Liposomal formulations for delivery of nucleic acids

Patent metadata

Field	Value
Publication number	US-2017128367-A1
Application number	US-201515320903-A
Country	US
Kind code	A1
Filing date	Jun 25, 2015
Priority date	Jun 26, 2014
Publication date	May 11, 2017
Grant date	—

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 comprising cationic lipid, membrane stabilizing lipid and PEG-amine conjugated to a phospholipid; b) nucleic acid encapsulated within the liposome; and c) glycosaminoglycan bound to said PEG amine.

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 further comprising 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).

5 . The liposome of claim 1 , further comprising one or more additional PEG derivative.

6 . The liposome of claim 5 , 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, or combinations thereof.

7 . The liposome of claim 1 , wherein the glycosaminoglycan is selected from the group consisting of: hyaluronic acid (HA), Chondroitin sulfate, Dermatan sulfate, Keratan sulfate, Heparin, Heparan sulfate, salts, and mixtures thereof.

8 . The liposome of claim 7 , wherein the hyaluronic acid is selected from a hyaluronic acid having a molecular weight in the range of about 5-20 KD, a hyaluronic acid having a molecular weight in the range of about 600-1000 KDa, and combinations thereof.

9 . The liposome of claim 1 , having a diameter of about 50 nm to about 300 nm.

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

11 . The liposome of claim 10 , wherein the RNA is selected from siRNA, miRNA, shRNA, antisense RNA, mRNA, modified mRNA and combinations thereof.

12 . A composition comprising a plurality of liposomes according to claim 1 .

13 . The composition of claim 12 , wherein the liposomes are capable of delivering the nucleic acid to a target site.

14 . The composition of claim 13 , wherein the target site is selected from a cell, a tissue, an organ, and a microorganism.

15 . The composition of claim 14 , wherein the target cell harbors a CD44 receptor.

16 . A pharmaceutical composition comprising the composition of claim 12 in a dosage form suitable for administration via a route selected from oral, parenteral and topical.

17 . The composition of claim 16 , wherein the administration is localized.

18 . A method for treating cancer, comprising the step of administering to a subject in need thereof a pharmaceutical composition according to claim 16 .

19 . The method of claim 18 , wherein the cancer is selected from Adenocarcinoma and Glioblastoma Multiforme (GBM).

20 . Use of the pharmaceutical composition of claim 16 for treating cancer.

21 . A method for the preparation of a glycosaminoglycan coated liposome for delivery of a nucleic acid, the method comprising the steps of:
a) forming a lipid phase comprising the step of mixing cationic lipid, membrane stabilizing lipid and PEG-Amine conjugated to a phospholipid, 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 glycosaminoglycan to the mixture.

22 . The method of claim 21 wherein the lipid particle has a diameter of about 100 nm to about 300 nm.

23 . The method of claim 21 , 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.

24 . The method of claim 21 , wherein the membrane stabilizing lipid is selected from cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids.

25 . The method of claim 21 , further comprising mixing a phosphatidylamine, wherein the phosphatidylamine is 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).

26 . The method of claim 21 , further comprising mixing one or more additional PEG derivatives.

27 . The method of

Assignees

• Ramot At Tel-Aviv Univ Ltd

Inventors

• Peer Dan

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61K9/1271

  Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N2320/32

  Special delivery means, e.g. tissue-specific · CPC title