Glyco-modified atrial natriuretic peptide

Abstract

The present invention provides a modified atrial natriuretic peptide that exhibits prolonged duration in blood and maintains cGMP elevating activity. The present invention provides a modified peptide in which at least one sugar substance is linked directly through a glycosidic bond or via a linker structure to at least one hANP peptide, or a pharmaceutically acceptable salt thereof, a medicament comprising the modified peptide or the salt thereof as an active ingredient, etc.

1 . A modified peptide in which at least one sugar substance is linked directly through a glycosidic bond or via a linker structure to at least one hANP peptide, or a pharmaceutically acceptable salt thereof. 
     
     
         2 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is linked directly through a glycosidic bond or via a linker structure to at least one of the N terminus of the hANP peptide, the C terminus of the hANP peptide, and the side chain of at least one amino acid constituting the peptide. 
     
     
         3 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the hANP peptide is hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
     
     
         4 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is selected from at least one type of monosaccharide, disaccharide, trisaccharide, and glycochain of 4 or more monosaccharides bonded through glycosidic bonds, and when a plurality of sugar substances are contained in one molecule, the sugar substances may be the same as or different from each other. 
     
     
         5 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a glycochain of 4 or more monosaccharides bonded through glycosidic bonds. 
     
     
         6 . The modified peptide according to  claim 5  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a glycoprotein-derived N-linked glycochain or 0-linked glycochain, or an altered glycochain thereof. 
     
     
         7 . The modified peptide according to  claim 6  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a N-linked glycochain comprising a glycochain structure represented by the following formula, or a glycochain altered at the reducing end thereof: 
       
         
           
           
               
               
           
         
         wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having the above structure are referred to as “AG(5)”, “AG(5-Glc)”, and “AG(5-Man)”, respectively, according to the type of GXX), and “O/N-L” represents binding to the linker structure or the hANP peptide through an O-glycosidic bond or a N-glycosidic bond. 
       
     
     
         8 . The modified peptide according to  claim 7  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a glycochain comprising a glycochain structure represented by the following formula: 
       
         
           
           
               
               
           
         
         wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having the above structure are referred to as “AG(7)”, “AG(7-Glc)”, and “AG(7-Man)”, respectively, according to the type of GXX), and “O/N-L” represents binding to the linker structure or the hANP peptide through an O-glycosidic bond or a N-glycosidic bond. 
       
     
     
         9 . The modified peptide according to  claim 8  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a glycochain comprising a glycochain structure represented by the following formula: 
       
         
           
           
               
               
           
         
         wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having the above structure are referred to as “AG(9)”, “AG(9-Glc)”, and “AG(9-Man)”, respectively, according to the type of GXX), and “O/N-L” represents binding to the linker structure or the hANP peptide through an O-glycosidic bond or a N-glycosidic bond. 
       
     
     
         10 . The modified peptide according to  claim 9  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is a glycochain comprising a glycochain structure represented by the following formula: 
       
         
           
           
               
               
           
         
         wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having the above structure are referred to as “SG”, “SG(Glc)”, and “SG(Man)”, respectively, according to the type of GXX), and “O/N-L” represents binding to the linker structure or the hANP peptide through an O-glycosidic bond or a N-glycosidic bond. 
       
     
     
         11 . The modified peptide according to  claim 7  or a pharmaceutically acceptable salt thereof, wherein in the sugar substance, Gxx is GlcNAc. 
     
     
         12 . The modified peptide according to  claim 11  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is SG. 
     
     
         13 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein 10 or less sugar substances are linked to one hANP peptide. 
     
     
         14 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein 1, 2, or 3 sugar substances are linked to one hANP peptide, 
     
     
         15 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein one molecule contains a divalent or higher hANP peptide. 
     
     
         16 . The modified peptide according to  claim 3  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is linked via a linker structure to the hANP peptide, and the linker structure is a chemical structure that has a linking chain of 3 or more atoms and is bonded at at least one site to the reducing end of the sugar substance through a glycosidic bond and bonded at at least one site to the hANP peptide. 
     
     
         17 . The modified peptide according to  claim 16  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is linked to either the N terminus or the C terminus, or both, of the hANP peptide via a linker structure. 
     
     
         18 . The modified peptide according to  claim 17  or a pharmaceutically acceptable salt thereof, wherein the linker structure is a structure having a linking chain of 15 or less atoms. 
     
     
         19 . The modified peptide according to  claim 18  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is SG-hANP(1-28) (compound 2-1), hANP(1-28)-SG (compound 2-2), SG-hANP(1-28)-SG (compound 2-7), AG(9)-hANP(1-28) (compound 2-10), SG-triazole-hANP(1-28) (compound 2-12), SG-thioacetamide-hANP(1-28) (compound 2-25), or AG(5)-hANP(1-28) (compound 2-26), or
 is derived from any of these modified peptides by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         20 . The modified peptide according to  claim 16  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one structure selected from a polyoxyalkylene chain, an amino acid, and an oligopeptide chain consisting of 2 or more amino acids. 
     
     
         21 . The modified peptide according to  claim 20  or a pharmaceutically acceptable salt thereof, wherein the polyoxyalkylene chain, the amino acid, and/or the oligopeptide chain contained in the linker structure is bonded through an amide bond to the N terminus and/or the C terminus of the hANP peptide. 
     
     
         22 . The modified peptide according to  claim 21  or a pharmaceutically acceptable salt thereof, wherein the polyoxyalkylene chain is PEG. 
     
     
         23 . The modified peptide according to  claim 21  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is SG-PEG(3)-(SG-)Asn-hANP(1-28) (compound 2-16), AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28) (compound 2-21), AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28) (compound 2-22), SG-PEG(3)-hANP(1-28)-PEG(3)-SG (compound 2-24), SG-(SG-)Asn-PEG(11)-hANP(1-28) (compound 2-27), SG-(SG-)Asn-PEG(11)-PEG(11)-hANP(1-28) (compound 2-28), SG-PEG(3)-hANP(1-28) (compound 2-29), SG-PEG(11)-hANP(1-28) (compound 2-30), SG-*(SG-)Gln-Mal-PEG(3)-hANP(1-28) (compound 2-31), SG-(SG-)Gln-PEG(3)-Mal-hANP(1-28) (compound 2-32), SG-(SG-)Asn-(Ser-Gly)3-hANP(1-28) (compound 2-36), or SG-(SG-)Asn-Gly 6 -hANP(1-28) (compound 2-37), or
 is derived from any of these modified peptides by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         24 . The modified peptide according to  claim 20  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having a functional group on the side chain selected from an amino acid having an amino group on the side chain, an amino acid having SH on the side chain, an amino acid having a carboxyl group on the side chain, an amino acid having a hydroxy group on the side chain, and an amino acid having phenol on the side chain and is linked at the side chain of the amino acid having a functional group on the side chain to the sugar substance or the hANP peptide. 
     
     
         25 . The modified peptide according to  claim 24  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having an amino group on the side chain and has a structure of the following general formula (C) in which the sugar substance is linked to the side chain of the amino acid having an amino group on the side chain: 
       
         
           
           
               
               
           
         
         wherein GLY represents the sugar substance; Lg represents a structure on the glycochain side in the linker structure and may be linear or have two or more branches; GLY and L are bonded through an O- or N-glycosidic bond; when Lg is branched, the same number of GLY as the number of branch ends is capable of being linked thereto; and N-(AA) represents a nitrogen atom derived from the side chain amino group of the amino acid having an amino group on the side chain. 
       
     
     
         26 . The modified peptide according to  claim 25  or a pharmaceutically acceptable salt thereof, wherein the side chain amino group and the α amino group of the amino acid having an amino group on the side chain form amide bonds with the α carboxyl groups of other amino acids. 
     
     
         27 . The modified peptide according to  claim 25  or a pharmaceutically acceptable salt thereof, wherein the amino acid having an amino group on the side chain is Lys. 
     
     
         28 . The modified peptide according to  claim 27  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is SG-(SG-)Lys-Gly-hANP(1-28) (compound 2-14), [(SG-) Cys-Gly] 3 -hANP(1-28) (compound 2-15), SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal)Lys-]Lys-PEG(3)-hANP(1-28) (compound 2-19), [SG 2 -Mal-(SG 2 -Mal-)Lys-[SG 2 -Mal-(SG 2 -Mal-)-Lys-]Lys-PEG(3)-hANP(1-28) (compound 2-20), SG-Mal-(SG-Mal-)Lys-hANP(1-28) (compound 2-33), SG-thioacetamide-(SG-thioacetamide-)Lys-PEG-(3)-hANP(1-28) (compound 2-34), or SG-(SG-)Lys-PEG(3)-hANP(1-28) (compound 2-35), or
 is derived from any of these modified peptides by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         29 . The modified peptide according to  claim 24  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having a SH group on the side chain and has a structure of the following general formula in which the sugar substance is linked to the side chain of the amino acid having an SH group on the side chain: 
       
         
           
           
               
               
           
         
         wherein GLY represents the sugar substance; Lg represents a structure on the glycochain side in the linker structure and may be linear or have two or more branches; GLY and L are bonded through an O- or N-glycosidic bond; when Lg is branched, the same number of GLY as the number of branch ends is capable of being linked thereto; and S represents a sulfur atom derived from the side chain SH group of the amino acid having a SH group on the side chain. 
       
     
     
         30 . The modified peptide according to  claim 29  or a pharmaceutically acceptable salt thereof, wherein the amino acid having a SH group on the side chain is Cys. 
     
     
         31 . The modified peptide according to  claim 30  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is [(SG-)Cys-Gly] 5 -hANP(1-28) (compound 2-17), [(SG 2 -) Cys-Gly] 5 -hANP(1-28) (compound 2-18), or SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal-)Lys-]Lys-PEG(11)-hANP(1-28) (compound 2-23), or
 is derived from any of these modified peptides by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         32 . The modified peptide according to  claim 24  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having a carboxyl group on the side chain and has a structure of the following general formula in which the sugar substance is linked to the side chain of the amino acid having carboxylic acid on the side chain: 
       
         
           
           
               
               
           
         
         wherein GLY represents the sugar substance; Lg represents a structure on the glycochain side in the linker structure and may be linear or have two or more branches; GLY and L are bonded through an O- or N-glycosidic bond; when Lg is branched, the same number of GLY as the number of branch ends is capable of being linked thereto; and CO represents CO derived from the side chain of the amino acid having carboxylic acid on the side chain. 
       
     
     
         33 . The modified peptide according to  claim 32  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is bonded through a N-glycosidic bond to both of the side chain carboxyl group and the α carboxyl group of the amino acid having a carboxyl group on the side chain and bonded to another linker structure or the hANP peptide via the α amino group of said amino acid. 
     
     
         34 . The modified peptide according to  claim 32  or a pharmaceutically acceptable salt thereof, wherein the amino acid having a carboxylic acid group on the side chain is Glu, Gln, Asp, or Asn. 
     
     
         35 . The modified peptide according to  claim 34  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is (SG-)Asn-hANP(1-28) (compound 2-3), (SG-)Asn-hANP(2-28) (compound 2-4), (SG-)Asn-hANP(3-28) (compound 2-8), SG-(SG-)Asn-hANP(1-28) (compound 2-9), or SG-(SG-)Asn-PEG(3)-hANP(1-28) (compound 2-13), or
 is derived from any of these modified peptides by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         36 . The modified peptide according to  claim 24  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having phenol on the side chain and has a structure of the following general formula in which the sugar substance is linked to the side chain of the amino acid having phenol on the side chain: 
       
         
           
           
               
               
           
         
         wherein GLY represents the sugar substance; Lg represents a structure on the glycochain side in the linker structure and may be linear or have two or more branches; GLY and L are bonded through an O- or N-glycosidic bond; when Lg is branched, the same number of GLY as the number of branch ends is capable of being linked thereto; and the phenol group represents a phenol group derived from the side chain of the amino acid having a phenol group on the side chain. 
       
     
     
         37 . The modified peptide according to  claim 36  or a pharmaceutically acceptable salt thereof, wherein the amino acid having a phenol group on the side chain is Tyr. 
     
     
         38 . The modified peptide according to  claim 37  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is hANP(1-27)-(SG-)Tyr (compound 2-6), or
 is derived from the modified peptide by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         39 . The modified peptide according to  claim 24  or a pharmaceutically acceptable salt thereof, wherein the linker structure comprises at least one amino acid having a hydroxy group on the side chain and has a structure of the following general formula in which the sugar substance is bonded through an O-glycosidic bond to the side chain of the amino acid having a hydroxy group on the side chain: 
       
         
           
           
               
               
           
         
         wherein GLY represents the sugar substance; and O represents an oxygen atom derived from the side chain hydroxy group of the amino acid having a hydroxy group on the side chain. 
       
     
     
         40 . The modified peptide according to  claim 39  or a pharmaceutically acceptable salt thereof, wherein the amino acid having a hydroxy group on the side chain is Ser. 
     
     
         41 . The modified peptide according to  claim 40  or a pharmaceutically acceptable salt thereof, wherein the modified peptide is (SG-)Ser-hANP(2-28) (compound 2-5), or
 is derived from the modified peptide by the replacement of the sugar substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). 
 
     
     
         42 . The modified peptide according to  claim 16  or a pharmaceutically acceptable salt thereof, wherein the modified peptide has one or two SG molecules as the sugar substance and one hANP(1-28) (SEQ ID NO: 1) as the hANP peptide, and the SG is linked to the N terminus of the hANP(1-28) via a linker structure having a linking chain of 10 or less atoms. 
     
     
         43 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the modified peptide has a structure represented by the formula of the following compound 2-1, 2-3, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-25, 2-26, 2-27, 2-29, or 2-30: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein hANP is hANP(1-28) consisting of the amino acid sequence of SEQ ID NO: 1 and is bonded at the N terminus of the amino acid sequence to the linker structure through an amide bond. 
       
     
     
         44 . The salt of the modified peptide according to  claim 42 , wherein the pharmaceutically acceptable salt is trifluoroacetate or an acetate. 
     
     
         45 . The modified peptide according to  claim 3  or a pharmaceutically acceptable salt thereof, wherein the sugar substance is linked to the side chain of an amino acid in the hANP peptide, and the linked amino acid is an amino acid other than amino acids at amino acid positions 7 to 23 of SEQ ID NO: 1 contained in the hANP peptide. 
     
     
         46 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the modified peptide or the pharmaceutically acceptable salt thereof exhibits a prolonged duration in blood compared with unmodified hANP(1-28) and maintains cGMP elevating activity. 
     
     
         47 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the modified peptide or the pharmaceutically acceptable salt thereof has resistance to the degradation of the hANP peptide by neutral endopeptidase. 
     
     
         48 . The modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof, wherein the modified peptide or the pharmaceutically acceptable salt thereof exhibits 3 or more times the water solubility of unmodified hANP(1-28). 
     
     
         49 . A medicament comprising a modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof. 
     
     
         50 . The medicament according to  claim 48 , wherein the medicament is an agent for treating or alleviating a cardiovascular disease. 
     
     
         51 . A method for treating or alleviating a cardiovascular disease, comprising administering an effective amount of a modified peptide according to  claim 1  or a pharmaceutically acceptable salt thereof. 
     
     
         52 . A method for producing a modified peptide according to  claim 1 , comprising the step of linking a hANP peptide, a sugar substance, and, if necessary, a linker molecule and an acceptor compound. 
     
     
         53 . The method according to  claim 52 , further comprising the step of transferring a glycochain to a GlcNAc compound, a Glc compound, or a Man compound by use of Endo-M or a mutant enzyme thereof.