molecule ion-pairs with the phosphate groups in said polynucleotide.

14. The method of claim 2, wherein said enhancer molecule comprises a guanidinium group or an amine group.

16. A permeability ligand-enhancer molecule comprising a targeting ligand covalently linked to a cationic group through a biological labile bond, said cationic group having a positive charge at pH 7.4, wherein cleavage of said biological labile bond yields a chemical group having a pK_a between 4.0 and 6.0 covalently linked to said cationic group.

17. The molecule of claim 16, wherein said ligand is selected from the group consisting of a monoclonal antibody or fragment thereof, a peptide, a protein, a carbohydrate and a vitamin.

18. The molecule of claim 16, wherein said biological labile bond is an ester bond, a carbamoyl bond or a peptide bond.

19. The molecule of claim 16, wherein said cationic group ion-pairs to a negatively charged polymer selected from the group consisting of a polyaminoacid and a polynucleotide.

20. The molecule of claim 16, wherein said cationic group ion-pairs to a negatively charged polynucleotide selected from the group consisting of RNA and DNA and protein.

21. The molecule of claim 20, wherein said RNA is enzymatic RNA.

22. The molecule of claim 16, wherein said cationic group is an amine group.

23. The molecule of claim 16 or 22, wherein said biological labile bond is covalently linked to said cationic group by a linker selected from the group consisting of a hydrocarbon chain comprising between 3 and 10 carbon atoms, a polyethoxy chain and an aromatic moiety.

24. A method for introducing a negatively charged polynucleotide into the cytoplasm of a cell, comprising the step of contacting said cell with said negatively charged polynucleotide ion-paired to an enhancer molecule, said enhancer molecule comprising a targeting ligand covalently linked to a cationic group through a biological labile bond, said cationic group having a positive charge at pH 7.4, wherein cleavage of said biological labile bond yields a chemical group having a pK_a between 4.0 and 6.0 covalently linked to said cationic group, wherein said negatively charged polynucleotide enters into said cytoplasm.

25. The method of claim 24, wherein said ligand is selected from the group consisting of a monoclonal antibody or fragment thereof, a peptide, a protein, a carbohydrate and a vitamin.

26. The method of claim 24, wherein said biological labile bond is an ester bond, a carbamoyl bond or a peptide bond.

27. The method of claim 24, wherein said negatively charged polynucleotide is selected from the group consisting of RNA and DNA.

28. The method of claim 27, wherein said RNA is enzymatic RNA.

29. The method of claim 24, wherein said cationic group is a terminal amine group.

30. The method of claim 24 or 29, wherein said biological labile bond is covalently linked to said cationic group by a linker selected from the group consisting of a hydrocarbon chain comprising between 3 and 10 carbon atoms, a polyethoxy chain and an aromatic moiety.