Insulinotropic peptide derivative with modified n-terminal charge

Abstract

The present invention relates to an insulinotropic peptide derivative with a modified N-terminal charge and a pharmaceutical composition including the same. Specifically, the insulinotropic peptide derivative is characterized in that the N-terminal positive charge of the insulinotropic peptide is modified to a neutral or net negative charge at neutral pH. The insulinotropic peptide derivative according to the present invention is rapidly dissociated from the GLP-1 receptor owing to the above modification in the N-terminal charge, and exhibits enhanced insulinotropic ability and blood glucose-lowering activity compared to the native insulinotropic peptide while maintaining its stability in blood. Accordingly, the insulinotropic peptide derivative of the present invention is very useful for the treatment of type 2 diabetes.

1 . An insulinotropic peptide derivative, wherein the insulinotropic peptide derivative is a native insulinotropic peptide having a modification on its N-terminal charge. 
     
     
         2 . The insulinotropic peptide derivative of  claim 1 , wherein the positive charge on the N-terminal amino group or N-terminal amino acid residue of the native insulinotropic peptide, or its analogue, or a fragment thereof is chemically modified to a neutral or net negative charge. 
     
     
         3 . The insulinotropic peptide derivative of  claim 2 , wherein the N-terminal residue of the native insulinotropic peptide, or its analogue, or a fragment thereof is histidine, and the modification is removal or substitution of the alpha-amino group of the histidine residue, or removal of the alpha-carbon, thereby having a neutral or net negative charge. 
     
     
         4 . The insulinotropic peptide derivative of  claim 3 , wherein the modification is at least one selected from the group consisting of:
 removing the N-terminal amino group of the native insulinotropic peptide, or its analogue or a fragment thereof;   substituting the N-terminal amino group of the native insulinotropic peptide, or its analogue or a fragment thereof with a hydroxyl group;   modifying the N-terminal amino group of the native insulinotropic peptide, or its analogue or a fragment thereof with two methyl groups;   substituting the N-terminal amino group of the native insulinotropic peptide, or its analogue or a fragment thereof with a carboxyl group;   removing the alpha-carbon of the N-terminal histidine residue of the native insulinotropic peptide, or its analogue or a fragment thereof thereby leaving only the imidazoacetyl group; and   removing the N-terminal amino group of the native insulinotropic peptide, or its analogue or a fragment thereof while substituting the C-terminal carboxyl group of the native insulinotropic peptide, or its analogue or a fragment thereof with propylamide.   
     
     
         5 . The insulinotropic peptide derivative of  claim 4 , wherein the N-terminal residue of the native insulinotropic peptide or its analogue or a fragment thereof is substituted with a material selected from the group consisting of desamino-histidyl, dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, and beta-carboxyimidazopropionyl. 
     
     
         6 . The insulinotropic peptide derivative of  claim 4 , wherein the N-terminal residue of the native insulinotropic peptide or its analogue or a fragment thereof is substituted with desamino-histidyl, and the C-terminal carboxyl group of the native insulinotropic peptide or its analogue or a fragment thereof is substituted with propylamide. 
     
     
         7 . The insulinotropic peptide derivative of  claim 1 , wherein the insulinotropic peptide derivative has a binding activity with a GLP-1 receptor. 
     
     
         8 . The insulinotropic peptide derivative of  claim 1 , wherein the native insulinotropic peptide derivative is GLP-represented by SEQ ID NO: 1, exendin-4 represented by SEQ ID NO: 2, exendin-3 represented by SEQ ID NO: 3, oxyntomodulin represented by SEQ ID NO: 5, GIP represented by SEQ ID NO: 6, an analogue thereof, or a fragment thereof. 
     
     
         9 . The insulinotropic peptide derivative of  claim 8 , wherein the native insulinotropic peptide derivative or its analogue is a GLP-1 analogue, and is selected from the group consisting of Arg 34 -GLP-1(7-37), Gly 8 -GLP-1(7-36)-amide, Gly 8 -GLP-1(7-37), Val 8 -GLP-1(7-36)-amide, Val 8 -GLP-1(7-37), Val 8 Asp 22 -GLP-1(7-36)-amide, Val 8 Asp 22 -GLP-1(7-37), Val 8 Glu 22 -GLP-1(7-36)-amide, Val 8 Glu 22 -GLP-1(7-37), Val 8 Lys 22 -GLP-1(7-36)-amide, Val 8 Lys 22 -GLP-1(7-37), Val 8 Arg 22 -GLP-1(7-36)-amide, Val 8 Arg 22 -GLP-1(7-37), Val 8 His 22 -GLP-1(7-36)-amide, Val 8 His 22 -GLP-1(7-37), Val 8 Trp 16 Glu 22 -GLP-1(7-37), Val 8 Glu 22 Val 25 -GLP-1(7-37), Val 8 Tyr 16 Glu 22 -GLP-1(7-37), Val 8 Trp 16 Glu 22 -GLP-1(7-37), Val 8 Leu 16 Glu 22 -GLP-1(7-37), Val 8 Tyr 18 Glu 22 -GLP-1(7-37), Val 8 Glu 22 His 37 -GLP-1(7-37), Val 8 Glu 22 Ile 33 -GLP-1(7-37), Val 8 Trp 16 Glu 22 Val 25 Ile 33 -GLP-1(7-37), Val 8 Trp 16 Glu 22 Ile 33 -GLP-1(7-37), Val 8 Glu 22 Val 25 Ile 33 -GLP-1(7-37), and Val 8 Trp 16 Glu 22 Val 25 -GLP-1(7-37). 
     
     
         10 . The insulinotropic peptide derivative of  claim 8 , wherein the native insulinotropic peptide derivative or its analogue is a ZP-10 (Ser 38 Lys 39 -exendin-4(1-39)-LysLysLysLysLys-amide), which is an exendin-4 analogue represented by SEQ ID NO: 4. 
     
     
         11 . The insulinotropic peptide derivative of  claim 1 , wherein the insulinotropic peptide derivative has a higher dissociation constant (Kd) for the GLP-1 receptor compared to the native insulinotropic peptide. 
     
     
         12 . The insulinotropic peptide derivative of  claim 5 , wherein the insulinotropic peptide derivative is represented by the following Formula 1:
   R1-X—R2  <Formula 1>
   wherein R1 is selected from the group consisting of desamino-histidyl, dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, and beta-carboxy imidazopropionyl;   R2 is —NH 2  or —OH,   X is Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;   Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser;   Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser; or   Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala.   
     
     
         13 . The insulinotropic peptide derivative of  claim 6 , wherein the N-terminal histidine residue of GLP-1, exendin-4, exendin-3, oxyntomodulin, GIP, or an analogue thereof is substituted with desamino-histidyl, and the C-terminal carboxyl group of GLP-1, exendin-4, exendin-3, oxyntomodulin, GIP, or an analogue thereof is substituted with propylamide. 
     
     
         14 . The insulinotropic peptide derivative of  claim 12 , wherein the insulinotropic peptide is selected from the group consisting of desamino-histidyl-exendin-4 prepared by removing the N-terminal amino group of exendin-4; beta-hydroxy imidazopropionyl-exendin-4 prepared by substituting the N-terminal amino group of exendin-4 with a hydroxyl group; beta-carboxyimidazopropionyl-exendin-4 prepared by substituting the N-terminal amino group of exendin-4 with a carboxyl group; dimethyl-histidyl-exendin-4 prepared by modifying the N-terminal amino group of exendin-4 with two methyl groups; and imidazoacetyl-exendin-4 prepared by removing the alpha-carbon of histidine, which is the first amino acid of exendin-4. 
     
     
         15 . The insulinotropic peptide derivative of  claim 13 , wherein the insulinotropic peptide derivative is DA-exendin-4-propyl-amide prepared by removing the N-terminal amino group of exendin-4 and substituting the C-terminal carboxyl group with propylamide. 
     
     
         16 . A pharmaceutical composition for the treatment of diabetes comprising as an active ingredient the insulinotropic peptide derivative as defined in  claim 1 . 
     
     
         17 . A method for treating diabetes comprising administering a therapeutically effective amount of the insulinotropic peptide derivative as defined in  claim 1  to a subject in need thereof.