Chemically and thermodynamically stable insulin analogues and improved methods for their production

Abstract

The subject matter of this invention is directed towards chemically and thermodynamically stable single-chain insulin (SCI) analogues that are resistant to deamidation and fibrillation. The invention further discloses improved methods for the recombinant expression, purification and refolding of SCI.

1 - 54 . (canceled) 
     
     
         55 . A compound comprising a B chain peptide, an A chain peptide and a C′ linker peptide; wherein
 the C′ linker peptide covalently links the B chain peptide and the A chain peptide, and has an amino acid sequence Y 1 Y 2 Y 3 Y 4 Y 5 Y 6 Y 7 ; wherein Y 1  is R or absent, Y 2  is R or absent, Y 3  is Y or V, Y 4  is P or absent, Y 5  is G or D, Y 6  is D or G, and Y 7  is any amino acid; and 
 the B chain peptide and the A chain peptide are each independently a therapeutic peptide; 
 wherein the B chain peptide and the A chain peptide are linked together by a disulfide bond. 
 
     
     
         56 . The compound of  claim 55 , wherein the amino acid sequence of the C′ linker peptide is Y 3 Y 5 Y 6 Y 7 . 
     
     
         57 . The compound of  claim 55 , wherein the compound has a Formula (I):
   Stable B chain-C′-Stable A chain  Formula (I).
   
     
     
         58 . The compound of  claim 57 , wherein the amino acid sequence of the C′ linker peptide is Y 3 Y 5 Y 6 Y 7 . 
     
     
         59 . The compound of  claim 55 , wherein the B chain peptide and the A chain peptide are linked together by two disulfide bonds. 
     
     
         60 . The compound of  claim 55 , wherein the C′ linker peptide links C-terminus of the B chain peptide to N-terminus of the A chain peptide. 
     
     
         61 . The compound of  claim 55 , wherein the B chain peptide is a peptide of 50 or fewer amino acids. 
     
     
         62 . The compound of  claim 55 , wherein the A chain peptide is a peptide of 50 or fewer amino acids. 
     
     
         63 . The compound of  claim 55 , wherein the B chain peptide is a peptide of 27-30 amino acids. 
     
     
         64 . The compound of  claim 55 , wherein the A chain peptide is a peptide of 21 amino acids. 
     
     
         65 . The compound of  claim 55 , wherein Y 7  is V or K. 
     
     
         66 . The compound of  claim 55 , wherein the C′ linker peptide is Y 3 GDY 7 . 
     
     
         67 . The compound of  claim 55 , wherein the C′ linker peptide is Y 3 DGY 7 . 
     
     
         68 . The compound of  claim 55 , wherein the C′ linker peptide is Y 3 DGV or Y 3 DGK. 
     
     
         69 . The compound of  claim 55 , wherein the C′ linker peptide is Y 3 GDV or Y 3 GDK. 
     
     
         70 . The compound of  claim 55 , wherein the C′ linker peptide is VDGK. 
     
     
         71 . The compound of  claim 55 , wherein the C′ linker peptide is YDGK. 
     
     
         72 . A polynucleotide comprising a nucleic acid sequence that encodes the compound of  claim 55 . 
     
     
         73 . A method for treating a disorder in a patient in need thereof, the method comprising administering to the patient a compound comprising a B chain peptide, an A chain peptide and a C′ linker peptide; wherein
 the C′ linker peptide covalently links the B chain peptide and the A chain peptide, and has an amino acid sequence Y 1 Y 2 Y 3 Y 4 Y 5 Y 6 Y 7 ; wherein Y 1  is R or absent, Y 2  is R or absent, Y 3  is Y or V, Y 4  is P or absent, Y 5  is G or D, Y 6  is D or G, and Y 7  is any amino acid; and 
 the B chain peptide and the A chain peptide are each independently a therapeutic peptide; 
 wherein the B chain peptide and the A chain peptide are linked together by a disulfide bond. 
 
     
     
         74 . The method of  claim 73 , wherein the disorder is a hormonal disorder.