Ester-based peptide prodrugs

Abstract

Prodrug formulations of bioactive polypeptides are provided wherein the bioactive polypeptide has been modified by the linkage of a dipeptide to the bioactive polypeptide through an ester linkage. The prodrugs disclosed herein in some embodiments have extended half lives of at least 1.5 hours (e.g., at least 10 hours), and more typically greater than 20 hours and less than 70 hours, and are converted to the active form at physiological conditions through a non-enzymatic reaction driven by chemical instability.

1 - 85 . (canceled) 
     
     
         86 . A prodrug comprising the general structure of Formula I: 
       
         
           
           
               
               
           
         
       
       wherein
 R 3  is selected from the group consisting of NH 2 , an amino acid sequence, and 
 
       
         
           
           
               
               
           
         
         R 4  is —OH, NH 2  or an amino acid sequence, 
         R 10  is selected from the group consisting of H, C i -C 4  alkyl, and (CH 2   n (C 6 -C 10  aryl); 
         W is C 6 -C 10  aryl or a bond; 
         n is an integer from 0 to 3; 
         R 12  is —OH, H or 
       
       
         
           
           
               
               
           
         
         R 1  and R 2  are independently selected from the group consisting of H, C 1 -C 4  alkyl, (C 1 -C 4  alkyl)OH, (C 1 -C 1  alkyl)SH, (C 2 -C 3  alkyl)SCH 3 , (C 1 -C 4  alkyl)CONH 2 , (C 1 -C 4  alkyl)COOH, (C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)NHC(NH 2   + )NH 2 , (C 4 -C 6 )cycloalkyl, (C 0 -C 4  alkyl)(C 6 - 10  aryl)R 9 , and CH 2 (C 5 -C 9  heteroaryl), 
         R 5  is OH or NH 2  and R 9  is C 1 -C 4  alkyl, NH 2  or OH, and 
         R 7  is —O-amino acid or O, 
       
       with the proviso that R 3  is not 
       
         
           
           
               
               
           
         
       
       when R 12  is 
       
         
           
           
               
               
           
         
       
       and with the further proviso that R 12  is 
       
         
           
           
               
               
           
         
       
       when R 3  is NH 2  or an amino acid sequence. 
     
     
         87 . The prodrug of  claim 86 , wherein R 3  is 
       
         
           
           
               
               
           
         
       
       and
 R 7  and R 4  each represent amino acid sequences selected from a bioactive peptide of glucagon or GLP-1. 
 
     
     
         88 . The prodrug of  claim 86 , wherein R 3  and R 4  each represent amino acid sequences selected from a bioactive peptide of glucagon or GLP-1; and
 R 12  is   
       
         
           
           
               
               
           
         
       
     
     
         89 . The prodrug of  claim 86 , wherein at least one of R 3  or R 4  is an amino acid sequence of a bioactive peptide selected from the group consisting of a glucagon superfamily peptide, osteocalcin, calcitonin, and an analog, derivative, or conjugate of one of the foregoing. 
     
     
         90 . The prodrug of  claim 89 , wherein the glucagon superfamily peptide is selected from the group consisting of Growth Hormone Releasing Hormone (GHRH; SEQ ID NO: 657), vasoactive intestinal peptide (VIP; SEQ ID NO: 658), Pituitary adenylate cyclase-activating polypeptide 27 (PACAP-27; SEQ ID NO: 659), peptide histidine methionine (PHM; SEQ ID NO: 660), Secretin (SEQ ID NO: 661), a glucagon related analog peptide, and an analog, derivative, or conjugate of one of the foregoing. 
     
     
         91 . A prodrug comprising a modified serine residue of the general structure of Formula III: 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  and R 2  are independently selected from the group consisting of H, C 1 -C 3  alkyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), (C 4 -C 5 )cycloalkyl, CH 2 (C 6 -C 10  aryl), and CH 2 (C 5 -C 9  heteroaryl); 
 R 3  comprises the amino acids of a bioactive protein located N-terminally to a serine residue present in said bioactive protein; 
 R 4  comprises the amino acids of the bioactive protein located C-terminally to the serine residue present in said bioactive protein; and 
 R 5  is OH or NH 2 , 
 optionally wherein R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 9  heteroaryl), and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and CH 2 (C 5 -C 9  heteroaryl). 
 
     
     
         92 . The prodrug of  claim 91  wherein the bioactive peptide is selected from the group consisting of glucagon or GLP-1, and said modified serine residue is located at position 2, 5, 7, 8, or 11 of the glucagon or GLP-1 prodrug, relative to the respective native position,
 optionally further comprising a hydrophilic moiety covalently linked to an amino acid at position 20, 21 or 24 of the glucagon or GLP-1 prodrug, relative to the respective native sequence, or wherein an additional amino acid is added to the carboxy terminus of the prodrug wherein said hydrophilic moiety is covalently linked to said additional amino acid. 
 
     
     
         93 . The prodrug of  claim 91  wherein the hydrophilic moiety is selected from the group consisting of a plasma protein, a polyethylene glycol chain and an Fc portion of an immunoglobin. 
     
     
         94 . A prodrug derivative of GLP-1 or glucagon comprising a polypeptide of the general structure of
   R 3 —Y—R 4  
   
       wherein Y is a structure selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein n is an integer selected from 0 to 3 and m is an integer selected from 1-4, R 3  comprises an amino acid sequence selected from the group consisting of X i X 2 X 3 G (SEQ ID NO: 621) or 
       
       
         
           
           
               
               
           
         
         wherein 
         X 1  is selected from the group consisting of histidine, desaminohistidine, homo-histidine, tyrosine and phenylalanine; 
         X 2  is an amino acid selected from the group consisting of glycine, alanine, serine, valine, d alanine, aminoisobutyric acid, N-methyl alanine; 
         X 3  is selected from the group consisting of glutamic acid, aspartic acid, glutamine and asparagine; 
         X 4  is selected from the group consisting of desaminohistidine, desaminohomo-histidine, desaminotyrosine and desaminophenylalanine, 
         R 4  comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 615, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, and SEQ ID NO: 619; or a peptide selected from the group consisting of FTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 603), FTSDVSSYLEGQAAKEFIAWLVKGR-amide (SEQ ID NO: 604), FTSDVSSYLEGQAAKEFIAWLVKGX 14 PSSGAPPPS-amide (SEQ ID NO: 605), wherein X 14  is Arg or Gly; FTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 618), and FTSDYSKYLDSRRAQDFVQWLMNTPSSGAPPPS-amide (SEQ ID NO: 620), optionally wherein R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 6  heteroaryl) and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and R 5  is OH or NH 2 ; 
         R 5  is NH 2  or HO; 
         R 6  is H or 
       
       
         
           
           
               
               
           
         
         R 7  is O or OX 4 X 2 X 3 G (SEQ ID NO: 634); and 
         R 1  and R 2  are independently selected from the group consisting of H, C 1 -C 3  alkyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), (C 4 -C 5 )cycloalkyl, CH 2 (C 6 -C 10  aryl), and CH 2 (C 5 -C 9  heteroaryl), 
       
       with the proviso that when R 3  is 
       
         
           
           
               
               
           
         
       
       R 6  is H and 
       when R 6  is H, R 3  is not X 1 X 2 X 3 G-(SEQ ID NO: 621). 
     
     
         95 . The prodrug derivative of  claim 94  wherein the prodrug comprises the structure of Formula III: 
       
         
           
           
               
               
           
         
       
       wherein
 R 3  is an amino acid sequence selected from the group consisting of R 14 HAEG (SEQ ID NO: 643), R 14 HAQG-(SEQ ID NO: 639), R 14 FAEG (SEQ ID NO: 644), R 14 FAQG (SEQ ID NO: 640); 
 R 4  comprises an amino acid sequence selected from the group consisting of FTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 603), FTSDVSSYLEGQAAKEFIAWLVKGR-amide (SEQ ID NO: 604), and FTSDVSSYLEGQAAKEFIAWLVKGRX 14 PSSGAPPPS-amide (SEQ ID NO: 605), wherein X 14  is Arg or Gly; 
 R 5  and R 14  are independently NH 2  or HO; and 
 R 1  and R 2  are independently selected from the group consisting of H, C i -C 3  alkyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 6  heteroaryl), and 
 R 5  is OH or NH 2 ; 
 
       optionally wherein
 a) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and CH 2 (C 5 -C 6 ) heteroaryl); 
 b) R 1  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 6  heteroaryl), and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and CH 2 (C 5 -C 6 ) heteroaryl); 
 c) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , and CH(CH 3 )(CH 2 CH 3 ), R 2  is selected from the group consisting of H, and C 1 -C 3  alkyl, and R 5  is NH 2 ; or 
 d) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 6  heteroaryl) and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), 
 optionally further comprising a hydrophilic moiety covalently bound to an amino acid residue at position 20, 21 or 24 of said prodrug derivative relative to the native sequence of GLP-1 or glucagon peptide, or wherein an additional amino acid is added to the carboxy terminus of the prodrug wherein said hydrophilic moiety is covalently linked to said additional amino acid; 
 optionally wherein the structure of Formula III comprises SEQ ID NO: 622 or SEQ ID NO: 623, each of SEQ ID NOs: 623 and 624 optionally comprising a carboxy terminal extension peptide, wherein said extension peptide comprises the sequence of GPSSGAPPPS (SEQ ID NO: 624). 
 
     
     
         96 . The prodrug derivative of  claim 95 , wherein said hydrophilic moiety is
 a) polyethylene glycol;   b) a polyethylene glycol having a molecular weight of at least about 40,000 Daltons;   (c) two or more polyethylene glycol chains covalently attached to two or more amino acids at positions selected from the group consisting of 20, 21, 24, or to a single amino acid added to the carboxy terminus; or   d) a plasma protein or the Fc portion of an immunoglobin.   
     
     
         97 . The prodrug derivative of  claim 94  having the structure 
       
         
           
           
               
               
           
         
       
       wherein
 R 4  is an amino acid sequence selected from the group consisting of FTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 603), FTSDVSSYLEGQAAKEFIAWLVKGR-amide (SEQ ID NO: 604), FTSDVSSYLEGQAAKEFIAWLVKGX 14 PSSGAPPPS-amide (SEQ ID NO: 605), wherein X 14  is Arg or Gly; and FTSDVSSYLEGQAAKEFIAWLVKGRGKRNRNNIA (SEQ ID NO: 606); 
 R 5  is NH 2  or HO; 
 R 7  is an amino acid sequence selected from the group consisting of O—HAEG-(SEQ ID NO: 640), O—HAQG-(SEQ ID NO: 639), O—FAEG-(SEQ ID NO: 644), and O—FAQG-(SEQ ID NO: 640); and 
 R 1  and R 2  are independently selected from the group consisting of H, C 1 -C 3  alkyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), (C 4 -C 5 )cycloalkyl, CH 2 (C 6 -C 10 aryl), and CH 2 (C 5 -C 9  heteroaryl); 
 optionally wherein
 a) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )(CH 2 CH 3 ), and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and CH 2 (C 5 -C 6 ) heteroaryl); 
 b) R 1  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl), and CH 2 (C 5 -C 6  heteroaryl), and R 2  is selected from the group consisting of (C 4 -C 5 )cycloalkyl, CH 2 (C 6  aryl) and CH 2 (C 5 -C 6 ) heteroaryl); 
 c) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , and CH(CH 3 )(CH 2 CH 3 ), R 2  is selected from the group consisting of H, and C 1 -C 3  alkyl, and R 5  is NH 2 ; 
 d) R 1  is selected from the group consisting of CH 2 (CH 3 ) 2 , and CH 2 (C 6  aryl); R 2  is CH 2 (C 6  aryl) and R 5  is OH; or 
 e) R 1  is CH 2 (CH 3 ) 2 , and R 2  is H or CH 2 (C 6  aryl) and R 5  is NH 2 . 
 
 
     
     
         98 . A pharmaceutical composition comprising the prodrug of  claim 86 , and a pharmaceutically acceptable carrier. 
     
     
         99 . A method of treating diabetes, suppressing appetite, reducing weight gain, or inducing weight loss, said method comprising administering an effective amount of a pharmaceutical composition comprising a prodrug of  claim 86 . 
     
     
         100 . The prodrug of  claim 90  wherein the glucagon related analog peptide
 a) is selected from the group consisting of glucagon (SEQ ID NO: 612), oxyntomodulin (SEQ ID NO: 665), exendin-4 (SEQ ID NO: 662), Glucagon-like peptide -1 (GLP-1) (amino acids 7-37 provided as SEQ ID NO: 601), Glucagon-like peptide -2 (GLP-2) (SEQ ID NO: 663) and GIP (SEQ ID NO: 664); 
 b) comprises an amino acid sequence at least 50% identical to native glucagon (SEQ ID NO: 612) that retains the alpha-helix conformation of the amino acids corresponding to amino acids 12-29 of SEQ ID NO: 612; 
 c) comprises 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: 739) with 1 to 3 amino acid modifications thereto, 
 wherein X1 and/or X2 is a non-native (relative to SEQ ID NO: 612) amino acid that reduces susceptibility of the glucagon related analog peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), 
 wherein Z is selected from the group consisting of —COOH, -Asn-COOH, Asn-Thr-COOH, and Y-COOH, wherein Y is 1 to 2 amino acids, and 
 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 analog peptide is substituted with an α,α-disubstituted amino acid; 
 and wherein the glucagon related analog peptide exhibits glucagon agonist activity; 
 d) comprises the amino acid sequence of SEQ ID NO: 612 with 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 Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid; 
 substitution at position 28 with Asn, Asp, or Glu; 
 substitution at position 28 with Asp; 
 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 Asp at position 15 with Glu, and 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 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 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; 
 deletion of amino acids at positions 27-29; 
 deletion of amino acids at positions 28-29; 
 deletion of the amino acid at positions 29; 
 or a combination thereof; 
 
 and wherein the glucagon related analog peptide exhibits glucagon agonist activity; 
 e) comprises the amino acid sequence of SEQ ID NO: 612, with the following modifications:
 (aa) an amino acid modification at position 1 that confers GIP agonist activity, 
 (bb) (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 glucagon related analog peptide is substituted with an α,α-disubstituted amino acid, 
 (cc) amino acid modifications at one, two or all of positions 27, 28 and 29, and 
 (dd) 1-6 further amino acid modifications, 
 
 wherein the EC50 of the glucagon related analog peptide for GIP receptor activation is about 10 nM or less; 
 f) comprises the sequence of SEQ ID NO: 55 or an analog of SEQ ID NO: 55, wherein said analog differs from SEQ ID NO: 55 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 analog peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor; 
 g) comprises an amino acid sequence that differs from SEQ ID NO: 612 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 analog peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor; 
 h) comprises the sequence of SEQ ID NO: 942, or an oxy derivative thereof and wherein the glucagon related analog peptide exhibits glucagon antagonist activity; 
 i) comprises the amino acid sequence of native glucagon modified by deletion of two to five amino acid residues from the N-terminus of SEQ ID NO: 612, and substitution of the aspartic acid residue at position nine of SEQ ID NO: 612 with glutamic acid, homoglutamic acid, β-homoglutamic acid, a sulfonic acid derivative of cysteine, or an alkylcarboxylate derivative of cysteine having the structure of: 
 
       
         
           
           
               
               
           
         
       
       wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl and wherein the glucagon related analog peptide exhibits glucagon antagonist activity;
 j) comprises the general structure of A-B-C, wherein A is selected from the group consisting of:
 (i) phenyl lactic acid (PLA); 
 (ii) an oxy derivative of PLA; 
 (iii) a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond; 
 
 B represents amino acids i to 26 of SEQ ID NO: 612, wherein i is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:
 (iv) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of: 
 
 
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl,
 (v) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; 
 (vi) substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety; 
 (vii) Asp at position 15 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
 (viii) Ser at position 16 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; and 
 (ix) substitution with AIB at one or more of positions 16, 20, 21, and 24 according to the amino acid numbering of SEQ ID NO: 612; 
 
         and C is selected from the group consisting of:
 (x) X; 
 (xi) X—Y; 
 (xii) X—Y—Z; and 
 (xiii) X—Y—Z—R10, 
 
       
       wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is selected from a group consisting of SEQ ID NOs: 919-921 and 953; and
   (xiv) any of (x) to (xiii) in which the C-terminal carboxylate is replaced with an amide;   
 and wherein the glucagon related analog peptide exhibits glucagon antagonist activity; 
 k) comprises the sequence of SEQ ID NO: 1051, wherein the amino acids at positions 4 and 7, positions 7 and 11, positions 11 and 15, positions 15 and 19, or positions 19 and 23 of SEQ ID NO: 1051 are linked via a lactam bridge, or an oxy derivative thereof, and wherein the glucagon related analog peptide exhibits glucagon antagonist activity and GLP-1 agonist activity; or 
 l) comprises a peptide comprising (1) an intramolecular bridge, or an alpha, alpha-di-substituted amino acid, or an acidic amino acid at position 16 (according to the numbering of SEQ ID NO: 612), or a combination thereof, (2) a C-terminal amide or ester in place of a C-terminal carboxylate, and (3) a general structure of A-B-C, 
 wherein A is selected from the group consisting of
 (i) PLA; 
 (ii) an oxy derivative of PLA; and 
 (iii) a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond; 
 
 wherein B represents amino acids p to 26 of SEQ ID NO: 612, wherein p is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:
 (iv) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of: 
 
 
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl;
 (v) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; 
 (vi) substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety; 
 (vii) Asp at position 15 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
 (viii) Ser at position 16 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
 (ix) Arg at position 17 is replaced with Gln, Arg at position 18 is replaced with Ala, Asp at position 21 is replaced with Glu, Val at position 23 is replaced with Ile, and Gln at position 24 is replaced with Ala (according to amino acid numbering of SEQ ID NO: 612); 
 (x) Ser at position 16 is replaced with Glu, Gln at position 20 is replaced with Glu, or Gln at position 24 is replaced with Glu(according to the amino acid numbering of SEQ ID NO: 612); 
 
         wherein C is selected from the group consisting of:
 (vii) X; 
 (viii) X—Y; 
 (ix) X—Y—Z; 
 (x) X—Y—Z—R10; 
 
       
       wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is selected from a group consisting of SEQ ID NOs: 1021, 1026, 1027, and 1050;
 and wherein the glucagon related analog peptide exhibits glucagon antagonist activity and GLP-1 agonist activity. 
 
     
     
         101 . The prodrug of  claim 100 , wherein the glucagon related analog peptide comprises 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: 739) with 1 to 3 amino acid modifications thereto,   wherein X1 and/or X2 is a non-native (relative to SEQ ID NO: 612) amino acid that reduces susceptibility of the glucagon related analog peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),   wherein Z is selected from the group consisting of —COOH, -Asn-COOH, Asn-Thr-COOH, and Y—COOH, wherein Y is 1 to 2 amino acids, and   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 analog peptide is substituted with an α,α-disubstituted amino acid;   and wherein the glucagon related analog peptide exhibits glucagon agonist activity, further wherein one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the glucagon related analog peptide is substituted with an α,α-disubstituted amino acid.   
     
     
         102 . The prodrug of  claim 100 , wherein the glucagon related analog peptide comprises the amino acid sequence of SEQ ID NO: 612 with
 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 Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid; 
 substitution at position 28 with Asn, Asp, or Glu; 
 substitution at position 28 with Asp; 
 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 Asp at position 15 with Glu, and 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 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 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; 
 deletion of amino acids at positions 27-29; 
 deletion of amino acids at positions 28-29; 
 deletion of the amino acid at positions 29; 
 or a combination thereof; 
   and wherein the glucagon related analog peptide exhibits glucagon agonist activity, further wherein the glucagon related analog peptide comprises a substitution of the amino acid at position 16, 20, or 24 with AIB, a deletion of the amino acid(s) at position(s) 27-29, at 28 and 29, or at 29, or a combination thereof.   
     
     
         103 . The prodrug of  claim 100 , wherein the glucagon related analog peptide comprises the amino acid sequence of native glucagon modified by deletion of two to five amino acid residues from the N-terminus of SEQ ID NO: 612, and substitution of the aspartic acid residue at position nine of SEQ ID NO: 612 with β-homoglutamic acid or an alkylcarboxylate derivative of cysteine having the structure of: 
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl. 
       
     
     
         104 . The prodrug of  claim 100 , wherein the glucagon related analog peptide comprises the general structure of A-B-C, wherein A is selected from the group consisting of:
 (i) phenyl lactic acid (PLA);   (ii) an oxy derivative of PLA;   (iii) a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond;   B represents amino acids i to 26 of SEQ ID NO: 612, wherein i is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:   (iv) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of:   
       
         
           
           
               
               
           
         
         wherein X 5  is C i -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl, 
         (v) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; 
         (vi) substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety; 
         (vii) Asp at position 15 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
         (viii) Ser at position 16 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; and 
         (ix) substitution with AIB at one or more of positions 16, 20, 21, and 24 according to the amino acid numbering of SEQ ID NO: 612; 
         and C is selected from the group consisting of: 
         (x) X; 
         (xi) X—Y; 
         (xii) X—Y—Z; and 
         (xiii) X—Y—Z—R10, 
         wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is selected from a group consisting of SEQ ID NOs: 919-921 and 953; and 
         (xiv) any of (x) to (xiii) in which the C-terminal carboxylate is replaced with an amide; 
         and wherein the glucagon related analog peptide exhibits glucagon antagonist activity, further wherein at least one of the following is true: 
         A is an oxyderivative of PLA or a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond; 
         B comprises one or more amino acid modifications selected from the group consisting of: 
         (i) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with β-homoglutamic acid or an alkylcarboxylate derivative of cysteine having the structure of: 
       
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl; 
         (ii) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; 
         (iii) substitution with AIB at one or more positions 16, 20, 21, and 24 according to the amino acid numbering of SEQ ID NO: 612; or C is X or X-Y. 
       
     
     
         105 . The prodrug of  claim 100 , wherein the glucagon related analog peptide is an oxyderivative of the glucagon related analog peptide comprising the sequence of SEQ ID NO: 1051 linked via a lactam bridge. 
     
     
         106 . The prodrug of  claim 100 , wherein the glucagon related analog peptide comprises a peptide comprising (1) an intramolecular bridge, or an alpha, alpha-di-substituted amino acid, or an acidic amino acid at position 16 (according to the numbering of SEQ ID NO: 612), or a combination thereof, (2) a C-terminal amide or ester in place of a C-terminal carboxylate, and (3) a general structure of A-B-C,
 wherein A is selected from the group consisting of
 (i) PLA; 
 (ii) an oxy derivative of PLA; and 
 (iii) a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond; 
   wherein B represents amino acids p to 26 of SEQ ID NO: 612, wherein p is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:
 (iv) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of: 
   
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl;
 (v) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; 
 (vi) substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety; 
 (vii) Asp at position 15 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
 (viii) Ser at position 16 (according to the numbering of SEQ ID NO: 612) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid; 
 (ix) Arg at position 17 is replaced with Gln, Arg at position 18 is replaced with Ala, Asp at position 21 is replaced with Glu, Val at position 23 is replaced with Ile, and Gln at position 24 is replaced with Ala (according to amino acid numbering of SEQ ID NO: 612); 
 (x) Ser at position 16 is replaced with Glu, Gln at position 20 is replaced with Glu, or Gln at position 24 is replaced with Glu(according to the amino acid numbering of SEQ ID NO: 612); 
 
         wherein C is selected from the group consisting of:
 (vii) X; 
 (viii) X—Y; 
 (ix) X—Y—Z; 
 (x) X—Y—Z—R10; 
 
         wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is selected from a group consisting of SEQ ID NOs: 1021, 1026, 1027, and 1050; 
         and wherein the glucagon related analog peptide exhibits glucagon antagonist activity and GLP-1 agonist activity, further wherein the at least one of the following is true:
 the glucagon related analog peptide comprisings an alpha, alpha-disubstituted amino acid, 
 
         A is an oxyderivative of PLA or a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond; 
         B comprises one or more amino acid modifications selected from the group consisting of:
 (i) Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 612) is substituted with β-homoglutamic acid or an alkylcarboxylate derivative of cysteine having the structure of: 
 
       
       
         
           
           
               
               
           
         
         wherein X 5  is C 1 -C 4  alkyl, C 2 -C 4  alkenyl, or C 2 -C 4  alkynyl;
 (ii) substitution of one or two amino acids at positions 10, 20, and 24, (according to the amino acid numbering of SEQ ID NO: 612) with an amino acid covalently attached to an acyl or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage; or 
 
         C is X or X—Y. 
       
     
     
         107 . A sterile pharmaceutical composition comprising the prodrug of any one of  claims 100 - 106 , and a pharmaceutically acceptable carrier. 
     
     
         108 . A method of suppressing appetite, reducing weight gain, inducing weight loss, or causing temporary paralysis of the intestinal tract in a patient in need thereof, said method comprising administering an effective amount of a pharmaceutical composition of  claim 107 . 
     
     
         109 . A complex comprising the general structure
 A-B-Q;   wherein   Q is a bioactive peptide, polypeptide, or protein;   A is an amino acid or a hydroxyl acid;   B is an amino acid; and A-B is a dipeptide that is linked to Q through formation of an ester bond between B and a hydroxyl group of Q, wherein the chemical cleavage half life (t 1/2 ) of A-B from Q is at least about 1 hour to about 1 week in standard PBS solution under physiological conditions.   
     
     
         110 . The complex of  claim 109  wherein
 a) A, B, or the amino group of Q to which A-B is linked is a non-coded amino acid; 
 b) a depot polymer is linked to the side chain of A or B; 
 c) a depot polymer selected from the group consisting of polyethylene glycol, dextran, polylactic acid, polyglycolic acid and a copolymer of lactic acid and glycolic acid is linked to the side chain of A or B; 
 d) a depot polymer is linked to the side chain of A or B wherein the molecular weight of said depot polymer is selected from a range of about 20,000 to about 120,000 Daltons; 
 e) a depot polymer is linked to the side chain of A or B wherein the molecular weight of said depot polymer is selected from a range of about 40,000 to 80,000 Daltons; or f) a depot polymer is linked to the side chain of A or B via linkage to a covalently bound C16 or C18 acyl or alkyl group.