Polyconjugates for in vivo delivery of polynucleotides

• US 8,137,695 B2
• Filed: 08/17/2007
• Issued: 03/20/2012
• Est. Priority Date: 08/18/2006
• Status: Active Grant

• Current Assignee: Arrowhead Pharmaceuticals, Inc.
• Original Assignee: Arrowhead Madison Inc (Arrowhead Pharmaceuticals, Inc.)
• Inventors: Rozema, David B., Hagstrom, James E., Klein, Jason, Lewis, David L., Wakefield, Darren H., Wolff, Jon A., Wong, So, Monahan, Sean D.
• Primary Examiner(s): Schnizer, Richard
• Application Number:
  US11/840,468
• Publication Number:
  US 20080152661A1
• Time in Patent Office: 1,677 Days
• Field of Search: None
• US Class Current: 424/486
• CPC Class Codes: A01K 2217/058 due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
• CPC Class Codes: A01K 2267/0362 Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

The present invention is directed to compounds, compositions, and methods useful for delivering polynucleotides or other cell-impermeable molecules to mammalian cells. Described are polyconjugates systems that incorporate targeting, anti-opsonization, anti-aggregation, and transfection activities into small biocompatible in vivo delivery vehicles. The use of multiple reversible linkages connecting component parts provides for physiologically responsive activity modulation.

Claims

1. A conjugate delivery system for delivering an RNA interference polynucleotide to a liver cell in vivo comprising:

2. The conjugate of claim 1 wherein the polynucleotide comprises siRNA or microRNA.

3. The conjugate of claim 1 wherein the polyamine is larger than 5 kDa.

4. The conjugate of claim 3 wherein the polyamine is larger than 7.5 kDa.

5. The conjugate of claim 4 wherein the polymer is larger than 10 kDa.

6. The conjugate of claim 1 wherein the conjugate has a zeta potential between 0 and −

20 mV at pH 9.

7. The conjugate of claim 6 wherein the conjugate has a zeta potential between 0 and −

10 mV at pH 9.

8. The conjugate of claim 7 wherein the conjugate has a zeta potential between 0 and −

5 mV at pH 9.

9. The conjugate of claim 1 wherein P-L²-M¹ has the structure:

10. The conjugate of claim 1 wherein P-L²-M¹ has the structure:

11. The conjugate of claim 1 wherein the conjugate is smaller than 20 nm in diameter.

12. The conjugate of claim 1 wherein the conjugate is smaller than 10 nm in diameter.

13. The conjugate of claim 1 wherein the membrane active polymer consists of a polyvinylether random copolymer.

14. The conjugate of claim 1 wherein the membrane active polymer contains two or more different monomers.

15. The conjugate of claim 14 wherein the membrane active polymer contains three or more different monomers.

16. The conjugate of claim 15 wherein the monomers are selected from the group consisting of:

amine-containing monomers, lower alkyl monomers, butyl monomers, higher alkyl monomers, dodecyl monomers, and octadecyl monomers.

17. The conjugate of claim 13 wherein the polyvinylether random copolymer is composed of amine-containing vinyl ether monomers, lower alkyl vinyl ether monomers, and higher alkyl vinyl ether monomers.

18. The conjugate of claim 17 wherein the lower alkyl vinyl ether consists of a butyl vinyl ether and the higher alkyl vinyl ether consists of an octadecyl vinyl ether or dodecyl vinyl ether.

19. The conjugate of claim 18 wherein the polyvinylether random copolymer is soluble in water.

20. The conjugate of claim 18 wherein the polyvinylether random copolymer is surface active.

21. The conjugate of claim 1 wherein the polymer is a biodegradable polymer.

22. The conjugate of claim 1 wherein the masking agents are reversibly linked to at least 70% of the amines on the polyamine.

23. The conjugate of claim 17 wherein the amine-containing vinyl ether monomers, lower alkyl vinyl ether monomers, and higher alkyl vinyl ether monomers are present in a ratio of 5.4-7.5 a amine-containing vinyl ether monomers:

3-3.5 lower alkyl vinyl ether monomers;

1 higher alkyl vinyl ether monomers.

24. The conjugate delivery system of claim 1 wherein the conjugate delivery system further comprises an excess of membrane active polyamine reversibly covalently linked to a plurality of masking agents via physiologically labile bonds wherein the excess polymer is not covalently linked to the RNA interference polynucleotide.

25. The conjugate delivery system of claim 1 wherein the conjugate is provided in a pharmaceutically acceptable carrier or diluent.

26. The conjugate of claim 2 wherein the polynucleotide is selected from the groups consisting of:

DNA, RNA, dsRNA, RNA interference polynucleotide, siRNA, and miRNA.

27. A conjugate delivery system for delivering an RNA interference polynucleotide to a liver cell in vivo formed by the process comprising:

a) forming a membrane active polyamine;

b) forming a first masking agent comprising a disubstituted maleic anhydride containing a galactose derivative;

c) forming a second masking agent comprising a disubstituted maleic anhydride containing a polyethyleneglycol;

d) linking the RNA interference polynucleotide to the polyamine via a physiologically labile covalent bond that is orthogonal to a maleamate linkage;

e) reversibly inhibiting membrane activity of the membrane active polyamine wherein the inhibiting consists of modifying 50% or more of the amines on the polyamine by reacting the polyamine with the first and second masking agents thereby linking a plurality of galactose derivatives and a plurality of polyethyleneglycols to the membrane active polymer via physiologically pH-labile disubstituted maleamate linkages.