Peptide inhibitors of inflammation mediated by selectins

Abstract

Peptides derived from three regions of the lectin domain of GMP-140 and the related selectins, ELAM-1 and the lymphocyte homing receptor, have been found to inhibit neutrophil adhesion to GMP-140. These and additional peptides have been synthesized, having as their core region portions of the 56-60 amino acid sequence of GMP-140, with residue 1 defined as the N-terminus of the mature protein after the cleavage of the signal peptide. Examples demonstrate the inhibition of the binding of neutrophils to GMP-140 of peptides in concentrations ranging from 5 to 1500 μmol. It has been found that alterations within the core sequence, as well as N-terminal and C-terminal flanking regions, do not result in loss of biological activity. The peptides are useful as diagnostics and, in combination with a suitable pharmaceutical carrier, for clinical applications in the modulation or inhibition of coagulation processes or inflammatory processes.

We claim:  
     
         1 . A peptide inhibiting binding of selectins selected from the group having the formula:  
       R 1 —X-A-B—C-D-E-Y—R 2   (I)  or a pharmaceutically acceptable acid- or based-addition salt thereof wherein: 
 A is D- or L-asparagine, D- or L-isoleucine or D- or L-valine;  
 B is D- or L-asparagine or glycine;  
 C is D- or L-lysine, D- or L-valine or glycine;  
 D is D- or L-valine, D- or L-threonine or D- or L-isoleucine;  
 E is D- or L-tryptophan;  
 X and Y are linear chains of from 0 to 10 amino acids;  
 R 1  is H (signifying a free N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl, aroyl, alkyloxycarbonyl or aryloxycarbonyl and  
 R 2  is OH (signifying a free C-terminal group) lower alkyl or aryl esters, or NR 3 R 4  where R 3  and R 4  each selected independently from H, lower alkyl or aryl.  
   
     
     
         2 . The peptide of  claim 1  wherein X is selected from the group consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys, Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys, D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.  
     
     
         3 . The peptide of  claim 1  wherein Y is selected from the group consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val, Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn, Thr-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys.  
     
     
         4 . The peptide of  claim 1  selected from the group consisting of peptides having the formula: 
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Lys-Asn-Asn-Lys-Thr-Trp-NH 2 .  
 Acetyl-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH 2 ;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-NH 2 ;  
 Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Glu-NH 2 ;  
 Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 FMOC-Asn-Asn-Lys-Thr-Trp-NH 2 ; and pharmaceutically acceptable acid- or base-addition salts thereof.  
 
     
     
         5 . The peptide of  claim 1  in combination with a pharmaceutical carrier selected from the group consisting of carriers suitable for parenteral administration, oral administration, topical administration, and controlled release formulations.  
     
     
         6 . A method for the preparation of peptides of the formula:  
       R 1 —X-A-B—C-D-E-Y—R 2   (I)  or a pharmaceutically acceptable acid- or based-addition salt thereof wherein: 
 A is D- or L-asparagine, D- or L-isoleucine or D- or L-valine;  
 B is D- or L-asparagine or glycine;  
 C is D- or L-lysine, D- or L-valine or glycine;  
 D is D- or L-valine, D- or L-threonine or D- or L-isoleucine;  
 E is D- or L-tryptophan;  
 X and Y are linear chains of from 0 to 10 amino acids;  
 R 1  is H (signifying a free N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl, aroyl, alkyloxycarbonyl or aryloxycarbonyl and  
 R 2  is OH (signifying a free C-terminal group) lower alkyl or aryl esters, or NR 3 R 4  where R 3  and R 4  each selected independently from H, lower alkyl or aryl.  
   
     
     
         7 . The method of  claim 6  wherein X is selected from the group consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys, Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys, D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.  
     
     
         8 . The method of  claim 6  wherein Y is selected from the group consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val, Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn, Thr-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys.  
     
     
         9 . The method of  claim 6  selected from the group consisting of peptides having the formula: 
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Lys-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Acetyl-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH 2 ;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-NH 2 ;  
 Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2;    
 Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Glu-NH 2 ;  
 Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 FMOC-Asn-Asn-Lys-Thr-Trp-NH 2 ; and pharmaceutically acceptable acid- or base-addition salts thereof.  
 
     
     
         10 . The method for preparation of the peptide of  claim 6  whereby the amino acids are assembled either singly or in preformed blocks in solution or suspension by chemical ligation techniques.  
     
     
         11 . The method for preparation of a peptide of  claim 6  whereby the amino acids are assembled either singly or in preformed blocks in solution or suspension by enzymatic ligation techniques.  
     
     
         12 . The method for preparation of a peptide of  claim 11  whereby the peptide is produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide.  
     
     
         13 . A method for modifying binding of a selectin comprising providing a peptide having the formula:  
       R 1 —X-A-B—C-D-E-Y—R 2   (I)  or a pharmaceutically acceptable acid- or based-addition salt thereof wherein: 
 A is D- or L-asparagine, D- or L-isoleucine or D- or L-valine;  
 B is D- or L-asparagine or-glycine;  
 C is D- or L-lysine, D- or L-valine or glycine;  
 D is D- or L-valine, D- or L-threonine or D- or L-isoleucine;  
 E is D- or L-tryptophan;  
 X and Y are linear chains of from 0 to 10 amino acids;  
 R 1  is H (signifying a free N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl, aroyl, alkyloxycarbonyl or aryloxycarbonyl and  
 R 2  is OH (signifying a free C-terminal group) lower alkyl or aryl esters, or NR 3 R 4  where R 3  and R 4  each selected independently from H, lower alkyl or aryl.  
   
     
     
         14 . The method of  claim 13  wherein X is selected from the group consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys, Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys, D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.  
     
     
         15 . The method of  claim 13  wherein Y is selected from the group consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val, Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn, Thr-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Gln, Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys.  
     
     
         16 . The method of  claim 13  selected from the group consisting of peptides having the formula: 
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-Glu-Cys-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Lys-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Acetyl-Asn-Asn-Lys-Thr-Trp-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH 2 ;  
 Arg-Lys-Val-Asn-Asn-Val-Trp-NH 2 ;  
 Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Glu-NH 2 ;  
 Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH 2 ;  
 D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH 2 ;  
 FMOC-Asn-Asn-Lys-Thr-Trp-NH 2 ; and pharmaceutically acceptable acid- or base-addition salts thereof.  
 
     
     
         17 . The method of  claim 13  wherein the pharmaceutical carrier is selected from the group consisting of carriers suitable for parenteral administration, oral administration, topical administration, and controlled release formulations.