Methods and compositions for the treatment of pancreatitis

Abstract

Methods and compositions for the treatment of acute pancreatitis in a mammal. Particular compositions comprise a binding element, a translocation element, and a therapeutic element able to prevent accumulation of digestive enzymes within the pancreas.

1 - 13 . (canceled)  
     
     
         14 . A method for making a polypeptide comprising: 
 a) expressing within a host cell a recombinant chimeric polypeptide comprising an amino terminal side extein and a carboxyl terminal side intein     wherein said extein comprises able to facilitate the transfer of a polypeptide across a vesicular membrane and a therapeutic element able, when present in the cytoplasm of a pancreatic cell, to inhibit enzymatic secretion by said pancreatic cell, and     wherein said intein comprises a binding element capable of affinity binding under selective conditions with a binding partner and an amino terminal end first amino acid selected from the group consisting of cysteine, serine or threonine.    b) contacting said chimeric polypeptide with a synthetic peptide and a nucleophilic reagent     wherein said synthetic peptide comprises a CCK binding element able to selectively bind a pancreatic cell surface marker under physiological conditions, a carboxyl terminal end amidated phenylalanine modification and an amino terminal end second amino acid selected from the group consisting of cysteine, serine or threonine,     wherein said nucleophilic reagent is able to cause cleavage of said intein from the said extein, and     wherein subsequent formation of a peptide bond occurs between carboxyl terminal end of said extein and amino terminal end of said synthetic peptide.    
     
     
         15 . The method of  claim 14  wherein said first and second amino acids are cysteine.  
     
     
         16 . The method of  claim 15  wherein said nucleophilic reagent is selected from the group consisting of phenol or thiphenol.  
     
     
         17 . The method of  claim 14  wherein said synthetic polypeptide further comprises a sulfated tyrosine at the position 7 amino acids from a natural C terminus of said sequence, and said therapeutic polypeptide preferentially binds a CCK-A receptor.  
     
     
         18 . The method of  claim 17  wherein said first and second amino acids are cysteine.  
     
     
         19 . The method of  claim 18  wherein said nucleophilic reagent is selected from the group consisting of phenol or thiphenol.  
     
     
         20 . The method of  claim 14  wherein said first and second amino acids are serine.  
     
     
         21 . The method of  claim 14  wherein said first and second amino acids are threonine.  
     
     
         22 . The method of  claim 17  wherein said first and second amino acids are serine.  
     
     
         23 . The method of  claim 17  wherein said first and second amino acids are threonine.  
     
     
         24 . A method for making a polypeptide comprising: 
 a) expressing within a host cell a recombinant chimeric polypeptide comprising an amino terminal side extein and a carboxyl terminal side intein     wherein said extein comprises a translocation element able to facilitate the transfer of a polypeptide across a vesicular membrane and a therapeutic element able, when present in the cytoplasm of a pancreatic cell, to inhibit enzymatic secretion by said pancreatic cell, and     wherein said intein comprises a binding element capable of affinity binding under selective conditions with a binding partner and an amino terminal end first amino acid selected from the group consisting of cysteine, serine or threonine.    b) contacting said chimeric protein with a synthetic peptide and a nucleophilic reagent     wherein said synthetic peptide comprises a binding element able to selectively bind a pancreatic cell surface marker under physiological conditions, a carboxyl terminal end amidated phenylalanine modification, and an amino terminal end second amino acid selected from the group consisting of cysteine, serine or threonine,     wherein said nucleophilic reagent is able to cause cleavage of said intein from the said extein, and     wherein subsequent formation of a peptide bond occurs between carboxyl terminal end of said extein and amino terminal end of said synthetic peptide.    
     
     
         25 . The method of  claim 24  wherein said therapeutic element will cleave a SNARE protein.  
     
     
         26 . The method of  claim 25  wherein said SNARE protein is selected from the group consisting of syntaxin, SNAP-25 and VAMP.  
     
     
         27 . The method of  claim 24  wherein said binding element of said synthetic peptide comprises a CCK sequence.  
     
     
         28 . The method of  claim 27  wherein said CCK sequence comprises a human CCK A amino acid sequence.  
     
     
         29 . The method of  claim 28  wherein said CCK A amino acid sequence comprises SEQ ID NO: 6.  
     
     
         30 . The method of  claim 28  wherein said CCK A amino acid sequence comprises SEQ ID NO: 5.  
     
     
         31 . The method of  claim 28  wherein said CCK A amino acid sequence comprises SEQ ID NO: 4.  
     
     
         32 . The method of  claim 28  wherein said CCK A amino acid sequence comprises SEQ ID NO: 3.  
     
     
         33 . The method of  claim 28  wherein said CCK A amino acid sequence comprises SEQ ID NO: 2.  
     
     
         34 . The method of  claim 24  wherein said first and second amino acids are cysteine.  
     
     
         35 . The method of  claim 24  wherein said nucleophilic reagent is selected from the group consisting of phenol or thiophenol.  
     
     
         36 . The method of  claim 24  wherein said first and second amino acids are serine.  
     
     
         37 . The method of  claim 24  wherein said first and second amino acids are threonine.  
     
     
         38 . The method of  claim 20  wherein said binding element of said synthetic peptide further comprises a sulfated tyrosine at the position 7 residues from the carboxyl terminal end.  
     
     
         39 . The method of  claim 38  wherein said binding element of said synthetic peptide comprises a CCK sequence.  
     
     
         40 . The method of  claim 39  wherein said CCK sequence comprises a human CCK A amino acid sequence.  
     
     
         41 . The method of  claim 39  wherein said CCK A amino acid sequence comprises SEQ ID NO: 6.  
     
     
         42 . The method of  claim 39  wherein said CCK A amino acid sequence comprises SEQ ID NO: 5.  
     
     
         43 . The method of  claim 39  wherein said CCK A amino acid sequence comprises SEQ ID NO: 4.  
     
     
         44 . The method of  claim 39  wherein said CCK A amino acid sequence comprises SEQ ID NO: 3.  
     
     
         45 . The method of  claim 39  wherein said CCK A amino acid sequence comprises SEQ ID NO: 2.