Polymyxin B Analogs for LPS Detoxification

Abstract

The invention relates to SAEP II peptide dimers that mimic polymyxin B i.a. in its ability to bind non-covalently the lipopolysaccharide (LPS) of Gram-negative bacteria with high affinity, and therefore to detoxify LPS as polymyxin B does. The dimeric structure is maintained by a pair of disulphide bonds involving the two cystein residues present in the peptide sequence, which does not exceed 17 amino acids and essentially comprises cationic and hydrophobic amino acid residues. In the dimers of the invention, peptides may have a parallel or anti-parallel orientation. As a matter of example, a dimer of the invention is constituted by a peptide of formula NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH, either in a parallel or antiparallel dimeric form. SAEP II dimers are useful for treating or preventing septic shock and related disorders generated by Gram-negative bacteria infection. The invention also relates to LPS-peptide complexes in which LPS and SAEP II dimers are non-covalently bound together. These complexes are useful as vaccinal agents against Gram-negative bacteria infection.

1 - 28 . (canceled) 
   
   
       29 . A method of preparing an SAEP II peptide dimer of formula (I),
   NH 2 -A-Cys1-B-Cys2-C-COOH     NH 2 -A′-Cys1-B′-Cys2-C′-COOH   wherein Cys1 and Cys2 are each a cysteine amino acid;   wherein the two Cys1 residues are linked together through an intermolecular disulphide bond and the two Cys2 residues are linked together through an intermolecular disulphide bond;   wherein A and A′ independently are a peptide moiety of from 2 to 5 amino acid residues, in which at least 2 amino acid residues, are independently selected from Lys, Hyl (hydroxy-Lysine), Arg and His;   wherein B and B′ independently are a peptide moiety of from 3 to 7 amino acid residues, which comprise at least two amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp; and   wherein C and C′ are optional and are independently an amino acid residue or a peptide moiety of from 2 to 3 amino acid residues;   provided that the cationic amino acid residues/hydrophobic amino acid residues ratio (cat/hydroph ratio) is from 0.4 to 2;   the method comprising separating the dimer of formula (I) from
 (a) corresponding dimers of formula (II),
   NH 2 -A-Cys1-B-Cys2-C-COOH 
   HOOC-C′-Cys2-B′-Cys1-A′-NH 2    
 wherein the Cys1 residues are linked to the Cys2 residues through intermolecular disulphide bonds; and/or 
 
 (b) corresponding monomers of formulae NH 2 -A-Cys1-B-Cys2-C-COOH and NH 2 -A′-Cys1-B′-Cys2-C′-COOH, and/or 
 (c) cyclic counterparts of the corresponding monomers. 
   
   
   
       30 . The method according to  claim 29 , wherein the SAEP II peptide dimer of formula (I) has a cat/hydroph ratio is from 0.5 to 1.5. 
   
   
       31 . The method according to  claim 30 , wherein the cat/hydroph ratio is from 0.6 to 1. 
   
   
       32 . The method according to  claim 31 , wherein the cat/hydroph ratio is from 0.6 to 0.8. 
   
   
       33 . The method according to  claim 29 , wherein the B and B′ peptide moieties comprise the sequence -X1-X2-X3-, in which X1 and X2; X2 and X3; or X1, X2 and X3 are independently selected from Val, Leu, Ile, Phe, Tyr and Trp. 
   
   
       34 . The method according to  claim 33  wherein the B and B′ peptide moieties comprise:
 (i) the sequence -X1-X2-X3-, in which:
 X1 is Lys, Hyl, His or Arg; 
 X2 is Phe, Leu, Ile, Tyr, Trp or Val; and 
 X3 is Phe, Leu, Ile, Tyr, Trp or Val; and 
   (ii) amino acid residues, if any, each being independently selected from the group consisting of Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His.   
   
   
       35 . The method according to  claim 29 , wherein the SAEP II peptide dimer of formula (I) is of formula (III)
   NH 2 -A-Cys1-B-Cys2-COOH     NH 2 -A′-Cys1-B′-Cys2-COOH   wherein the two Cys1 residues are linked together through a disulphide bond and the two Cys2 residues are linked together through a disulphide bond;   and the SAEP II peptide dimer of formula (II) is of formula (IV)
   NH 2 -A-Cys1-B-Cys2-COOH 
   HOOC-Cys2-B′-Cys1-A′-NH 2 , 
   wherein the Cys1 residues are linked to the Cys2 residues through a disulphide bond, and
 the monomers are of formulae NH 2 -A-Cys1-B-Cys2-COOH or NH 2 -A′-Cys1-B′-Cys2-COOH. 
   
   
   
       36 . The method of  claim 29  wherein the SAEP II peptide dimer of formulae I is a homologous peptide dimer. 
   
   
       37 . The method of  claim 29  wherein the SAEP II peptide dimer of formula I is a parallel dimer of formula (VII)
   NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH     NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH   wherein the two Cys1 residues are linked together through a disulphide bond and the two Cys2 residues are linked together through a disulphide bond, and
 the SAEP II peptide dimer of formula II is an antiparallel dimer form of formula (VI)
   NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH 
   COOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH 2 , 
 
   wherein the Cys1 residues are linked to the Cys2 residues through a disulphide bond, and
 and the monomers are of formulae NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH and NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH. 
   
   
   
       38 . A method of preparing an SAEP II peptide dimer of formula (II),
   NH 2 -A-Cys1-B-Cys2-C-COOH     HOOC-C′-Cys2-B′-Cys1-A′-NH 2      wherein Cys1 and Cys2 are each a cysteine amino acid;   wherein the Cys1 residues are linked to the Cys2 residues through intermolecular disulphide bonds;   wherein the two Cys1 residues are linked together through an intermolecular disulphide bond and the two Cys2 residues are linked together through an intermolecular disulphide bond;   wherein A and A′ independently are a peptide moiety of from 2 to 5 amino acid residues, in which at least 2 amino acid residues, are independently selected from Lys, Hyl (hydroxy-Lysine), Arg and His;   wherein B and B′ independently are a peptide moiety of from 3 to 7 amino acid residues, which comprise at least two amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp; and   wherein C and C′ are optional and are independently an amino acid residue or a peptide moiety of from 2 to 3 amino acid residues;   provided that the cationic amino acid residues/hydrophobic amino acid residues ratio (cat/hydroph ratio) is from 0.4 to 2;   the method comprising separating the dimer of formula (I) from
 (a) corresponding dimers of formula (I),
   NH 2 -A-Cys1-B-Cys2-C-COOH 
   NH 2 -A′-Cys1-B′-Cys2-C′-COOH 
 wherein Cys1 and Cys2 are each a cysteine amino acid; and/or 
 
 (b) corresponding monomers of formulae NH 2 -A-Cys1-B-Cys2-C-COOH and NH 2 -A′-Cys1-B′-Cys2-C′-COOH, and/or 
 (c) cyclic counterparts of the corresponding monomers. 
   
   
   
       39 . The method according to  claim 38 , wherein the SAEP II peptide dimer of formula (II) has a cat/hydroph ratio is from 0.5 to 1.5. 
   
   
       40 . The method according to  claim 39 , wherein the cat/hydroph ratio is from 0.6 to 1. 
   
   
       41 . The method according to  claim 40 , wherein the cat/hydroph ratio is from 0.6 to 0.8. 
   
   
       42 . The method according to  claim 38 , wherein the B and B′ peptide moieties comprise the sequence -X1-X2-X3-, in which X1 and X2; X2 and X3; or X1, X2 and X3 are independently selected from Val, Leu, Ile, Phe, Tyr and Trp. 
   
   
       43 . The method according to  claim 42  wherein the B and B′ peptide moieties comprise:
 (i) the sequence -X1-X2-X3-, in which:
 X1 is Lys, Hyl, His or Arg; 
 X2 is Phe, Leu, Ile, Tyr, Trp or Val; and 
 X3 is Phe, Leu, Ile, Tyr, Trp or Val; and 
   (ii) amino acid residues, if any, each being independently selected from the group consisting of Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His.   
   
   
       44 . The method according to  claim 38 , wherein the SAEP II peptide dimer of formula (I) is of formula (III)
   NH 2 -A-Cys1-B-Cys2-COOH     NH 2 -A′-Cys1-B′-Cys2-COOH   wherein the two Cys1 residues are linked together through a disulphide bond and the two Cys2 residues are linked together through a disulphide bond;   and the SAEP II peptide dimer of formula (II) is of formula (IV)
   NH 2 -A-Cys1-B-Cys2-COOH 
   HOOC-Cys2-B′-Cys1-A′-NH 2 , 
   wherein the Cys1 residues are linked to the Cys2 residues through a disulphide bond, and
 the monomers are of formulae NH 2 -A-Cys1-B-Cys2-COOH or NH 2 -A′-Cys1-B′-Cys2-COOH. 
   
   
   
       45 . The method of  claim 38  wherein the SAEP II peptide dimer of formulae II is a homologous peptide dimer. 
   
   
       46 . The method of  claim 38  wherein the SAEP II peptide dimer of formula II is an antiparallel dimer form of formula (VI)
   NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH     COOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH 2 ,   wherein the Cys1 residues are linked to the Cys2 residues through a disulphide bond, and the SAEP II peptide dimer of formula I is a parallel dimer of formula (VII)
   NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH 
   NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH 
   wherein the two Cys1 residues are linked together through a disulphide bond and the two Cys2 residues are linked together through a disulphide bond, and
 the monomers are of formulae NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH and NH 2 -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.