Insulin-incretin conjugates

Abstract

Disclosed herein are insulin agonist peptides conjugated to incretins wherein the insulin/incretin conjugate has agonist activity at both the insulin receptor and the corresponding incretin receptor. Insulin is a proven therapy for the treatment of juvenile-onset diabetes and later-stage adult-onset diabetes. The peptide is biosynthesized as a larger linear precursor of low potency (approximately 2% to 9% of native insulin), named proinsulin. Proinsulin is proteolytically converted to insulin by the selective removal of a 35-residue connecting peptide (C peptide).

1 . An insulin agonist/incretin conjugate comprising
 a glucagon related peptide; and   an insulin peptide, wherein the C-terminal region of the glucagon related peptide is linked either directly or through a linker to the insulin peptide at a position independently selected from   i) the side chain of an amino acid at a position selected from A9, A14 or A15 of the A chain, or positions B1, B2, B10, B22, B28 or B29 of the B chain; or   ii) the N-terminal alpha amine of the B chain; or   iii) the carboxy terminus of the B chain; or   iv) at the side chain of an amino acid at any position of a linking moiety that links the A chain and B chain of a single chain insulin analog.   
     
     
         2 .- 6 . (canceled) 
     
     
         7 . The conjugate of  claim 1  wherein the glucagon related peptide comprises
 (i) the amino acid sequence:
 X1-X2-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-Z (SEQ ID NO: 839) with 1 to 3 amino acid modifications thereto, wherein 
 X1 and/or X2 is a non-native (relative to SEQ ID NO: 701) amino acid that reduces susceptibility of the glucagon related peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), 
 Z is selected from the group consisting of Asn-Thr-COOH, and Y—COOH, wherein Y is 1 to 2 amino acids, and further wherein 
 (1) a lactam bridge connects the side chains of an amino acid at position i and an amino acid at position i+4, wherein i is 12, 16, 20 or 24 or 
 (2) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the glucagon related peptide is substituted with an α,α-disubstituted amino acid; 
 
 and said glucagon related peptide has glucagon agonist activity; 
 (ii) the amino acid sequence of SEQ ID NO: 701 modified to comprise at least one amino acid modification selected from the group consisting of:
 substitution of Asn at position 28 with a charged amino acid; 
 substitution of Asn at position 28 with a charged amino acid selected from the group consisting of Asp, Glu, cysteic acid, and homocysteic acid; 
 substitution at position 28 with Asn, Asp, or Glu; 
 substitution at position 28 with Glu; 
 substitution of Thr at position 29 with a charged amino acid; 
 substitution of Thr at position 29 with a charged amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid; 
 substitution at position 29 with Asp, Glu, or Lys; 
 substitution at position 29 with Glu; 
 insertion of 1-3 charged amino acids after position 29; 
 insertion after position 29 of Glu or Lys; 
 insertion after position 29 of Gly-Lys or Lys-Lys; or a combination thereof; 
 and at least one amino acid modification selected from Group A or Group B, or a combination thereof; 
 
 wherein Group A is an amino acid modification selected from the group consisting of substitution of Ser at position 16 with Thr or AIB; and 
 wherein Group B is an amino acid modification selected from the group consisting of: 
 substitution of His at position 1 with a non-native amino acid that reduces susceptibility of the glucagon related peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), 
 substitution of Ser at position 2 with a non-native amino acid that reduces susceptibility of the glucagon related peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), 
 substitution of Tyr at position 10 with Phe or Val; 
 substitution of Lys at position 12 with Arg; 
 substitution of Gln at position 20 with Ala or AIB; 
 substitution of Asp at position 21 with Glu; 
 substitution of Gln at position 24 with Ala or AIB; 
 substitution of Met at position 27 with Leu or Nle; 
 or a combination thereof; 
 and wherein said glucagon related peptide has glucagon agonist activity; 
 (iii) a glucagon related peptide of SEQ ID NO: 701, modified to comprise
 (a) an amino acid modification at position 1 that confers GIP agonist activity, 
 (b) (1) a lactam bridge between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17, or
 (2) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an α,α-disubstituted amino acid, 
 
 (c) amino acid modifications at one, two or all of positions 27, 28 and 29, and 
 (d) 1-6 further amino acid modifications, 
 
 wherein the EC50 of the analog for GIP receptor activation is about 10 nM or less; 
 (iv) the sequence of SEQ ID NO: 72 or an analog of SEQ ID NO: 72, wherein said analog differs from SEQ ID NO: 72 by 1 to 3 amino acid modifications, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 24, 27, 28, and 29, wherein said glucagon related peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor; optionally, wherein the glucagon related peptide comprises an intramolecular bridge between the side chains of the amino acids at positions 12 and 16, 16 and 20, 20 and 24, or 24 and 28 or a pharmaceutically acceptable salt thereof; optionally a salt bridge or a lactam bridge between amino acids at positions 16 and 20; 
 (v) an amino acid that differs from SEQ ID NO: 701 by no more than ten amino acid modifications, comprising one or more amino acid substitutions with AIB at positions 16, 20, 21, and/or 24, and an amino acid modification at position 1 and/or 2 that provides reduced susceptibility to cleavage by dipeptidyl peptidase IV, wherein said glucagon related peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor. 
 
     
     
         8 .- 9 . (canceled) 
     
     
         10 . The conjugate of  claim 7  wherein said insulin peptide comprises an A chain and a B chain wherein
 I) said A chain comprises a sequence GIVX 4 X 5 CCX 8 X 9 X 10 CX 12 LX 14 X 15 LX 17 X 18 YCX 21 -R 13  (SEQ ID NO: 19), and said B chain comprises a sequence R 22 -X 25 LCGX 20 X 30 LVX 33 X 34 LYLVCGX 41 X 42 GFX 45  (SEQ ID NO: 20), wherein 
 X 4  is glutamic acid or aspartic acid; 
 X 5  is glutamine or glutamic acid 
 X 8  is histidine, threonine or phenylalanine; 
 X 9  is serine, arginine, lysine, ornithine or alanine; 
 X 10  is isoleucine or serine; 
 X 12  is serine or aspartic acid; 
 X 14  is tyrosine, arginine, lysine, ornithine or alanine; 
 X 15  is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine; 
 X 17  is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine; 
 X 18  is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine; 
 X 21  is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine; 
 X 25  is histidine or threonine; 
 X 29  is selected from the group consisting of alanine, glycine and serine; 
 X 30  is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid; 
 X 33  is selected from the group consisting of aspartic acid and glutamic acid; 
 X 34  is selected from the group consisting of alanine and threonine; 
 X 41  is selected from the group consisting of glutamic acid, aspartic acid or asparagine; 
 X 42  is selected from the group consisting of alanine, ornithine, lysine and arginine; 
 X 45  is tyrosine or phenylalanine; 
 R 22  is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and 
 R 13  is COOH or CONH 2 ; 
 II) said A chain comprises the sequence GIVEQCCX 8 X 9 ICSLYQLENYCX 21 -R 13  (SEQ ID NO: 73) said B chain comprises the sequence R 22 -X 25 LCGX 29 X 30 LVX 33 X 34 LYLVCGX 41 X 42 GFX 45  (SEQ ID NO: 20) 
 X 8  is histidine or threonine; 
 X 9  is serine, lysine, or alanine; 
 X 21  is alanine, glycine or asparagine; 
 X 25  is histidine or threonine; 
 X 29  is selected from the group consisting of alanine, glycine and serine; 
 X 30  is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid; 
 X 33  is selected from the group consisting of aspartic acid and glutamic acid; 
 X 34  is selected from the group consisting of alanine and threonine; 
 X 41  is selected from the group consisting of glutamic acid, aspartic acid or asparagine; 
 X 42  is selected from the group consisting of alanine, ornithine, lysine and arginine; 
 X 45  is tyrosine or phenylalanine; 
 R 22  is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine; and 
 R 13  is COOH or CONH 2 ; 
 III) said A chain comprises a sequence GIVDECCX 8 X 9 SCDLRRLEMX 19 CX 21 -R 13  (SEQ ID NO: 74) and said B chain comprises a sequence R 22 -X 25 LCGAX 30 LVDALYLVCGDX 42 GFY (SEQ ID NO: 75), wherein 
 X 8  is phenylalanine or histidine; 
 X 9  is arginine, ornithine or alanine; 
 X 19  is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine; 
 X 21  is alanine or asparagine; 
 X 25  is histidine or threonine; 
 X 30  is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid; 
 X 42  is selected from the group consisting of alanine ornithine and arginine; and R 13  is COOH or CONH 2 ; 
 R 22  is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and 
 R 13  is COOH or CONH 2 ; 
 IV) said A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R 13  (SEQ ID NO: 1) and said B chain comprises a sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2). 
 
     
     
         11 . The conjugate of  claim 7  wherein said B chain comprises a sequence R 22 -X 25 LCGX 29 X 30 LVX 33 X 34 LYLVCGX 41 X 42 GFX 45 YT-Z 1 -B 1  (SEQ ID NO: 142), wherein
 Z 1  is a dipeptide selected from the group consisting of aspartate-lysine, lysine-proline, and proline-lysine; and 
 B 1  is selected from the group consisting of threonine, alanine or a threonine-arginine-arginine tripeptide. 
 
     
     
         12 . (canceled) 
     
     
         13 . The conjugate of claim  8  wherein the insulin peptide is a single chain insulin and the peptide linker joining the B and A chains is selected from the group consisting of SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52), SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51), GAGSSSX 57 X 58  (SEQ ID NO: 76), GYGSSSX 57 X 58  (SEQ ID NO: 21) and GYGSSSX 57 X 58 APQT; (SEQ ID NO: 77), wherein
 X 57  and X 58  are independently arginine, lysine or ornithine. 
 
     
     
         14 . The conjugate of  claim 13  wherein the peptide linker is selected from the group consisting of GYGSSSRR (SEQ ID NO: 18) and GAGSSSRR (SEQ ID NO: 22). 
     
     
         15 .- 21 . (canceled) 
     
     
         22 . The conjugate of  claim 1  wherein the glucagon related peptide comprises an amino acid sequence selected from the group consisting of:
 i. SEQ ID NO: 81; 
 j. SEQ ID NO: 83; 
 k. SEQ ID NO: 89; 
 l. any one of SEQ ID NOs: 84-88; 
 m. any one of SEQ ID Nos: 100-103; 
 n. SEQ ID NO: 108, wherein the amino acid at position 20 is selected from the group consisting of arginine, ornithine, and citrulline; 
 o. any one of SEQ ID Nos: 98, 99, 109-112, 104-106, and SEQ ID NO: 72, wherein the Xaa at position 28 of the peptide is asparagine or aspartic acid; the Xaa at position 29 of the peptide is threonine or glycine; and C-terminus of the peptide further comprises SEQ ID NO: 78, SEQ ID NO: 79, COOH or CONH 2 ; and 
 p. any one of SEQ ID Nos: 251, 319 and 510. 
 
     
     
         23 . The conjugate of  claim 1  wherein the glucagon related peptide comprises an analog of glucagon (SEQ ID NO: 701) having GIP agonist activity, said analog comprising one or more of the following modifications:
 (a) an amino acid modification at position 1 that confers GIP agonist activity, optionally, wherein the amino acid at position 1 is an amino acid lacking an imidazole side chain; 
 (b) an amino acid substitution of Ser at position 16 with an amino acid of Formula IV: 
 
       
         
           
           
               
               
           
         
         wherein n is 1 to 7, wherein each of R1 and R2 is independently selected from the group consisting of H, C 1 -C 18  alkyl, (C 1 -C 18  alkyl)OH, (C 1 -C 18  alkyl)NH 2 , (C 1 -C 18  alkyl)SH, (C 0 -C 4  alkyl)(C 3 -C 6 )cycloalkyl, (C 0 -C 4  alkyl)(C 2 -C 5  heterocyclic), (C 0 -C 4  alkyl)(C 6 -C 10  aryl)R 7 , and (C 1 -C 4  alkyl)(C 3 -C 9  heteroaryl), wherein R 7  is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group, the amino acid of Formula IV optionally being homoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab), 
         (c) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an α,α-disubstituted amino acid, 
         (d) amino acid modifications at one, two or all of positions 27, 28 and 29, and 
         (e) 1-9 further amino acid modifications relative to the glucagon sequence (SEQ ID NO: 701), 
       
       wherein the EC50 of the analog for GIP receptor activation is about 10 nM or less. 
     
     
         24 . The conjugate of  claim 23 , wherein the glucagon related peptide comprises the following modifications: (a) the amino acid at position 1 is a large, aromatic amino acid, optionally, Tyr, and (b) wherein (i) the Met at position 27 is substituted with a large, aliphatic amino acid, optionally Leu, (ii) the Asn at position 28 is substituted with a small aliphatic amino acid, optionally Ala, or (iii) the Thr at position 29 is substituted with a small aliphatic amino acid, optionally Gly, or wherein the analog comprises a combination of (i), (ii), and (iii); and (c) the glucagon related peptide further comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 95) or XGPSSGAPPPS (SEQ ID NO: 96) linked to said peptide at a position located C-terminal to the amino acid at position 29. 
     
     
         25 . (canceled) 
     
     
         26 . The conjugate of  claim 24 , wherein the glucagon related peptide further comprises one or more of the following modifications:
 (a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid;   (b) Gln at position 3 substituted with Glu;   (c) substitution of the amino acid Tyr at position 10 with an amino acid, optionally an amino acid of Formula I:   
       
         
           
           
               
               
           
         
         wherein n=1 to 4, 
         comprising a side chain covalently linked to an acyl group or alkyl group; 
         (d) addition of an amino acid, optionally an amino acid of Formula I, comprising a side chain covalently linked to an acyl group or alkyl group as the C-terminal amino acid of the analog; 
         (e) Lys at position 12 substituted with Ile; 
         (f) Arg at position 17 substituted with Gln; 
         (g) Arg at position 18 substituted with Ala; 
         (h) Asp at position 21 substituted with Glu; 
         (i) Gln at position 24 substituted with Asn; and 
         (j) replacement of the carboxylic acid of the C-terminal amino acid with a charge-neutral group, optionally, an amide. 
       
     
     
         27 .- 30 . (canceled) 
     
     
         31 . The conjugate of  claim 1  wherein the glucagon related peptide comprises the sequence of HAEGTFTSDVSSYLEEQAAREFIAWLVRGRG (SEQ ID NO: 700), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 703), HAEGTFTSDVSSYLEGQAAKEFICWLVKGR (SEQ ID NO: 717) HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQID NO: 701) or HSQGTFTSDYSKYLDERRAQDFVQWLMNT (SEQ ID NO: 699). 
     
     
         32 . The conjugate of  claim 31  wherein the insulin peptide is a single chain insulin analog comprising the sequence GPEX 25 LCGAX 30 LVDALYLVCGDX 42 GFYFNX 48 X 49 GAGSSSRRGIVDECCX 8 RSCDLRRLENYCN-R 13  (SEQ ID NO: 144), FVNQHLCGSHLVEALYLVCGERGFFYTPKTGAGSSSRRGIVEQCCTSICSLYQLENYCN-R 13  (SEQ ID NO: 143) or GPEHLCGAHLVDALYLVCGDRGFYFNDRGAGSSSRRGIVDECCHRSCDLRRLENYCN (SEQ ID NO: 145) wherein
 X 8  is phenylalanine or histidine; 
 X 25  is histidine or threonine; 
 X 30  is histidine, aspartic acid, glutamic acid, homocysteic acid or cysteic acid; 
 X 42  is alanine ornithine or arginine; 
 X 48  is lysine or aspartic acid; 
 X 49  is proline, ornithine or arginine; and 
 R 13  is COOH or CONH 2 . 
 
     
     
         33 . A derivative of the conjugate of  claim 1  further comprising the structure U-J, wherein
 U is an amino acid or a hydroxy acid; 
 J is an N-alkylated amino acid linked to said conjugate through an amide bond between a carboxyl moiety of J and an amine of the conjugate, further wherein the chemical cleavage half-life (t 1/2 ) of U-J from the conjugate is at least about 1 hour to about 1 week in PBS under physiological conditions. 
 
     
     
         34 .- 35 . (canceled) 
     
     
         36 . The conjugate of  claim 1  wherein a hydrophilic moiety is covalently linked to the side chain of an amino acid of said conjugate at one or more positions corresponding to A14, A15, B0, B1, B10, B22, B28, B29 or positions 16, 17, 20, 21, 24, or 29 of native glucagon (SEQ ID NO: 701), or at the C-terminal region of the glucagon related peptide. 
     
     
         37 .- 38 . (canceled) 
     
     
         39 . The conjugate of  claim 1 , further comprising an acyl group or alkyl group covalently linked to an amino acid side chain. 
     
     
         40 . The conjugate of  claim 39  wherein said acyl group or alkyl group is covalently linked to a position of the glucagon related peptide that corresponds to position 10 of native glucagon (SEQ ID NO: 701), or at one or more positions selected from A14, A15, B0, B1, B10, B22, B28, B29 of the insulin peptide. 
     
     
         41 . A pharmaceutical composition comprising a conjugate of  claim 1 , or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
         42 . (canceled) 
     
     
         43 . A method for treating diabetes, said method comprising the step of administering the composition of  claim 41 , or pharmaceutically acceptable salt thereof, to a patient in need thereof.