Single-chain insulin

Abstract

The present invention is related to single-chain insulin having insulin activity comprising a B- and an A-chain or a modified B- and A-chain connected by a connecting peptide of from 6-11 amino acids. The single-chain insulins will have biological insulin activity and an IGF-1 receptor affinity similar to or lower than that of human insulin and a high physical stability. The single-chain insulin may contain at least one basic amino acid residues in the connecting peptide. The single-chain insulins may also be acylated in one or more Lys residues.

1 . A single-chain insulin polypeptide comprising the B-chain and the A-chain of human insulin, or analogues or derivatives thereof, wherein the B-chain and A-chain are connected by a connecting peptide having from 5-11 amino acid residues, wherein the connecting peptide does not contain two adjacent basic amino acid residues and wherein the single-chain insulin has an affinity to the human insulin receptor of at least about 20% of that of human insulin if the single-chain insulin molecule is not chemically modified by acylation.  
   
   
       2 . A single-chain insulin polypeptide comprising the B-chain and the A-chain of human insulin or analogues or derivatives thereof, wherein the B-chain and A-chain are connected by a connecting having from 5-11 amino acid residues, provided that, if the connecting peptide contains two adjacent basic amino acid residues: 
 (i) at least one of the natural amino acid residues in the B and/or A chain is substituted with another amino acid residue or    (ii) at least one lysine residue in the A-chain, in the B-chain or in the connecting peptide has been chemically modified by acylation or    (ii) the connecting peptide is not one of the following sequences Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO:1), Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO:2), Gly-Gly-Gly-Gly-Gly-Lys-Arg (SEQ ID NO:3), Arg-Arg-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:4), Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO:5), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO:6), Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO:7), Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO:8), Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO:9), or Arg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO:10).    
   
   
       3 . A single-chain insulin polypeptide comprising the B-chain and the A-chain of human insulin, or analogues or derivatives thereof, wherein the B-chain and A-chain are connected by a connecting peptide having from 5-11 amino acid residues, provided that, if the polypeptide has a pI above about 6.5, it comprises (i) at least one amino acid residue substitution and/or deletion in the A-chain and/or B chain compared to the human insulin A-chain and B-chain or (i) at least one lysine residue in the A-chain, in the B-chain, or in the connecting peptide has been chemically modified by acylation.  
   
   
       4 . A single-chain insulin polypeptide comprising the B-chain and the A-chain of human insulin, or analogues or derivatives thereof, wherein the B-chain and A-chain are connected by a connecting peptide having from 5-14 amino acid residues and wherein at least one lysine residue in the A-chain, in the B-chain or in the connecting peptide has been chemically modified by acylation.  
   
   
       5 . A polypeptide according to  claim 1 , wherein said peptide is acylated in at least one lysine group.  
   
   
       6 . A polypeptide according to  claim 5 , wherein said polypeptide is acylated at residue B29.  
   
   
       7 . A polypeptide according to  claim 6 , wherein said polypeptide is acylated with a fatty acid having from 6 to 24 C-atoms.  
   
   
       8 . A polypeptide according to  claim 1 , wherein said polypeptide is soluble at neutral pH and has a pI below about 6.5.  
   
   
       9 . A polypeptide according to  claim 8 , wherein said polypeptide has a pI from about 4.5 to below about 6.5.  
   
   
       10 . A polypeptide according to  claim 1 , wherein said polypeptide has an affinity to the human insulin receptor of at least 30% of that of human insulin.  
   
   
       11 . A polypeptide according to  claim 1 , wherein the connecting peptide comprises a sequence selected from the group consisting of AGRGSGK (SEQ ID NO:15); AGLGSGK (SEQ ID NO:33); AGMGSGK (SEQ ID NO:45); ASWGSGK (SEQ ID NO:48); TGLGSGQ (SEQ ID NO:22); TGLGRGK (SEQ ID NO:23); TGLGSGK (SEQ ID NO:21); HGLYSGK (SEQ ID NO:50); KGLGSGQ (SEQ ID NO:51); VGLMSGK (SEQ ID NO:56); VGLSSGQ (SEQ ID NO:27); VGLYSGK (SEQ ID NO:28), VGLSSGK (SEQ ID NO:30); VGMSSGK (SEQ ID NO:65); VWSSSGK (SEQ ID NO:76), VGSSSGK (SEQ ID NO:16), and VGMSSGK (SEQ ID NO:106).  
   
   
       13 . A polypeptide according to  claim 1  having the formula  
       B(1-26)-X 1 -X 2 -X 3 -X 4 -A(1-21)  wherein    X 1  is Thr, Lys, Arg, or a peptide bond; X 2  is Pro, Lys, Asp, or a peptide bond; X 3  is Lys, Pro, Glu, or a peptide bond; X 4  is a peptide sequence of 6-11 amino acid residues, B(1-26) is a peptide chain consisting of the first 26 amino acid residues of the B chain of human insulin counted from the N-terminal end of the B chain or an analogue or derivative thereof, and A(1-21) is the natural insulin A chain or an analogue thereof or derivative thereof, wherein X 4  does not contain two adjacent basic amino acid residues.    
   
   
       14 . A polypeptide according to  claim 2  having the formula  
       B(1-26)-X 1 -X 2 -X 3 -X 4 -A(1-21)  wherein    X 1  is Thr, Lys, Arg, or a peptide bond; X 2  is Pro, Lys, Asp, or a peptide bond; X 3  is Lys, Pro, Glu, or a peptide bond; X 4  is a peptide sequence of 6-11 amino acid residues, B(1-26) is a peptide chain consisting of the first 26 amino acid residues of the B chain of human insulin counted from the N-terminal end of the B chain or an analogue or derivative thereof, and A(1-21) is the natural insulin A chain or an analogue thereof or derivative thereof.    
   
   
       15 . A polypeptide according to  claim 3  having the formula  
       B(1-26)-X 1 -X 2 -X 3 -X 4 -A(1-21)  wherein    X 1  is Thr, Lys, Arg, or a peptide bond; X 2  is Pro, Lys, Asp, or a peptide bond; X 3  is Lys, Pro, Glu, or a peptide bond; X 4  is a peptide sequence of 6-11 amino acid residues, B(1-26) is a peptide chain consisting of the first 26 amino acid residues of the B chain of human insulin counted from the N-terminal end of the B chain or an analogue or derivative thereof, and A(1-21) is the natural insulin A chain or an analogue thereof or derivative thereof, provided that single-chain insulins with a pI above about 6.5 will comprise at least one amino acid residue substitution and/or deletion in the A- and/or B chain compared to the human insulin A- and B-chains or at least one lysine residue in the A-chain, in the B-chain or in the connecting peptide has been chemically modified by acylation.    
   
   
       16 . A polypeptide according to  claim 4  having the formula  
       B(1-26)-X 1 -X 2 -X 3 -X 4 -A(1-21)  wherein    X 1  is Thr, Lys, Arg, or a peptide bond; X 2  is Pro, Lys, Asp, or a peptide bond; X 3  is Lys, Pro, Glu, or a peptide bond; X 4  is a peptide sequence of 5-11 amino acid residues, B(1-26) is a peptide chain consisting of the first 26 amino acid residues of the B chain of human insulin counted from the N-terminal end of the B chain or an analogue or derivative thereof, and A(1-21) is the natural insulin A chain or an analogue thereof or derivative thereof, wherein at least one lysine residue in the A-chain, in the B-chain or in the connecting peptide has been chemically modified by acylation.    
   
   
       17 . A polypeptide according to  claim 1 , wherein the amino acid residue in position A21 is substituted by another amino acid residue except Cys.  
   
   
       18 . A polypeptide according to  claim 13 , wherein X 4  is a peptide sequence with the following formula X a -X b -X c -X d -X e -X f -X g  (SEQ ID NO:129) wherein 
 X a  is selected from the group consisting of L, R, T, A, H, Q, G, S, V, and a peptide bond;    X b  is selected from the group consisting of W, G, S, A, H, R, T, and a peptide bond;    X c  is selected from the group consisting of L, Y, M, H, R, T, Q, K, V, S, A, G, P, and a peptide bond;    X d  is selected from the group consisting of R, A, Y, M, S, N, H, G, and a peptide bond;    X e  is selected from the group consisting of S, R, A, T, K P, N M, H, Q, V, G, and a peptide bond;    X f  is selected from the group consisting of G, A, and a peptide bond; and    X g  is selected from the group consisting of K, R, P, H, F, T, I, Q, W, A, and a peptide bond.    
   
   
       19 . A polypeptide according to according to  claim 13 , wherein X 4  comprises the sequence SGK.  
   
   
       20 . A polypeptide according to  claim 13 , wherein X 4  comprises the sequence GSGK (SEQ ID NO:131)  
   
   
       21 . A polypeptide according to  claim 13 , wherein X 4  comprises the sequence SSSGK (SEQ ID NO:132)  
   
   
       22 . A polypeptide according to  claim 1 , wherein at least one of the natural amino acid residues in the positions B1, B3, B10, B22, B28, B29, A8, A15, A18 and A21 is substituted by another amino acid residue.  
   
   
       23 . A polypeptide according to  claim 1  selected from the group consisting of: desB1, desB27, desB28, and desB29 insulin analogues.  
   
   
       24 . A polynucleotide sequence encoding a polypeptide according to  claim 1 .  
   
   
       25 . An expression vector containing a polynucleotide sequence according to  claim 24 .  
   
   
       26 . A transformed host cell containing an expression vector according to  claim 25 .  
   
   
       27 . A pharmaceutical preparation comprising a biologically active amount of a polypeptide according to  claim 1 .  
   
   
       28 . A pharmaceutical preparation according to  claim 28 , further comprising a second biologically active compound.  
   
   
       29 . A pharmaceutical preparation according to  claim 28 , wherein the second compound is selected from the group consisting of rapid acting insulin analogues, protracted insulin analogues, GLP-1, GLP-2 and exendin or analogues or derivatives thereof.  
   
   
       30 . A method of reducing the blood glucose level in mammalians, said method comprising administrating a therapeutically active dose of a pharmaceutical preparation according to  claim 27  to a patient in need of such treatment.