Liposomal formulations for delivery of nucleic acids

Patent metadata

Field	Value
Publication number	US-10583084-B2
Application number	US-201515320903-A
Country	US
Kind code	B2
Filing date	Jun 25, 2015
Priority date	Jun 26, 2014
Publication date	Mar 10, 2020
Grant date	Mar 10, 2020

Abstract

There are provided liposomes, comprising cationic lipids, a membrane stabilizing lipid and at least one lipid conjugated to a polyethylene glycol (PEG) derivative, in particular PEG-amine, the liposomes are coated with a glycosaminoglycan, in particular, Hyaluronic Acid (HA), compositions comprising the same, methods for their preparation and uses thereof for the efficient delivery of nucleic acids, such as, si RNA molecules and for treating various conditions, such as cancer.

First claim

What is claimed is:

1. A liposome for delivery of a nucleic acid comprising a lipid membrane encapsulating the nucleic acid, wherein the lipid membrane is composed of a cationic lipid; a membrane stabilizing lipid; a phospholipid conjugated to a PEG-amine; one or more additional PEG derivatives; and optionally one or more phosphatidylamine, wherein said lipid membrane is coated with hyaluronic acid having a molecular weight in the range of about 600-1000 KDa which is bound to said PEG-amine, and wherein said liposome has a diameter of about 50 nm to about 300 nm.

2. The liposome of claim 1 , wherein the cationic lipid is selected from DLinDMA, DLin-MC3-DMA and DLin-KC2-DMA; monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane (DOTAP), BCAT O-(2R-1,2-di-O-(1′Z, 9′Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC (Bis-guanidinium-spermidine-cholesterol), BGTC (Bis-guanidinium-tren-cholesterol), CDAN (N′ -cholesteryl oxycarbony 1-3,7-diazanonane-1,9-diamine), CHDTAEA (Cholesteryl hemidithiodiglycolyl tris(amino(ethyl)amine), DCAT (O-(1,2-di-O-(9′Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate), DC-Chol (3β [N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol), DLKD (O,O′-Dilauryl N-lysylaspartate), DMKD (O,O′-Dimyristyl N-lysylaspartate), DOG (Diolcylglycerol, DOGS (Dioctadecylamidoglycylspermine), DOGSDSO (1,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine, DOSN (Dioleyl succinyl ethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleyl succinyl tobramycin), DOTAP (1,2-Uiolcoyl-3-trimethyl ammoniopropane), DOTMA (N′[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethvlammonium chloride), DPPES (Di-palmitoyl phosphatidyl ethanolamidosperminc), DDAB, and DODAP.

3. The liposome of claim 1 , wherein the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids, and glycoglycerolipids.

4. The liposome of claim 1 , wherein the phospholipid conjugated to a PEG-amine bound to hyaluronic acid constitutes about 0.25 mole% to 3 mole% of the lipid membrane.

5. The liposome of claim 1 containing one or more phosphatidylamine selected from 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE) 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin-Phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine(DMPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and Dipalmitoylphosphatidylethanolamine (DPPE).

6. The liposome of claim 1 , wherein the additional PEG derivative is selected from DMG-PEG, PEG-cDMA, 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine; PEG-cDSA, 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-distearyloxy-propylamine, and combinations thereof.

7. The liposome of claim 1 , wherein the nucleic acid is selected from DNA, RNA, and modified forms thereof.

8. The liposome of claim 7 , wherein the RNA is selected from siRNA, miRNA, shRNA, antisense RNA, mRNA, modified mRNA, and combinations thereof.

9. A composition comprising a plurality of liposomes according to claim 1 , wherein the liposomes are capable of delivering the nucleic acid to a target site, wherein the target site is selected from a cell, a tissue, an organ, and a microorganism.

10. The composition of claim 9 , wherein the target cell harbors a CD44 receptor.

11. The composition of claim 9 in a dosage form suitable for localized administration or for administration via a route selected from oral, parenteral and topical.

12. A method for treating cancer, comprising the step of administering to a subject in need thereof a composition according to claim 11 .

13. The method of claim 12 , wherein the cancer is selected from Adenocarcinoma, and Glioblastoma Multiforme (GBM).

14. A method for the preparation of the liposome of claim 1 , the method comprising the steps of:
a) forming a lipid phase comprising the step of mixing a cationic lipid, a membrane stabilizing lipid, a phospholipid conjugated to a PEG-amine, one or more additional PEG derivatives, and optionally one or more phosphatidylamine, in an organic solvent at a desired ratio and forming a lipid mixture,
b) generating the liposome by the step of: introducing a nucleic acid in an aqueous solution into the lipid mixture of step (a); and
c) adding an activated hyaluronic acid to the mixture.

15. The method of claim 14 , wherein the hyaluronic acid is activated by dissolving a hyaluronic acid in an acidic buffer and adding a crosslinker to form an activated hyaluronic acid.

16. A method for the preparation of the liposome of claim 1 , the method comprising the steps of:
a) forming a lipid phase comprising the steps of:
i) mixing a cationic lipid, a membrane stabilizing lipid, a phospholipid conjugated to a PEG-amine, one or more additional PEG derivatives, and optionally one or more phosphatidylamine, in an organic solvent at a desired ratio and forming a lipid mixture,
ii) suspending the lipid mixture in a buffer to generate multilamellar vesicles;

b) generating the liposome by the steps of:
i) incubating the lipid phase of step (a) with the nucleic acid; and
ii) adding an activated hyaluronic acid to the mixture.

17. A liposome for delivery of a nucleic acid comprising a lipid membrane encapsulating the nucleic acid, wherein the lipid membrane is composed of a plurality of lipids comprising a cationic lipid, a membrane stabilizing lipid, a phospholipid conjugated to a PEG-amine, optionally one or more additional PEG derivatives and optionally one or more phosphatidylamine; wherein the external surface of said lipid membrane is coated with hyaluronic acid covalently attached to said PEG-amine; wherein the hyaluronic acid has a molecular weight in the range of about 600-1000 KDa; and wherein said liposome has a diameter of about 50 nm to about 300 nm.

18. The liposome of claim 17 , wherein the cationic lipid is selected from: DLinDMA, DLin-MC3-DMA and DLin-KC2-DMA; monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane (DOTAP), BCAT O-(2R-1,2-di-O-(′Z, 9′Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC (Bis-guanidinium-spermidine-cholesterol), BGTC (Bis-guanidinium-tren-cholesterol), CDAN (N′ -cholesteryl oxycarbony 1-3,7-diazanonane-1,9-diamine), CHDTAEA (Cholesteryl hemidithiodiglycolyl tris(amino(ethyl)amine), DCAT (O-(1,2-di-O-( 9 ′Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate), DC-Chol (3β [N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol), DLKD (O,O′-Dilauryl N-lysylaspartate), DMKD (O,O′-Dimyristyl N -lysylaspartate), DOG (Diolcylglycerol, DOGS (Dioctadecylamidoglycylspermine), DOGSDSO (1,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-Dioleoyl-sn-glycerol -3-phosphoethanolamine, DOSN (Dioleyl succinyl ethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleyl succinyl tobramycin), DOTAP (1,2-Uiolcoyl-3-trimethyl ammoniopropane), DOTMA (N′[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethvlammonium chloride), DPPES (Di-palmitoyl phosphatidyl ethanolamidosperminc), DDAB, and DODAP.

19. The liposome of claim 17 , wherein the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholi

Assignees

• Univ Ramot

Inventors

• Peer Dan

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/11

  Antisense · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title