Novel lipid complexes for transferring at least one therapeutically active substance, in particular a polynucleotide, into a target cell, and use in gene therapy

Abstract

The present invention relates to a complex comprising at least a lipid and at least a therapeutically active substance for transferring the therapeutically active substance into a target cell. The present invention also relates to a method of transferring a therapeutically active substance into cells.

1 . Complex comprising at least one lipid and at least one therapeutically active substance, which can be used for transferring said subtance into a target cell, characterized in that said lipid is of formula I:  
       
         
           
           
               
               
           
         
       
       in which: 
 n 1  and n 2 , which may be identical or different, are integers chosen from 0 and 1,  
 R 1  and R 2 , which may be identical or different, are: 
 a) chosen from the group consisting of a hydrogen atom and alkyl radicals of 1 to 6 carbon atoms which are optionally substituted, independently of each other, with a hydroxyl radical, or  
 b) according to a specific case for which n 1 =n 2 =1, R 1  and R 2  can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3), R 3  and R 4 , which may be identical or different, are alkyl radicals of 1 to 6 carbon atoms or can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3),  
 
 m and p, which may be identical or different, are integers between 1 and 10,  
 R 5  and R 6 , which may be identical or different, are chosen from the group consisting of the radicals of formula: 
 1) R 7  C(═O)—X— in which: 
 X═NH, O or S,  
 R 7  is a linear or branched, alkyl or alkenyl radical of 6 to 23 carbon atoms (C 6 -C 23 ),  
 
 2) R 8 R 9 NC(═O)— in which: 
 R 8  and R 9 , which may be identical or different, are chosen from the group consisting of a hydrogen atom or linear or branched, alkyl or alkenyl radicals of 6 to 23 carbon atoms, on condition that R 8  and R 9  cannot simultaneously correspond to a hydrogen atom, and  
 
 3) it also being possible for one of the radicals R 5  and R 6  to correspond to a hydrogen atom.  
 
 
     
     
         2 . Complex according to  claim 1 , characterized in that said lipid is a lipid of formula VII:  
       
         
           
           
               
               
           
         
       
       in which: 
 n 1  and n 2 , which may be identical or different, are integers chosen from 0 and 1,  
 R 1  and R 2 , which may be identical or different, are: 
 a) chosen from the group consisting of a hydrogen atom and alkyl radicals of 1 to 6 carbon atoms which are optionally substituted, independently of each other, with a hydroxyl radical, or  
 b) according to a specific case for which n 1 =n 2 =1, R 1  and R 2  can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3),  
 
 R 3  and R 4 , which may be identical or different, are alkyl radicals of 1 to 6 carbon atoms or can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3),  
 m and p, which may be identical or different, are integers between 1 and 10,  
 R 8  and R 9 , which may be identical or different, are chosen from the group consisting of a hydrogen atom or linear or branched, alkyl or alkenyl radicals of 6 to 23 carbon atoms, on condition that R 9  and R 9  cannot simultaneously correspond to a hydrogen atom, and the radicals NR 8 R 9  on each side of the molecule can be different.  
 
     
     
         3 . Complex according to  claim 1 , characterized in that said lipid is a lipid of formula II:  
       
         
           
           
               
               
           
         
       
       in which: 
 n 1  and n 2 , which may be identical or different, are chosen from the integers 0 and 1,  
 R 1  and R 2  which may be identical or different, are chosen from the group consisting of a hydrogen atom and alkyl radicals of 1 to 6 carbon atoms, optionally substituted, independently of each other, with hydroxyl radicals, where, in the specific case in which n 1 =n 2 =1, R 1  and R 2  can together form a divalent alkylene chain of 2 to 3 carbon atoms,  
 m and p, which may be identical or different, are integers between 1 and 10,  
 R 7 , which may be identical or different, is a linear or branched, alkyl or alkenyl radical of 6 to 23 carbon atoms.  
 
     
     
         4 . Complex according to one of  claims 1  to  3 , characterized in that 
 R 1  and/or R 2  or,  
 R 3  and/or R 4    
 is a methyl radical.  
 
     
     
         5 . Complex according to one of  claims 1  to  3 , characterized in that 
 R 1  and R 2  and/or  
 R 3  and R 4    
 together form an ethylene chain.  
 
     
     
         6 . Complex according to one of  claims 1  to  3 , characterized in that 
 R 1  and R 2  and/or  
 R 3  and R 4    
 together form a propylene chain.  
 
     
     
         7 . Complex according to one of  claims 1  to  6 , characterized in that m and/or p are integers between 1 and 6.  
     
     
         8 . Complex according to claims  1  or  3 , characterized in that the radicals R 7  C(═O), which may be identical or different, are radicals of 13 to 18 carbon atoms, in particular oleoyl, stearoyl, palmitoyl or myristoyl radicals.  
     
     
         9 . Complex according to claims  1 ,  3  or  8 , characterized in that said lipid is chosen from the group consisting of the compounds of the following formula:  
       
         
           
           
               
               
           
         
       
     
     
         10 . Complex according to claims  1  or  3 , characterized in that the R 8  and R 9  are chosen from alkyl or alkenyl radicals of 6 to 23 carbon atoms.  
     
     
         11 . Complex according to claims  1 ,  3  or  10 , characterized in that said lipids are chosen from the group consisting of the compounds of the following formulae:  
       
         
           
           
               
               
           
         
       
     
     
         12 . Complex according to  claim 1 , characterized in that said lipid is of formula X:  
       
         
           
           
               
               
           
         
       
       in which 
 n 1  and n 2 , which may be identical or different, are integers chosen from 0 and 1,  
 R 1  and R 2 , which may be identical or different, are: 
 a) chosen from the group consisting of a hydrogen atom and alkyl radicals of 1 to 6 carbon atoms which are optionally substituted, independently of each other, with a hydroxyl radical, or  
 b) according to a specific case for which n 1 =n 2 =1, R 1  and R 2  can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3),  
 
 m and p, which may be identical or different, are integers between 1 and 10,  
 R 8  and R 9 , which may be identical or different, are chosen from the group consisting of a hydrogen atom or linear or branched, alkyl or alkenyl radicals of 6 to 23 carbon atoms, on condition that R 8  and R 9  cannot simultaneously correspond to a hydrogen atom.  
 
     
     
         13 . Complex according to  claim 12 , characterized in that: 
 R 1 , R 2  or    R 3 , R 4      are a methyl or hydroxyethyl radical.    
     
     
         14 . Complex according to  claim 12 , characterized in that: 
 R 1  and R 2  or    R 1  and R 4      together form an ethylene chain.    
     
     
         15 . Complex according to  claim 12 , characterized in that: 
 R 1  and R 2  or    R 3  and R 4      together form a propylene chain.    
     
     
         16 . Complex according to one of  claims 12  to  15 , characterized in that m is an integer between 1 and 6 and p=1.  
     
     
         17 . Complex according to one of  claims 12  to  16 , characterized in that R 8  and R 9  are chosen from alkyl or alkenyl radicals of 6 to 23 carbon atoms.  
     
     
         18 . Complex according to one of  claims 12  to  17 , characterized in that said lipids are chosen from the group consisting of the compounds of the following formulae:  
       
         
           
           
               
               
           
         
       
     
     
         19 . Complex according to one of  claims 1  to  18 , characterized in that said lipid is one, the other or a mixture of the two diastereoisomers (cis and/or trans) corresponding to said lipid.  
     
     
         20 . Complex according to one of  claims 1  to  19 , characterized in that it also comprises at least one adjuvant capable of improving the formation of the complexes between a said lipid and a said therapeutically active substance, or of improving the functioning of these complexes toward the cell.  
     
     
         21 . Complex according to  claim 20 , characterized in that said adjuvant is a neutral or zwitterionic lipid, such as, for example, a triglyceride, a diglyceride, cholesterol, which is derived from a phosphatidylethanolamine (PE), phosphatidylcholine, phosphocholine, sphingomyelin, ceramide or cerebroside, preferably dioleoylphosphatidylethanolamine (DOPE) or phosphatidylethanolamine (PE).  
     
     
         22 . Complex according to  claim 21 , characterized in that the weight ratio between the lipid and said adjuvant is between 0.1 and 10.  
     
     
         23 . Complex according to  claims 1  to  22 , characterized in that said therapeutically active substance is chosen from nucleic acids and proteins, preferably; the active substance in the complex is a polynucleotide.  
     
     
         24 . Complex according to  claims 1  to  23 , characterized in that the ratio between the number of positive charges in the cationic lipid and the number of negative charges in the therapeutically active substance is between 0.05 and 20, in particular between 0.1 and 15 and preferably between 5 and 10.  
     
     
         25 . Complex according to  claim 1 , characterized in that the lipid is chosen from the group consisting of: 
 lipid according to  claim 1  for which R 1 ═R 2 =methyl; R 3  and R 4  together form an ethylene chain, m=3, p=3, R 5 ═R 6 ═NHC(═O)C 13 H 27  (pcTG24);    lipid according to  claim 1  for which R 1 ═R 2 ═R 3 ═R 4 =methyl, R 5 ═R 6 ═C(═O)—N(C 8 H 17 ) 2  (pcTG42).    
     
     
         26 . Use of a complex according to  claims 1  to  25 , for the preparation of a medicinal product for curative, preventive or vaccinal purposes, intended for treating the human or animal body, in particular by gene therapy.  
     
     
         27 . Use of a complex according to  claims 1  to  25  for transferring at least one therapeutically active substance, more particularly a nucleic acid, into target cells in vitro.  
     
     
         28 . Use according to  claim 26 , characterized in that said medicinal product is intended to be administered via the intramuscular, intratracheal, intranasal, intracerebral, intrapleural, intratumoral, intracardiac, intragastric, intraperitoneal, epidermal, intravenous or intraarterial route.  
     
     
         29 . Process for introducing a therapeutically active substance containing negative charges into a cell, characterized in that cells cultured on a suitable medium are placed in contact with a suspension of complex according to one of  claims 1  to  25 , and in that, after a certain period of incubation, the cells are washed and recovered.  
     
     
         30 . Pharmaceutical preparation comprising at least one complex according to one of  claims 1  to  25  and optionally containing at least one adjuvant capable of stabilizing said pharmaceutical preparation for the purpose of storing it, for example, and/or of enhancing the transfecting power of said complex.  
     
     
         31 . Pharmaceutical preparation according to claim  30 , characterized in that said adjuvant is chosen from the group consisting of chloroquine, a polar protic compound chosen in particular from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof, or a polar aprotic compound chosen in particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea and acetonitrile, or derivatives thereof.