Lipid compounds and compositions containing them which can be used for the transfer of at least one active substance, in particular a polynucleotide, into a target cell and use in gene therapy

Abstract

The invention relates to new lipid compounds of formula: R—HN—[—(CH 2 ) m —NR—] n−1 —(CH 2 ) m —NH—R  I in which: the R residues are, independently of each other, a hydrogen atom or a group of formula II:  for which: R 1 and R 2 are, independently of each other, C 6 -C 23 alkyl or alkenyl radicals, which are linear or branched, or radicals —C(═O)—(C 6 -C 23 ) alkyl or —C(═O)—(C 6 -C 23 ) alkenyl, which are linear or branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals, polyethylene glycol groups, oxyethylene or oxymethylene groups which are optionally repeated, linear or branched, optionally substituted, p is a positive integer from 1 to 4, n is a positive integer from 1 to 6, m is a positive integer from 1 to 6 which may be different for each motif —(CH 2 ) m , and more particularly for each motif —(CH 2 ) m —NR— when n>1, the number of R groups of formula II being between 1 and 4 said compounds being optionally in a cationic form and being combined with one or more biologically acceptable anions. It also relates to new complexes comprising at least one said cationic compound and an active substance comprising negative charges allowing the introduction of said active substances into cells. It relates in particular to new complexes, in which the active substance consists of one or more nucleic acids, useful for transfecting cells.

1 . Lipid compound of formula: 
       R—HN—[—(CH 2 ) 0 —NR—] n−1 —(CH 2 ) m —NH—R  I in which: 
 the R residues are, independently of each other, a hydrogen atom or a group of formula II:  
                     
  for which: 
 R 1  and R 2  are, independently of each other, C 6 -C 23  alkyl or alkenyl radicals, which are linear or branched, or radicals —C(═O)—(C 6 -C 23 ) alkyl or —C(═O)—(C 6 -C 23 ) alkenyl, which are linear or branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals, polyethylene glycol groups, oxyethylene or oxymethylene groups which are optionally repeated, linear or branched, optionally substituted,  
 p is a positive integer from 1 to 4,  
 n is a positive integer from 1 to 6,  
 m is a positive integer from 1 to 6 which may be different for each motif —(CH 2 ) m , and more particularly for each motif —(CH 2 ) m —NR— when n>1,  
 the number of R groups of formula II being between 1 and 4.  
 
   
     
     
         2 . Compound according to  claim 1 , chosen from the compounds of formulae: 
       H 2 N—[—(CH 2 ) m —NH—] n R  IIIRNH—[—(CH 2 ) m —NH] n H  IIIa in which: 
 R is a group of formula II as defined in  claim 1 ,  
 n is a positive integer from 1 to 6,  
 m is a positive integer from 1 to 6 which may be different for each motif —(CH 2 ) m .  
   
     
     
         3 . Compound according to  claim 1  of formula: 
       H 2 N—[—(CH 2 ) m —NR] n−1 —(CH 2 ) m —NH 2   IIIb in which: 
 R has one of the meanings indicated for the formula I of  claim 1  provided that at least one R group is of formula II,  
 n is a positive integer from 1 to 6,  
 m is a positive integer from 1 to 6 which may be different for each motif —(CH 2 ) m , and more particularly for each motif —(CH 2 ) m —NR— when n>1.  
   
     
     
         4 . Compound according to  claim 3 , characterized in that it contains one or two R groups of formula II.  
     
     
         5 . Compound according to  claims 1  to  4 , characterized in that R 1  and R 2  are, independently of each other, linear —C(═O)-alkyl or linear —C(═O)-alkenyl radicals.  
     
     
         6 . Compound according to  claim 5 , characterized in that said alkyl or alkenyl comprises from 12 to 20 carbon atoms and in that said compound comprises 1 or 2 R groups of formula II.  
     
     
         7 . Compound according to  claim 6 , characterized in that said alkyl or alkenyl comprises 12, 16 or 18 carbon atoms.  
     
     
         8 . Compound according to  claims 1  to  7 , characterized in that n is an integer chosen from the numbers 2, 3 or 4.  
     
     
         9 . Compound according to  claims 1  to  8 , characterized in that m is an integer chosen from the numbers 2, 3 or 4.  
     
     
         10 . Compound according to  claim 1 , characterized in that it is chosen from the group consisting of the compounds of the following formulae:  
       
         
           
                 
               
                     
                 
                     
                 
                   IV 
                 
                 
               
                     
                 
                   
                     
                       
                       
                           
                           
                       
                     
                   
                 
                     
                 
                 
               
                   IVa 
                 
                 
               
                     
                 
                   
                     
                       
                       
                           
                           
                       
                     
                   
                 
                     
                 
                     
                 
             
                
                
               
               
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
                
                
                
               
            
           
         
         in which R 1  and R 2  are identical and are chosen from the stearoyl and oleoyl radicals.  
       
     
     
         11 . Compound according to one of  claims 1  to  10 , characterized in that it is conjugated with one or more ligands of interest via one of the secondary or primary nitrogen atoms of the polyamine chain or of the diaminocarboxylic acid.  
     
     
         12 . Compound according to  claim 11 , characterized in that said targeting component is chosen from the group consisting of all or part of sugars, peptides, oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds.  
     
     
         13 . Compound according to one of  claims 1  to  12 , characterized in that it is in a cationic form.  
     
     
         14 . Composition characterized in that it comprises at least one compound according to one of the preceding claims and optionally at least one adjuvant capable in particular of enhancing the formation of the complex between said compound and an active substance.  
     
     
         15 . Composition according to  claim 14 , characterized in that said adjuvant is a neutral or zwitterionic lipid.  
     
     
         16 . Composition according to  claim 15 , characterized in that said neutral or zwitterionic lipid is or is derived from a triglyceride, a diglyceride, cholesterol, a phosphatidylethanolamine (PE), phosphatidylcholine, phosphocholine, sphyngo-myelin, ceramide or cerebroside.  
     
     
         17 . Composition according to  claim 16 , characterized, in that said neutral or zwitterionic lipid is dioleylphosphatidylethanolamine (DOPE).  
     
     
         18 . Composition according to  claims 14  to  17 , characterized in that the compound/adjuvant weight ratio is between 0.1 and 10.  
     
     
         19 . Complex comprising at least one compound according to  claim 13  or at least one composition according to one of  claims 14  to  18  and at least one active substance, in particular a therapeutically active substance, comprising at least one negative charge.  
     
     
         20 . Complex according to  claim 19 , characterized in that said active substance is chosen from nucleic acids and proteins.  
     
     
         21 . Complex according to  claim 20 , characterized in that said active substance is a nucleic acid chosen from the group consisting of a cDNA, a genomic DNA, a plasmid DNA, an antisense polynucleotide, a messenger RNA, a ribosomal RNA, a ribozyme, a transfer RNA, or a DNA encoding such RNAs.  
     
     
         22 . Complex according to  claim 21 , characterized in that said nucleic acid comprises a gene of interest and components allowing the expression of said gene of interest.  
     
     
         23 . Complex according to one of  claims 19  to  22 , characterized in that it has a size of less than 500 nm, advantageously less than 200 nm.  
     
     
         24 . Complex according to one of claims  23 , characterized in that it has a size of less than 100 nm.  
     
     
         25 . Complex according to one of  claims 19  to  24 , characterized in that the ratio between the number of positive charges of the cationic compound(s) and/or composition(s) and the number of negative charges of said active substance varies from 0.05 to 20, more particularly from 0.1 to 15, and preferably from 5 to 10.  
     
     
         26 . Process for preparing a complex according to  claims 19  to  25 , characterized in that one or more compounds according to  claim 13  and/or at least one composition according to one of  claims 14  to  18  are brought into contact with one or more active substances comprising at least one negative charge and in that said complex is recovered, optionally after a purification step.  
     
     
         27 . Process of preparation according to  claim 26 , characterized in that said compounds and/or compositions are dissolved beforehand in a solvent which is miscible with water, in particular ethanol or dimethyl sulfoxide or a mixture of both.  
     
     
         28 . Process of preparation according to  claim 26 , characterized in that said compounds and/or compositions are suspended beforehand in a detergent solution.  
     
     
         29 . Process of preparation according to  claim 28 , characterized in that, in addition, a step of purification of said complex by dialysis is carried out.  
     
     
         30 . Use of a compound according to any one of  claims 1  to  13 , of a composition according to any one of  claims 14  to  18 , of a complex according to any one of  claims 19  to  25  to transfer at least one active substance, especially a therapeutically active substance, in particular a nucleic acid, into target cells in vitro, ex vivo or in vivo, more particularly in vivo.  
     
     
         31 . Use according to  claim 30 , characterized in that said target cell is a mammalian cell.  
     
     
         32 . Use according to  claim 31 , characterized in that said target cell is selected from a muscle cell, a hematopoietic stem cell, a cell of the airways, more particularly a tracheal or pulmonary cell.  
     
     
         33 . Process for transferring an active substance comprising at least one negative charge, into a cell, characterized in that said cell is brought into contact with a complex according to any one of  claims 19  to  25 .  
     
     
         34 . Complex according to one of  claims 19  to  25 , as a medicament for curative, preventive or vaccinal purposes.  
     
     
         35 . Complex according to one of  claims 19  to  25  for carrying out a method of therapeutic treatment which consists in transferring at least one therapeutically active substance, in particular a nucleic acid, into target cells.  
     
     
         36 . Complex according to  claim 35 , characterized in that said target cell is a mammalian cell.  
     
     
         37 . Complex according to  claim 36 , characterized in that said target cell is selected from a muscle cell, a hematopoietic stem cell, a cell of the airways, more particularly a tracheal or pulmonary cell, a cell of the respiratory epithelium.  
     
     
         38 . Use of a compound according to any one of  claims 1  to  13 , of a composition according to any one of  claims 14  to  18 , of a complex according to any one of  claims 19  to  25  for the preparation of a medicament for curative, preventive or vaccinal purposes, intended for the treatment of the human or animal body, in particular by gene therapy.  
     
     
         39 . Use according to  claim 38 , characterized in that the medicament is intended to be administered by intramuscular injection, by inhalation, by intratracheal injection, by instillation, by aerosolization, by the topical route or by the oral route.  
     
     
         40 . Pharmaceutical preparation characterized in that it comprises at least one complex according to any one of  claims 19  to  25 .  
     
     
         41 . Preparation according to  claim 40 , characterized in that it comprises, in addition, at least one adjuvant capable of enhancing the transfecting power of said complex.  
     
     
         42 . Preparation according to  claim 41 , characterized in that said adjuvant is chosen from the group consisting of chloroquine, a protic polar compound chosen in particular from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof, or an aprotic polar compound chosen in particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof.  
     
     
         43 . Preparation according to one of  claims 40  to  42 , characterized in that it comprises, in addition, a pharmaceutically acceptable carrier allowing its administration to humans or animals.  
     
     
         44 . Cell transfected with a complex according to any one of  claims 19  to  25 .