Long-acting conjugate of triple glucagon/glp-1/gip receptor agonist

Abstract

A long-acting conjugate of a triple agonist which has activities to all of glucagon, GLP-1, and GIP receptors and uses thereof.

1 . A conjugate represented by Chemical Formula 1 below:
   [Chemical Formula 1]     X—La—F
   wherein,   X is a peptide having activities to a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor;   L is a linker;   a is 0 or a positive integer, with the proviso that when a is 2 or greater, each L is independent from each other; and   F is a material capable of increasing the half-life of X.   
     
     
         2 . The conjugate of  claim 1 , wherein X is an analog of native glucagon with a variation selected from the group consisting of substitution, addition, deletion, modification, and a combination thereof, on at least one amino acid of the native glucagon sequence. 
     
     
         3 . The conjugate of  claim 2 , wherein the amino acid sequence with addition is derived from a native GLP-1 amino acid sequence, native GIP amino acid sequence, or native exendin-4 amino acid sequence. 
     
     
         4 . The conjugate of  claim 1 , wherein X is a peptide comprising an amino acid sequence represented by General Formula 1 below: 
       
         
           
                 
               
                   (General Formula 1, SEQ ID NO: 103) 
                 
                   Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10- 
                 
                     
                 
                   Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18- 
                 
                     
                 
                   Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27- 
                 
                     
                 
                   Xaa28-Xaa29-Xaa30-R1 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
         wherein, in General Formula 1, 
         Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y); 
         Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or aminoisobutyric acid (Aib); 
         Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q); 
         Xaa7 is threonine (Thr, T) or isoleucine (Ile, I); 
         Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or valine (Val, V); 
         Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I); 
         Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine (Cys, C); 
         Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y); 
         Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L); 
         Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S); 
         Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K); 
         Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H); 
         Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), or valine (Val, V); 
         Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R); 
         Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D); 
         Xaa23 is isoleucine (Ile, I) or valine (Val, V); 
         Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E); 
         Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M); 
         Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D); 
         Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H); 
         Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or is absent; and 
         R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; 
         wherein: 
         m is -Cys-, -Pro-, or -Gly-Pro-; and 
         n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent. 
       
     
     
         5 . The conjugate of  claim 4 ,
 wherein Xaa14 is leucine or methionine; and   Xaa15 is cysteine, aspartic acid, or leucine.   
     
     
         6 . The conjugate of  claim 4 ,
 wherein, in General Formula 1,   Xaa2 is glycine, α-methyl-glutamic acid, or Aib;   Xaa7 is threonine,   Xaa10 is tyrosine, cysteine, or valine;   Xaa12 is lysine or isoleucine;   Xaa13 is tyrosine, alanine, glutamine, or cysteine;   Xaa14 is leucine, cysteine, or methionine;   Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;   Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;   Xaa18 is alanine, glutamine, arginine, or histidine;   Xaa19 is alanine, glutamine, valine, or cysteine;   Xaa20 is lysine, arginine, or glutamine;   Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;   Xaa23 is isoleucine or valine;   Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and   Xaa27 is leucine or lysine.   
     
     
         7 . The conjugate of  claim 4 , wherein X is a peptide comprising an amino acid sequence represented by General Formula 2 below: 
       
         
           
                 
               
                   (General Formula 2, SEQ ID NO: 104) 
                 
                   Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser- 
                 
                     
                 
                   Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19- 
                 
                     
                 
                   Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28- 
                 
                     
                 
                   Xaa29-Xaa30-Xaa31- Ser-Ser-Gly-Gln-Pro-Pro-Pro- 
                 
                     
                 
                   Ser-Xaa40  
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein, in General Formula 2, 
         Xaa1 is 4-imidazoacetyl, histidine, or tyrosine; 
         Xaa2 is glycine, α-methyl-glutamic acid, or Aib; 
         Xaa10 is tyrosine or cysteine; 
         Xaa13 is alanine, glutamine, tyrosine, or cysteine; 
         Xaa14 is leucine, methionine, or tyrosine; 
         Xaa15 is aspartic acid, glutamic acid, or leucine; 
         Xaa16 is glycine, glutamic acid, or serine; 
         Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine; 
         Xaa18 is alanine, glutamine, arginine, or histidine; 
         Xaa19 is alanine, glutamine, cysteine, or valine; 
         Xaa20 is lysine, glutamine, or arginine; 
         Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid; 
         Xaa23 is isoleucine or valine; 
         Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid; 
         Xaa28 is lysine, cysteine, asparagine, or aspartic acid; 
         Xaa29 is glycine, glutamine, cysteine, or histidine; 
         Xaa30 is cysteine, glycine, lysine, or histidine; 
         Xaa31 is proline or cysteine; and 
         Xaa40 is cysteine or is absent. 
       
     
     
         8 . The conjugate of  claim 4 ,
 wherein, in General Formula 1,   Xaa2 is glycine, α-methyl-glutamic acid, or Aib;   Xaa7 is threonine;   Xaa10 is tyrosine, cysteine, or valine;   Xaa12 is lysine or isoleucine;   Xaa13 is tyrosine, alanine, or cysteine;   Xaa14 is leucine or methionine;   Xaa15 is cysteine or aspartic acid;   Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;   Xaa18 is alanine, arginine, or histidine;   Xaa19 is alanine, glutamine, or cysteine;   Xaa20 is lysine or glutamine;   Xaa21 is glutamic acid, cysteine, or aspartic acid;   Xaa23 is valine;   Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and   Xaa27 is leucine or lysine.   
     
     
         9 . The conjugate of  claim 7 ,
 wherein, in General Formula 2,   Xaa13 is alanine, tyrosine, or cysteine;   Xaa15 is aspartic acid or glutamic acid;   Xaa17 is glutamine, arginine, cysteine, or lysine;   Xaa18 is alanine, arginine, or histidine;   Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;   Xaa23 is isoleucine or valine;   Xaa24 is cysteine, glutamine, or asparagine;   Xaa28 is cysteine, asparagine, or aspartic acid;   Xaa29 is glutamine, cysteine, or histidine; and   Xaa30 is cysteine, lysine, or histidine.   
     
     
         10 . The conjugate of  claim 4 ,
 herein, in General Formula 1,   Xaa2 is α-methyl-glutamic acid or Aib;   Xaa7 is threonine;   Xaa10 is tyrosine or cysteine;   Xaa12 is lysine or isoleucine;   Xaa13 is tyrosine, alanine, or cysteine;   Xaa14 is leucine or methionine;   Xaa15 is cysteine or aspartic acid;   Xaa16 is glutamic acid;   Xaa17 is arginine, isoleucine, cysteine, or lysine;   Xaa18 is alanine, arginine, or histidine;   Xaa19 is alanine, glutamine, or cysteine;   Xaa20 is lysine or glutamine;   Xaa21 is glutamic acid or aspartic acid;   Xaa23 is valine;   Xaa24 is glutamine, asparagine, or aspartic acid;   Xaa27 is leucine; and   Xaa28 is cysteine, alanine, asparagine, or aspartic acid.   
     
     
         11 . The conjugate of  claim 4 ,
 wherein, in General Formula 1,   Xaa1 is histidine or 4-imidazoacetyl;   Xaa2 is α-methyl-glutamic acid or Aib;   Xaa3 is glutamine;   Xaa7 is threonine;   Xaa10 is tyrosine;   Xaa12 is isoleucine;   Xaa13 is alanine or cysteine;   Xaa14 is methionine;   Xaa15 is aspartic acid;   Xaa16 is glutamic acid;   Xaa17 is isoleucine or lysine;   Xaa18 is alanine or histidine;   Xaa19 is glutamine or cysteine;   Xaa20 is lysine;   Xaa21 is aspartic acid;   Xaa23 is valine;   Xaa24 is asparagine;   Xaa27 is leucine;   Xaa28 is alanine or asparagine;   Xaa29 is glutamine or threonine; and   Xaa30 is cysteine or lysine, or is absent.   
     
     
         12 . The conjugate of  claim 4 ,
 wherein X is a peptide comprising an amino acid sequence represented by General Formula 3 below:   
       
         
           
                 
               
                   (General Formula 3, SEQ ID NO: 105) 
                 
                   Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 
                 
                     
                 
                   Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys- 
                 
                     
                 
                   Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29- 
                 
                     
                 
                   Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser- 
                 
                     
                 
                   Xaa40  
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein, in General Formula 3, 
         Xaa1 is histidine or tyrosine; 
         Xaa2 is α-methyl-glutamic acid or Aib; 
         Xaa13 is alanine, tyrosine, or cysteine; 
         Xaa17 is arginine, cysteine, or lysine; 
         Xaa18 is alanine or arginine; 
         Xaa19 is alanine or cysteine; 
         Xaa21 is glutamic acid or aspartic acid; 
         Xaa24 is glutamine or asparagine; 
         Xaa28 is cysteine or aspartic acid; 
         Xaa29 is cysteine, histidine, or glutamine; 
         Xaa30 is cysteine or histidine; 
         Xaa31 is proline or cysteine; and 
         Xaa40 is cysteine or is absent. 
       
     
     
         13 . The conjugate of  claim 4 , wherein R1 is cysteine, GKKNDWKHNIT (SEQ ID NO:
 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or is absent.   
     
     
         14 . The conjugate according to  claim 4 , wherein, in General Formulas 1 to 3, the 16 th  amino acid and the 20 th  amino acid from the N-terminus together form a ring. 
     
     
         15 . The conjugate of  claim 1 , wherein X is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 102. 
     
     
         16 . The conjugate according to  claim 1 , wherein F is selected from the group consisting of a polymer, fatty acid, cholesterol, albumin and a fragment thereof, an albumin-binding material, a polymer of repeating units of particular amino acid sequences, an antibody, an antibody fragment, an FcRn-binding material, an in vivo connective tissue, a nucleotide, fibronectin, transferrin, a saccharide, heparin, and elastin. 
     
     
         17 . The conjugate of  claim 16 , wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitins, hyaluronic acid, an oligonucleotide, and a combination thereof. 
     
     
         18 . The conjugate according to  claim 1 , wherein F is an immunoglobulin Fc region. 
     
     
         19 . The conjugate of  claim 18 , wherein F is an IgG Fc region. 
     
     
         20 . The conjugate according to  claim 1 , wherein L is a peptide, fatty acid, a saccharide, a polymer, a low molecular weight compound, a nucleotide, or a combination thereof 
     
     
         21 . The conjugate of  claim 20 , wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitins, hyaluronic acid, an oligonucleotide, and a combination thereof. 
     
     
         22 . The conjugate according to  claim 1 , wherein L is polyethylene glycol. 
     
     
         23 . A pharmaceutical composition for preventing or treating metabolic syndrome comprising a conjugate according  claim 1 . 
     
     
         24 . The pharmaceutical composition of  claim 23 , wherein the metabolic syndrome comprises impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary heart disease.