Means and methods of enhancing delivery to biological systems

Abstract

This invention relates to a peptide or polypeptide (a) which is esterified or thio-esterified (i) at the carboxylate of the C-terminus with a guanidinium alkanol, a guanidinium alkanethiol, a PEG substituted with a guanidinium group and having a free hydroxyl group, or a PEG substituted with a guanidinium group and a sulfhydryl group; (ii) at a side-chain carboxylate of one or more Asp or Glu residues, if present, with a guanidinium alkanol, a guanidinium alkanethiol, a PEG substituted with a guanidinium group and having a free hydroxyl group, or a PEG substituted with a guanidinium group and a sulfhydryl group; (iii) at a hydroxyl group of one or more Ser, Thr or Tyr residues, if present, with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group; (iv) at a sulfhydryl group of one or more Cys residues, if present, with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group; and/or (v) at the N-terminus with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group, wherein said N-terminus is previously amidated with an alpha- or beta-hydroxy acid, and wherein the ester is formed between the hydroxy group of said alpha- or beta-hydroxy acid and the carboxylic group of said guanidinium alkanoic acid or said PEG substituted with a guanidinium group and a carboxyl group; and/or (b) which contains one or more disulfides, the disulfide being formed between the sulfhydryl group of a Cys reside, if present, and a guanidinium alkanethiol or a PEG substituted with a guanidinium group and a sulfhydryl group.

1 . A peptide or polypeptide
 (a) which is esterified or thio-esterified
 (i) at the carboxylate of the C-terminus with a guanidinium alkanol, a guanidinium alkanethiol, a PEG substituted with a guanidinium group and having a free hydroxyl group, or a PEG substituted with a guanidinium group and a sulfhydryl group; 
 (ii) at a side-chain carboxylate of one or more Asp or Glu residues, if present, with a guanidinium alkanol, a guanidinium alkanethiol, a PEG substituted with a guanidinium group and having a free hydroxyl group, or a PEG substituted with a guanidinium group and a sulfhydryl group; 
 (iii) at a hydroxyl group of one or more Ser, Thr or Tyr residues, if present, with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group; 
 (iv) at a sulfhydryl group of one or more Cys residues, if present, with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group; and/or 
 (v) at the N-terminus with a guanidinium alkanoic acid or a PEG substituted with a guanidinium group and a carboxyl group, wherein said N-terminus is previously amidated with an alpha- or beta-hydroxy acid, and wherein the ester is formed between the hydroxy group of said alpha- or beta-hydroxy acid and the carboxylic group of said guanidinium alkanoic acid or said PEG substituted with a guanidinium group and a carboxyl group; 
    and/or   (b) which contains one or more disulfides, the disulfide being formed between the sulfhydryl group of a Cys reside, if present, and a guanidinium alkanethiol or a PEG substituted with a guanidinium group and a sulfhydryl group.   
     
     
         2 . The peptide or polypeptide of  claim 1 , wherein the guanidinium alkanol or guanidinium alkanethiol has the following structure:
   HX—(CR 1 R 2 ) n —N(R 3 )—C(═NR 4 )—NHR 5   (Formula I)
   wherein:   X is O or S;   each occurrence of R 1  and R 2  is independently selected from H; halogen; NH 2 ; alkyl with 1 to 5 carbon atoms; aryl; and heteroaryl;   n is an integer number between 2 and 15;   R 3  is H or alkyl with 1 to 5 carbon atoms; and   R 4  and R 5  are independently selected from H; alkyl with 1 to 5 carbon atoms; aryl; heteroaryl, wherein the N bearing the R 4  or R 5  group may be a ring atom of the heteroaryl ring; and/or R 4  and R 5  may be selected together to give a heteroaryl ring.   
     
     
         3 . The peptide or polypeptide of  claim 1  or  2 , wherein the guanidinium alkanol or guanidinium alkanethiol has the following structure:
   HX—(CH 2 ) n —NR—C(═NH)—NH 2   (Formula II)
 
 wherein: 
 X is O or S; 
 n is an integer number between 2 and 10; and 
 R is H or alkyl with 1 to 5 carbon atoms. 
 
     
     
         4 . The peptide or polypeptide of any one of  claims 1  to  3 , wherein the guanidinium alkanoic acid has the following structure:
   HOOC—(CR 1 R 2 ) n —N(R 3 )—C(═NR 4 )—NHR 5   (Formula III)
 
 wherein: 
 each occurrence of R 1  and R 2  is independently selected from H; halogen; NH 2 ; alkyl with 1 to 5 carbon atoms; aryl; and heteroaryl; 
 n is an integer number between 2 and 15; 
 R 3  is H or alkyl with 1 to 5 carbon atoms; and 
 R 4  and R 5  are independently selected from H; alkyl with 1 to 5 carbon atoms; aryl; heteroaryl, wherein the N bearing the R 4  or R 5  group may be a ring atom of the heteroaryl ring; and/or R 4  and R 5  may be selected together to give a heteroaryl ring. 
 
     
     
         5 . The peptide or polypeptide of any one of  claims 1  to  4 , wherein the guanidinium alkanoic acid has the following structure:
   HOOC—CHR 1 —(CH 2 ) n-1 —N(R 2 )—C(═NH)—NH 2   (Formula IV)
 
 wherein: 
 R 1  is H or NH 2 ; 
 n is an integer number between 2 and 10; and 
 and R 2  is H or alkyl with 1 to 5 carbon atoms. 
 
     
     
         6 . The peptide or polypeptide of any one of  claims 2  to  5 , wherein n is between 4 and 9, preferably between 4 and 6. 
     
     
         7 . The peptide or polypeptide of any one of  claims 2  to  6 , wherein said PEG substituted with a guanidinium group and having a free hydroxyl group or said PEG substituted with a guanidinium group and a sulfhydryl group, respectively, has the following structure
   HX—CH 2 CH 2 (OCH 2 CH 2 ) k —N(R 3 )—C(═NR 4 )—NHR 5   (Formula V)
 
 wherein k is an integer number between 1 and 12 and X, R 3 , R 4  and R 5  are as defined in  claim 2 . 
 
     
     
         8 . The peptide or polypeptide of any one of  claims 2  to  7 , wherein said PEG substituted with a guanidinium group and a carboxyl group has the following structure
   HOOC—CH 2 CH 2 (OCH 2 CH 2 ) k —N(R 3 )—C(═NR 4 )—NHR 5   (Formula VI)
 
 wherein k is an integer number between 1 and 12 and X, R 3 , R 4  and R 5  are as defined in  claim 4 . 
 
     
     
         9 . The peptide or polypeptide of any one of  claims 2  to  8 , wherein k is 1 to 3. 
     
     
         10 . The peptide or polypeptide of any one of  claims 2  or  4  to  9 , wherein —C(═NR 4 )—NHR 5  is pyrimidin-2-yl, quinazolin-2-yl, imdidazol-2-yl or benzimidazol-2-yl. 
     
     
         11 . The peptide or polypeptide of any one of the preceding claims, wherein
 (a) the carboxylate of the C-terminus, if not esterified or thioesterified as defined in the preceding claims, is esterified with a fatty alcohol; and/or   (b) the amino group of the N-terminus, if not amidated with a alpha- or beta-hydroxy acid, is amidated with a fatty acid.   
     
     
         12 . The peptide or polypeptide of  claim 11 , wherein said fatty alcohol and/or said fatty acid contains between 5 and 15 carbon atoms, preferably 9 carbon atoms. 
     
     
         13 . The peptide or polypeptide of any one of the preceding claims, wherein the number of guanidinium groups including those of any Arg residue(s), if present, is between 4 and 20, more preferred between 7 and 15, and most preferred 9, wherein at least one guanidinium group is comprised in a guanidinium alkanol, guanidinium alkanethiol, guanidinium alkanoic acid, PEG substituted with a guanidinium group and having a free hydroxyl group, PEG substituted with a guanidinium group and a sulfhydryl group or PEG substituted with a guanidinium group and a carboxyl group as defined in any one of  claims 1  to  10 . 
     
     
         14 . The peptide or polypeptide of any one of  claims 1  to  13 , wherein said peptide or polypeptide is part of a peptide-PNA chimera. 
     
     
         15 . A pharmaceutical composition comprising a peptide or polypeptide of any one of  claims 1  to  13 .