Amphoteric liposomes

Abstract

A serum-stable mixture of lipids capable of encapsulating an active agent to form a liposome, said mixture comprising phosphatidylcholine and phosphatidylethanolamine in a ratio in the range of about 0.5 to about 8. The mixture may also include pH sensitive anionic and cationic amphiphiles, such that the mixture is amphoteric, being negatively charged or neutral at pH 7.4 and positively charged at pH 4. Amphoteric liposomes comprising such a mixture may be used for encapsulating nucleic acid therapeutics, such as oligonucleotides and DNA plasmids. The drug/lipid ratio may be adjusted to target the liposomes to particular organs or other sites in the body.

1 . A mixture of lipids capable of encapsulating an active agent to form a liposome, said mixture comprising phosphatidylcholine and phosphatidylethanolamine in a ratio of phosphatidylethanolamine to phosphatidylcholine in the range of about 0.5 to about 8.  
     
     
         2 . The mixture as claimed in  claim 1 , wherein said ratio is in the range of about 0.75 to about 5.  
     
     
         3 . The mixture as claimed in  claim 1 , wherein said ratio is in the range of about 1 to about 4.  
     
     
         4 . The mixture as claimed in  claim 1 , wherein said phosphatidylcholine is selected from DMPC, DPPC, DSPC, POPC, DOPC, soy bean PC or egg PC.  
     
     
         5 . The mixture as claimed in  claim 1 , wherein said phosphatidylethanolamine is selected from DOPE or DMPE or DPPE.  
     
     
         6 . The mixture as claimed in  claim 1 , wherein said mixture is neutral.  
     
     
         7 . Neutral liposomes comprising a mixture of lipids as claimed in  claim 1 .  
     
     
         8 . The mixture as claimed in  claim 1 , further comprising one or more charged amphiphiles.  
     
     
         9 . The mixture as claimed in  claim 8 , wherein said one or more charged amphiphiles are amphoteric, being negatively charged or neutral at pH 7.4 and positively charged at pH 4.  
     
     
         10 . The mixture as claimed in  claim 9 , wherein said mixture comprises a plurality of charged amphiphiles which in combination with one another have amphoteric character.  
     
     
         11 . The mixture as claimed in  claim 10 , wherein said one or more charged amphiphiles comprise at least one pH sensitive anionic lipid and at least one pH sensitive cationic lipids.  
     
     
         12 . The mixture as claimed in  claim 11 , wherein said anionic lipid is selected from DOGSucc, POGSucc, DMGSucc, DPGSucc and CHEMS.  
     
     
         13 . The mixture as claimed in  claim 11 , wherein said cationic lipid is selected from MoChol, H is Chol and CHIM.  
     
     
         14 . The mixture as claimed in  claim 11 , wherein the ratio between the cationic and the anionic lipids (the charge ratio) is in the range of 4:1 to 1:4.  
     
     
         15 . The mixture as claimed in  claim 11 , wherein the ratio of cationic lipids to anionic lipids is in the range of 3:1 to 2:1, and said mixture comprises 5 to 95 mol. % charged lipids and 95 to 5 mol % phosphatidylcholine and phosphatidylethanolamine.  
     
     
         16 . The mixture as claimed in  claim 11 , wherein the ratio of cationic lipids to anionic lipids is about 1:1, and said mixture comprises 5 to 75 mol. % charged lipids and 95 to 25 mol % phosphatidylcholine and phosphatidylethanolamine.  
     
     
         17 . The mixture as claimed in  claim 11 , wherein the ratio of cationic lipids to anionic lipids is in the range of 1:3 to 1:2, and said mixture comprises 40 to 75 mol. % charged lipids and 60 to 25 mol % phosphatidylcholine and phosphatidylethanolamine.  
     
     
         18 . The mixture as claimed in  claim 14 , wherein said mixture comprises: 
 70 to 20 mol. % of POPC and DOPE in a ratio in the range of 1:1 to 1:4; and    30 and 80 mol. % of an amphoteric pair of charged lipids, said pair being selected from MoChol and CHEMS, MoChol and DMGSucc, MoChol and DOGSucc, CHIM and CHEMS or CHIM and DMGSucc, wherein the ratio of cationic to anionic lipid is in the range of 3:1 to 1:1.    
     
     
         19 . The mixture as claimed in  claim 18 , wherein said mixture consists of a formulation selected from:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   6:24:47:23 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   15:45:20:20 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   10:30:30:30 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   6:24:47:23 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   16:24:30:30 (mol. %) 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         20 . The mixture as claimed in  claim 11 , wherein said mixture comprises: 
 70. to 20 mol. % of POPC and DOPE in a ratio in the range of 1:1 to 1:4; and    and 80 mol. % of MoChol and DMGSucc or DOGSucc, wherein the molar amount of DMGSucc or DOGSucc exceeds the molar amount of MoChol.    
     
     
         21 . The mixture as claimed in  claim 20 , wherein the ratio of cationic to anionic lipid is in the range of 1:3 to 1:2, and said mixture comprises 30 to 50 mol. % POPC and DOPE and 70 to 50 mol. % charged lipids.  
     
     
         22 . The mixture as claimed in  claim 21 , wherein said mixture consists of a formulation selected from:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   6:24:23:47 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   10:30:20:40 (mol. %) 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
               
            
           
         
       
     
     
         23 . Amphoteric liposomes comprising a mixture of lipids as claimed in  claim 9 .  
     
     
         24 . The amphoteric liposomes as claimed in  claim 23 , wherein said liposomes have a size in the range of 50 to 500 mm.  
     
     
         25 . The amphoteric liposomes as claimed in  claim 23 , wherein said liposomes encapsulate at least one active agent.  
     
     
         26 . The amphoteric liposomes as claimed in  claim 25 , wherein said active agent comprises a nucleic acid that is capable of being transcribed in a vertebrate cell into one or more RNAs, said RNAs being mRNAs, shRNAs, miRNAs or ribozymes, said mRNAs coding for one or more proteins or polypeptides.  
     
     
         27 . The amphoteric liposomes as claimed in  claim 26 , wherein said nucleic acid is a circular DNA plasmid, a linear DNA construct or an mRNA.  
     
     
         28 . The amphoteric liposomes as claimed in  claim 25 , wherein said active agent is an oligonucleotide.  
     
     
         29 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a decoy oligonucleotide, an antisense oligonucleotide, a siRNA, an agent influencing transcription, an agent influencing splicing, Ribozymes, DNAzymes or Aptamers.  
     
     
         30 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotides comprise modified nucleosides such as DNA, RNA, locked nucleic acids (LNA), peptide nucleic acids (PNA), 2′O-methyl RNA (2′Ome), 2′ O-methoxyethyl RNA (2′MOE) in their phosphate or phosphothioate forms.  
     
     
         31 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is an antisense oligonucleotide of 15 to 30 basepairs length.  
     
     
         32 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a siRNA of 15 to 30 basepairs length.  
     
     
         33 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a decoy oligonucleotide of 15 to 30 basepairs length.  
     
     
         34 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is an agent influencing the transcription of 15 to 30 basepairs length.  
     
     
         35 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a DNAzyme of 25 to 50 basepairs length.  
     
     
         36 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a Ribozyme of 25 to 50 basepairs length.  
     
     
         37 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is a Aptamer of 15 to 60 basepairs length.  
     
     
         38 . The amphoteric liposomes as claimed in  claim 28 , wherein said oligonucleotide is adapted to target a nucleic acid encoding CD40 gene, its sense or antisense strand, any exons or introns or untranslated regions thereof thereby to modulate expression of CD40 in mammalian cells.  
     
     
         39 . The amphoteric liposomes as claimed in  claim 38 , wherein said oligonucleotide is directed against any mRNA of CD40, wherein such mRNAs include pre-mRNA and their subsequently matured forms.  
     
     
         40 . The amphoteric liposomes as claimed in  claim 38 , wherein said mixture of lipids consists of a formulation selected from:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   6:24:47:23 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   15:45:20:20 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/CHEMS 
                   10:30:30:30 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   6:24:47:23 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   16:24:30:30 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   6:24:23:47 (mol. %) 
                 
                     
                   POPC/DOPE/MoChol/DMGSucc 
                   10:30:20:40 (mol. %) 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         41 . The amphoteric liposomes as claimed in  claim 28 , wherein at least 80 wt. % of said oligonucleotide is disposed inside said liposomes.  
     
     
         42 . The amphoteric liposomes as claimed in  claim 28 , wherein said liposomes comprise non-encapsulated oligonucleotides.  
     
     
         43 . A pharmaceutical composition comprising active agent-loaded amphoteric liposomes as claimed in  claim 25  and a pharmaceutically acceptable vehicle therefor.  
     
     
         44 . The pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of greater than about 150 nm.  
     
     
         45 . The pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of less than about 150 nm.  
     
     
         46 . The pharmaceutical composition as claimed in  claim 43 , said composition further comprising empty liposomes having a similar composition and size to said active agent-loaded amphoteric liposomes.  
     
     
         47 . A method of using the amphoteric liposomes as claimed in  claim 28  for preventing or treating an inflammatory, immune or autoimmune disorder of a human or non-human animal.  
     
     
         48 . The method as claimed in  claim 28  for preventing or treating graft rejection, graft-versus-host disease, diabetes type I, multiple sclerosis, systemic lupus erythematosous, rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis or thyroiditis, wherein said amphoteric liposomes are formulated for systemic administration.  
     
     
         49 . A method as claimed in  claim 28  for preventing or treating graft rejection, graft-versus-host disease, inflammatory bowel disease, asthma, Crohn's disease or ulcerative colitis, wherein said amphoteric liposomes is formulated for local administration.  
     
     
         50 . A method of treating a human or non-human animal by administering systemically thereto at a low lipid dose a pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of greater than 150 nm, thereby targeting said active agent to the liver.  
     
     
         51 . A method of treating a human or non-human animal by administering systemically thereto at a high lipid dose a pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of greater than 150 nm, thereby targeting said active agent to the spleen, sites of infections and inflammations or solid tumours.  
     
     
         52 . A method of treating a human or non-human animal by administering systemically thereto at a low lipid dose a pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of less than 150 mm, thereby targeting the active agent to the liver.  
     
     
         53 . A method of treating a human or non-human animal by administering systemically thereto at a high lipid dose a pharmaceutical composition as claimed in  claim 43 , wherein said liposomes have a size of less than 150 nm, thereby targeting the active agent to sites of infections and inflammations or solid tumours, excluding the spleen  
     
     
         54 . The method as claimed in  claim 51  or  claim 53 , further comprising lowering the drug/lipid ratio to the desired lipid concentration.  
     
     
         55 . The method as claimed in  claim 51  or  claim 53 , further comprising including in said composition empty liposomes having a similar size and composition to said active agent-loaded liposomes.