WO2017210168A1 - Agonistes hydrosolubles et chimiquement stables du depsi-glucagon - Google Patents

Agonistes hydrosolubles et chimiquement stables du depsi-glucagon Download PDF

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WO2017210168A1
WO2017210168A1 PCT/US2017/034961 US2017034961W WO2017210168A1 WO 2017210168 A1 WO2017210168 A1 WO 2017210168A1 US 2017034961 W US2017034961 W US 2017034961W WO 2017210168 A1 WO2017210168 A1 WO 2017210168A1
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amino acid
seq
glucagon
analog
acid
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Richard D. Dimarchi
John P. Mayer
Piotr Mroz
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 735 kilobytes acii (text) file named "265654seqlist_ST25.txt,” created on May 26, 2017.
  • Pre-proglucagon is a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP- 2) and oxyntomodulin (OXM), that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake.
  • GLP-1 glucagon-like peptide-1
  • GLP- 2 glucagon-like peptide-2
  • OXM oxyntomodulin
  • Glucagon is a 29-amino acid peptide that corresponds to amino acids 33 through 61 of pre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of pre-proglucagon.
  • hypoglycemia occurs when blood glucose levels drops too low to provide enough energy for the body's activities. In adults or children older than 10 years, hypoglycemia is uncommon except as a side effect of diabetes treatment, but it can result from other medications or diseases, hormone or enzyme deficiencies, or tumors.
  • glucagon a hormone produced by the pancreas, signals the liver to break down glycogen and release glucose, causing blood glucose levels to rise toward a normal level.
  • glucagon's most recognized role in glucose regulation is to counteract the action of insulin and maintain blood glucose levels. However for diabetics, this glucagon response to hypoglycemia may be impaired, making it harder for glucose levels to return to the normal range.
  • hypoglycemia is a life threatening event that requires immediate medical attention.
  • the administration of glucagon is an established medication for treating acute hypoglycemia and it can restore normal levels of glucose within minutes of administration.
  • glucagon is used in the acute medical treatment of
  • hypoglycemia a crystalline form of glucagon is solubilized with a dilute acid buffer and the solution is injected intramuscularly. While this treatment is effective, the methodology is cumbersome and dangerous for someone that is semi-conscious. Accordingly, there is a need for a glucagon analog that maintains the biological performance of the parent molecule but is sufficiently soluble and stable, under relevant physiological conditions, that it can be pre- formulated as a solution, ready for injection.
  • hypoglycemia in their diabetic patients Accordingly, improved pharmaceuticals and methodologies are needed for treating diabetes that are less likely to induce hypoglycemia than current insulin therapies.
  • high potency glucagon agonists are provided that exhibit enhanced aqueous solubility.
  • glucagon agonists are provided that have pharmacokinetic and pharmacodynamics profiles comparable to native hormone but with solubility at physiological pH and chemical stability, once commercially formulated, to constitute a ready-to-use medicine.
  • glucagon peptides are provided that retain glucagon receptor activity and exhibit improved solubility relative to the native glucagon peptide (SEQ ID NO: 1).
  • Native glucagon exhibits poor solubility in aqueous solution, particularly at physiological pH, with a tendency to aggregate and precipitate over time.
  • the glucagon peptides disclosed herein exhibit at least 2-fold, 5-fold, or even higher solubility compared to native glucagon at a pH between 6 and 7.
  • glucagon analogs having enhanced solubility and activity at the GLP-1 and/or GIP receptors are also provided.
  • the enhanced solubility is derived from the discovery that single amide bonds within the glucagon amino acid sequence can be replaced by ester bonds that spontaneously rearrange, after administration to a patient and exposure to physiological conditions, to generate the parent native peptide sequence or a very similar structural analog.
  • the ester-based analogs are biologically inactive but of significantly enhanced solubility relative to the native hormone in physiological buffers.
  • the enhanced soluble glucagon analogs as disclosed herein can be further modified to delay the conversion of the ester bonds to amide bonds.
  • a glucagon agonist peptide comprising the sequence XiX 2 X3GTFTSDYSXi2YLXi5SRRAQX2iFVX2 4 WLX 2 7X28X29
  • Xi is selected from the group consisting of His, D-His, N-methyl-His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo- His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA);
  • X2 IS selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Gly, N- methyl-Ser, amino iso-butyric acid (Aib), Val, or a-amino-N-butyric acid;
  • X 3 is an amino acid comprising a side chain of Structure I, II, or III:
  • R 1 is C0-3 alkyl or C0-3 heteroalkyl
  • R 2 is NHR 4 or C 1-3 alkyl
  • R 3 is C 1-3 alkyl
  • R 4 is H or C1-3 alkyl
  • X is NH, O, or S
  • Y is NHR 4 , SR 3 , or OR 3 ;
  • X12 is Lys or Arg
  • Xi5 is Asp, Glu, cysteic acid, homoglutamic acid or homocysteic acid;
  • X21 is Asp, Lys, Cys, Orn, homocysteine or acetyl phenylalanine;
  • X 24 is Gin, Lys, Cys, Orn, homocysteine or acetyl phenylalanine;
  • X27 is Met, Leu or Nle
  • X 2 8 is Asn, Lys, Arg, His, Asp or Glu
  • X29 is Thr, Lys, Arg, His, Gly, Asp or Glu, optionally wherein SEQ ID NO: 933 is further modified by one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 being substituted with an ⁇ , ⁇ -disubstituted amino acid.
  • an enhanced soluble analog of glucagon wherein the analog comprises one or more amino acids, located at a position selected from positions 5, 7, 8, 11 or 16, linked to the immediately preceding amino acid (i.e., at positions 4, 6, 7, 10 or 15, respectively ) via an ester bond.
  • an enhanced soluble analog of glucagon is provided wherein the analog comprises one or more amino acids, located at a position selected from positions 13, 17, 18, 19, 20, 21, 25 or 28, linked to the immediately preceding amino acid (i.e., at positions 12, 16, 17, 18, 19, 20, 24 or 27, respectively) via an ester bond.
  • glucagon analog comprising one or more ester linked amino acids comprising the structure of
  • Ri5 is H or CH 3 and the ester linked amino acid is present at a position selected from 5, 7, 8, 11, 13, 16, 17, 18, 19, 20, 21, 25 or 28.
  • a glucagon analog is provided comprising 1, 2, or 3 ester linked amino acids comprising the structure of
  • Ri5 is H or CH 3 and the ester linked amino acid is present at a position selected from 5, 7, 8, 11, 13, 16, 17, 18, 19, 20, 21, 25 or 28, relative to the native glucagon sequence.
  • R15 is CH 3 and an ester linked amino acid is present at a position selected from 5, 7, 13, 17, 18, 19, 20, 21, 25 or 28, relative to the native glucagon sequence.
  • R15 is CH 3 and the ester linked amino acid is present at a position selected from 5 or 7.
  • R15 is H and the ester linked amino acid is present at a position selected from 8, 11 or 16, relative to the native glucagon sequence.
  • the enhanced soluble glucagon analogs of the present invention can be further modified to stabilize the ester bond configuration at physiological pH, so the ester bonds are not converted to amides until after administration to a patient.
  • the alpha amine of the ester linked amino acid is covalently bound to a dipeptide that is either susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • glucagon analog comprising one, two, three or more ester linked amino acids comprising the structure of
  • Ri5 is H or CH 3 ;
  • Ri6 is an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • R 1 ⁇ 2 is a dipeptide that is susceptible to cleavage by Dipeptidyl Peptidase IV (DPP-IV).
  • Ri6 is a dipeptide having the general structure of Formula I:
  • Ri is C1-C4 alkyl or (C1-C4 alkyl)NH 2;
  • R 2 is H or is Ci-C 4 alkyl
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and Ci-C 4 alkyl
  • R 8 is H
  • R5 is NH 2 or OH.
  • the dipeptide is selected form the group consisting of -Pro-Glu or -Azetidine-Lys.
  • solubility of glucagon peptide analogs is further improved by amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide, and in one embodiment at a position C-terminal to position 27 of SEQ ID NO: 1.
  • amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide, and in one embodiment at a position C-terminal to position 27 of SEQ ID NO: 1.
  • one, two or three charged amino acids may be introduced within the C- terminal portion, and in one embodiment C-terminal to position 27 relative to native glucagon.
  • the native amino acid(s) at positions 28 and/or 29 are substituted with a charged amino acid, and/or one to three charged amino acids are added to the C-terminus of the peptide, after position 29.
  • one, two or all of the charged amino acids are negatively charged. Additional modifications, e.g. conservative substitutions, may be made to the glucagon peptide that still allow it to retain glucagon activity.
  • the present invention further encompasses pharmaceutically acceptable salts of said glucagon agonists.
  • modifications at position 1 and/or 2 of the glucagon peptide can increase the peptide's resistance to dipeptidyl peptidase IV (DPP IV) cleavage.
  • DPP IV dipeptidyl peptidase IV
  • the amino acid at position 2 may be substituted with D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, or amino isobutyric acid.
  • the amino acid at position 1 may be substituted with D-histidine (D-His), desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine, N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA).
  • D-His D-histidine
  • desaminohistidine desaminohistidine
  • hydroxyl-histidine hydroxyl-histidine
  • acetyl-histidine acetyl-histidine
  • homo-histidine N-methyl histidine
  • alpha-methyl histidine alpha-methyl histidine
  • imidazole acetic acid imidazole acetic acid
  • alpha-dimethyl imidiazole acetic acid DIA
  • modifications at position 2 may reduce glucagon activity, sometimes significantly. This reduction in glucagon activity can be restored by stabilization of the alpha-helix structure in the C-terminal portion of glucagon (around amino acids 12-29).
  • stabilization is via a covalent bond between amino acids at positions "i" and "i+4", wherein i is any integer from 12 to 25.
  • "i" and "i+4" are 12 and 16, 16 and 20, or 20 and 24, or 24 and 28.
  • this covalent bond is a lactam bridge between a glutamic acid at position 16 and a lysine at position 20.
  • the bridge or linker is about 8 (or about 7-9) atoms in length.
  • stabilization is via a covalent bond between amino acids at positions "j" and "j+3,” wherein j is any integer between 12 and 27.
  • the bridge or linker is about 6 (or about 5-7) atoms in length.
  • stabilization is via a covalent bond between amino acids at positions "k" and "k+7,” wherein k is any integer between 12 and 22.
  • this covalent bond is an intramolecular bridge other than a lactam bridge.
  • suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, the use of a, ⁇ -diaminoalkane tethers, the formation of metal-atom bridges, and other means of peptide cyclization.
  • the helix is stabilized by non-covalent bonds (i.e., non-covalent intramolecular bridges), including but not limited to hydrogen-bonding, ionic interactions, and salt bridges.
  • non-covalent bonds i.e., non-covalent intramolecular bridges
  • stabilization of the alpha-helix structure in the C-terminal portion of the glucagon peptide is achieved through purposeful introduction of one or more a, a-disubstituted amino acids at positions that retain the desired activity.
  • one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of a glucagon peptide is substituted with an a, a-disubstituted amino acid.
  • substitution of position 16 of a glucagon peptide with amino iso-butyric acid (Aib) provides a stabilized alpha helix in the absence of a salt bridge or lactam.
  • stabilization of the alpha-helix is accomplished by introducing one or more a, a- disubstituted amino acids without introduction of a covalent intramolecular bridge, e.g., a lactam bridge, a disulfide bridge.
  • a covalent intramolecular bridge e.g., a lactam bridge, a disulfide bridge.
  • Such peptides are considered herein as a peptide lacking a covalent intramolecular bridge.
  • one, two, three or more of positions 16, 20, 21 or 24 are substituted with Aib.
  • glucagon peptide comprising the amino acid sequence:
  • XI and/or X2 is a non-native amino acid that reduces susceptibility of (or increases resistance of) the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV).
  • DPP-IV dipeptidyl peptidase IV
  • XI is selected from the group consisting of His, D- His, (Des-amino)His, hydroxyl-His, acetyl-His, homo-His or alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl His, alpha-methyl His, and imidazole acetic acid
  • X2 is selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Val, Gly, N-methyl Ser, Aib and N-methyl Ala; wherein Z is selected from the group consisting of -COOH (the naturally occurring C-terminal carboxylate), -Asn-COOH,
  • the intramolecular bridge is a lactam bridge.
  • the amino acids at positions i and i+4 of SEQ ID NO: 940 are Lys and Glu, e.g., Glul6 and Lys20.
  • XI is selected from the group consisting of: D-His, N-methyl- His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl- His, homo-His, and alpha, alpha-dimethyl imidiazole acetic acid (DMIA).
  • X2 is selected from the group consisting of: D-Ser, D-Ala, Gly, N- methyl-Ser, Val, and alpha, amino isobutyric acid (Aib).
  • a glucagon peptide is provided, optionally formulated with a pharmaceutically acceptable carrier, wherein the peptide comprises the amino acid sequence HSQGTFTSDYSXi 2 YLDSXi 7 Xi 8 Xi9 X 20 X 2 iFVX 24 WLM X 28 T (SEQ ID NO: 934), wherein
  • Xi 2 is Lys, Arg, Tyr or an ester linked amino acid
  • X 17 is Arg or an ester linked amino acid
  • Xis is Arg or an ester linked amino acid
  • Xi9 is Ala or an ester linked amino acid
  • X 2 o is Gin or an ester linked amino acid
  • X 2 i is Asp or an ester linked amino acid
  • X 24 is Gin or an ester linked amino acid
  • X 28 is Asn or an ester linked amino acid.
  • the ester linked amino acid comprises the structure
  • Ri5 is H or CH 3 .
  • the glucagon peptide is covalently linked to a hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, within a C- terminal extension (e.g., at position 40 of a C-terminal extension of SEQ ID NO: 26), or at the C-terminal amino acid.
  • this hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine residue at any of these positions.
  • Exemplary hydrophilic moieties include polyethylene glycol (PEG), for example, of a molecular weight of about 1,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons.
  • the invention provides a glucagon peptide, comprising the amino acid sequence: Xl-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: 940),
  • amino acids selected from amino acids at positions 7, 8, 11 or 16 are linked to the immediately preceding amino acid via an ester bond;
  • XI and/or X2 is a non-native amino acid that reduces susceptibility of (or increases resistance of) the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
  • Z is selected from the group consisting of -COOH (the naturally occurring C-terminal carboxylate), -Asn-COOH, Asn-Thr-COOH, and Y-COOH, wherein Y is 1 to 2 amino acids.
  • XI is selected from the group consisting of His, D-His, (Des-amino)His, hydroxyl-His, acetyl-His, homo-His or alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl His, alpha- methyl His, and imidazole acetic acid and
  • X2 is selected from the group consisting of Ser, D- Ser, Ala, D-Ala, Val, Gly, N-methyl Ser, Aib and N-methyl Ala; optionally in one embodiment XI is His or DMIA and/or X2 is Ser, D-Ser or Aib.
  • glucagon peptides or analogs include substitution of Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He, optionally, in combination with
  • substitution of Asp at position 21 with Glu substitution of Gin at position 24 with Ser, Thr, Ala or Aib; substitution of Met at position 27 with Leu or Nle; 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 at position 30 (i.e., after position 29) of Glu or Lys; optionally with insertion at position 31 of Lys; addition of SEQ ID NO: 820 to the C- terminus, optionally, wherein the amino acid at position 29 is Thr or Gly; substitution or addition of an amino acid covalently attached to a hydrophilic moiety; or a combination thereof.
  • any of the foregoing peptides can be further modified to improve stability by modifying the amino acid at position 15 of SEQ ID NO: 1 to reduce degradation of the peptide over time, especially in acidic or alkaline buffers.
  • Asp at position 15 is substituted with a Glu, homo-Glu, cysteic acid, or homo-cysteic acid.
  • any of the glucagon peptides described herein can be further modified to improve stability by modifying the amino acid at position 16 of SEQ ID NO: 1.
  • Ser at position 16 is substituted with Thr, or any of the amino acids substitutions described above which enhance potency at the glucagon receptor. Such modifications reduce cleavage of the peptide bond between Aspl5-Serl6.
  • Maintained or enhanced activity at the glucagon receptor may be achieved by modifying the Gin at position 3 with a glutamine analog.
  • a glucagon peptide comprising a glutamine analog at position 3 may exhibit about 5%, about 10%, about 20%, about 50%, or about 85% or greater the activity of native glucagon (e.g. SEQ ID NO: 1) at the glucagon receptor.
  • the glutamine analog is a naturally occurring or a non-naturally occurring amino acid comprising a side chain of Structure I, II or III:
  • R 1 is C 0 - 3 alkyl or C 0 - 3 heteroalkyl
  • R 2 is NHR 4 or Ci_ 3 alkyl
  • R 3 is Ci_ 3 alkyl
  • R 4 is H or Ci_ 3 alkyl
  • X is NH, O, or S
  • Y is NHR 4 , SR 3 , or OR 3 .
  • X is NH or Y is NHR 4 .
  • R 1 is C0-2 alkyl or Ci heteroalkyl.
  • R 2 is NHR 4 or Ci alkyl.
  • R 4 is H or C 1 alkyl.
  • Enhanced activity at the glucagon receptor of the glucagon peptide also may be achieved by covalently attaching an acyl or alkyl group, e.g., an acyl or alkyl group which is non-native to a naturally occurring amino acid (e.g., a C4 to C30 fatty acyl group, a C4 to C30 alkyl group), to the side chain of an amino acid of the glucagon peptide.
  • the acylated or alkylated glucagon peptides lack an intramolecular bridge, e.g., a covalent intramolecular bridge (e.g., a lactam).
  • the acyl or alkyl group is attached to the side chain of the amino acid of the glucagon peptide through a spacer, e.g., a spacer which is 3 to 10 atoms in length. In some embodiments, the acyl or alkyl group is attached to the side chain of the amino acid at position 10 of the glucagon peptide through a spacer. In specific embodiments, the acylated or alkylated glucagon peptides further comprise a modification which selectively decreases the activity of the peptide at the GLP-1 receptor.
  • the acylated or alkylated glucagon peptide may comprise a C- terminal alpha carboxylate, a substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He, a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28, yielding a 27- or 28-amino acid peptide, or a combination thereof.
  • any of the glucagon peptides described herein can be further modified to reduce degradation at various amino acid positions by modifying any one, two, three, or all four of positions 20, 21, 24, or 27.
  • embodiments include substitution of Gin at position 20 with Ala or Aib, substitution of Asp at position 21 with Glu, substitution of Gin at position 24 with Ala or Aib, substitution of Met at position 27 with Leu or Nle.
  • Removal or substitution of methionine reduces degradation due to oxidation of the methionine.
  • Removal or substitution of Gin or Asn reduces degradation due to deamidation of Gin or Asn.
  • Removal or substitution of Asp reduces degradation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to iso- aspartate.
  • the glucagon peptide may be part of a dimer, trimer or higher order multimer comprising at least two, three, or more peptides bound via a linker, wherein at least one or both peptides is a glucagon peptide.
  • the dimer may be a homodimer or heterodimer.
  • the linker is selected from the group consisting of a bifunctional thiol crosslinker and a bi-functional amine crosslinker.
  • the linker is PEG, e.g., a 5 kDa PEG, 20 kDa PEG. In some
  • the linker is a disulfide bond.
  • each monomer of the dimer may comprise a Cys residue (e.g., a terminal or internally positioned Cys) and the sulfur atom of each Cys residue participates in the formation of the disulfide bond.
  • the monomers are connected via terminal amino acids (e.g., N-terminal or C-terminal), via internal amino acids, or via a terminal amino acid of at least one monomer and an internal amino acid of at least one other monomer.
  • the monomers are not connected via an N-terminal amino acid.
  • the monomers of the multimer are attached together in a "tail-to-tail" orientation in which the C-terminal amino acids of each monomer are attached together.
  • a conjugate moiety may be covalently linked to any of the glucagon peptides described herein, including a dimer, trimer or higher order multimer. Fusion peptides comprising the amino acid sequence of any of SEQ ID NOs: 820 to 822 are also contemplated.
  • a pharmaceutical composition comprising any of the novel glucagon peptides disclosed herein, preferably sterile and preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • compositions may contain a glucagon peptide at a concentration of at least A, wherein A is 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher.
  • A is 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg
  • compositions may contain a glucagon peptide at a concentration of at most B, wherein B is 30 mg/ml, 25 mg/ml, 24 mg/ml, 23, mg/ml, 22 mg/ml, 21 mg/ml, 20 mg/ml, 19 mg/ml, 18 mg/ml, 17 mg/ml, 16 mg/ml, 15 mg/ml, 14 mg/ml, 13 mg/ml, 12 mg/ml, 11 mg/ml 10 mg/ml, 9 mg/ml, 8 mg/ml, 7 mg/ml, 6 mg/ml, 5 mg/ml, 4 mg/ml, 3 mg/ml, 2 mg/ml, 1 mg/ml, or 0.1 mg/ml.
  • B is 30 mg/ml, 25 mg/ml, 24 mg/ml, 23, mg/ml, 22 mg/ml, 21 mg/ml, 20 mg/ml, 19 mg/ml, 18 mg/ml, 17 mg/ml, 16
  • the compositions may contain a glucagon peptide at a concentration range of A to B mg/ml, for example, 0.001 to 30.0 mg/ml.
  • the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored within various containers. Such solutions can be used in accordance with one embodiment to prepare pre-formulated solutions ready for injection.
  • the pharmaceutical compositions comprise a lyophilized powder.
  • the pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient. Devices may include a syringe and needle, or a pre-filled syringe.
  • the containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.
  • a method of rapidly increasing glucose level, normalizing blood glucose level, stabilizing blood glucose level, or preventing or treating hypoglycemia using a pre-formulated aqueous composition of a glucagon peptide of the invention comprises the step of administering an effective amount of an aqueous solution comprising a novel modified glucagon peptide of the present disclosure.
  • the aqueous composition is pre-packaged in a device that is used to administer the composition to the patient.
  • a method is provided for inducing the temporary paralysis of the intestinal tract. The method comprises the step of administering one or more of the glucagon peptides disclosed herein to a patient in need thereof.
  • a method of reducing weight gain or inducing weight loss involves administering an effective amount of an aqueous solution comprising a depsi glucagon peptide of the present disclosure.
  • Methods for reducing weight gain or inducing weight loss are expected to be useful to treat obesity of various causes, including drug-induced obesity, and reducing complications associated with obesity including vascular disease (coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia and musculoskeletal diseases.
  • Hyperglycemia includes diabetes, diabetes mellitus type I, diabetes mellitus type II, or gestational diabetes, either insulin-dependent or non- insulin-dependent, and reducing complications of diabetes including nephropathy, retinopathy and vascular disease.
  • Co-administration of insulin and a depsi glucagon peptide of the invention can reduce nocturnal hypoglycemia and/or buffer the hypoglycemic effects of insulin, allowing the same or higher doses of short-acting or long-acting insulin to be administered with fewer adverse hypoglycemic effects.
  • compositions comprising insulin together with a glucagon peptide of the invention are also provided.
  • an improved method of regulating blood glucose levels in insulin dependent patients comprises the steps of administering insulin in an amount therapeutically effective for the control of diabetes and administering a novel modified glucagon peptide of the present disclosure in an amount therapeutically effective for the prevention of hypoglycemia, wherein said administering steps are conducted within twelve hours of each other.
  • the glucagon peptide and the insulin are co-administered as a single composition.
  • glucagon peptides glucagon agonist analogs, glucagon agonists, or glucagon analogs includes all pharmaceutically acceptable salts or esters thereof.
  • Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acid sequence of glucagon (i.e. SEQ ID NO: 1) followed by an 8 amino acid carboxy terminal extension of SEQ ID NO: 821 (KRNRNNIA). While the present invention contemplates that glucagon analogs described herein may optionally be joined to this 8 amino acid carboxy terminal extension (SEQ ID NO: 821), the invention in some embodiments also specifically contemplates glucagon analogs and uses of glucagon analogs lacking the 8 contiguous carboxy amino acids of SEQ ID NO: 821.
  • any known glucagon analog including any of those disclosed herein, can be modified by the substitution of a depsi amino acid at a position selected from positions 16, 17, 20, 21, 24, 29, or within a C-terminal extension (e.g., at position 40) relative to the native glucagon sequence (SEQ ID NO: 1) to improve the solubility of the glucagon analog.
  • the invention includes any one or all embodiments of the invention that are narrower in scope in any way than the variations defined by specific paragraphs herein.
  • a genus it should be understood that every member of a genus is, individually, an embodiment of the invention, and that combinations of two or more members of the genus are embodiments of the invention.
  • Fig. 1 is a schematic representation of a depsi-glucagon analog in accordance with one embodiment and its conversion to the native peptide structure at pH 7.4.
  • Fig. 2 is a schematic representation of the locations in native glucagon (SEQ ID NO: 1) where an amide bond can be substituted with an ester bond.
  • Figs. 3A & 3B represents the in vitro conversion of depsi glucagon analog 2 [D15:S 16] (HSQGTFTSDYSKYLDXi 6 RRAQDFVQWLMNT-Ri 3 ; SEQ ID NO: 1263) comprising a depsi-Ser at position 16 (Fig. 3A), and depsi glucagon analog 3 [D15:T16] (SEQ ID NO: 935) comprising a depsi-Thr at position 16 (Fig. 3B).
  • Fig. 4 represents data showing the in vitro kinetics of O-to-N acyl shift for depsi glucagon analogs 2 [D15:S 16] (SEQ ID NO: 1263), 3 [D15:T16] (SEQ ID NO: 935), 4 [F6:T7] (SEQ ID NO: 1264), 5 [G4:T5] (SEQ ID NO: 1265), 6 [F15:T16] (SEQ ID NO: 1269) and 7 [D6:T7] (SEQ ID NO: 1270).
  • Fig. 4 represents data showing the in vitro kinetics of O-to-N acyl shift for depsi glucagon analogs 2 [D15:S 16] (SEQ ID NO: 1263), 3 [D15:T16] (SEQ ID NO: 935), 4 [F6:T7] (SEQ ID NO: 1264), 5 [G4:T5] (SEQ ID NO: 1265), 6 [F15:T16] (SEQ
  • 5A and 5B are graphs demonstrating the ability of three depsi-glucagon analogs, 2 [D15:S 16] (HS QGTFTS D YS KYLDX 1 6 RR AQDFVQWLMNT ; SEQ ID NO: 1263, wherein a depsi-Ser is present at position 16); 5 [G4:T5]
  • [D15:S 16] (SEQ ID NO: 1263), 3 [D15:T16] (SEQ ID NO: 935) and 5 [G4:T5] (SEQ ID NO: 1265) each increase blood glucose upon administration in vivo.
  • the three depsi peptides show equivalent activity as native glucagon (SEQ ID NO: 1).
  • Fig. 6 is a schematic representation of modifications to the depsi-glucagon peptides to enhance the stability of the peptides at physiological pH. More particularly, a secondary trigger is added to the amino group that must first be removed before the depsi peptide can be converted.
  • Fig.7 A and 7B provide two separate synthetic schemes for preparation of depsi-peptides extended with an enzymatically susceptible substrate either on [G4:T5] (SEQ ID NO: 1265) (Fig. 7A), or an [F6:T7] (SEQ ID NO: 1264) (Fig. 7B) backbone.
  • Fig. 9 is a bar graph demonstrating the solubility of depsi-glucagon 10
  • [G4:T5(KZ)] (SEQ ID NO: 1267) at different buffers and pH ranging from 5 to 7.7.
  • the solid line at concentration 1.0 reflects a target concentration for an injectable emergency glucagon formulation.
  • the dotted line at concentration 0.2 represents the solubility of native glucagon in PBS.
  • PBS physiologically buffered saline
  • NaPB sodium phosphate
  • CADSPB 0.1M citric acid with 0.2M disodium phosphate buffer.
  • Fig. 10 is a bar graph representing the solubility of the glucagon and depsi- peptides 10-15 in PBS pH 7.4, at room temperature (see Example 4 for structures of compounds 10-15). [black line represent pharmaceutically relevant concentration level for use of emergency glucagon treatment] .
  • Fig. 11 is a bar graph of data demonstrating the temperature dependent aggregation of native glucagon (1) and depsi-peptides 8 [D15:T16(Ac)] (SEQ ID NO: 1266) and 10 [G4:T5(KZ)] (SEQ ID NO: 1267) incubated in pH 3 and pH 4 buffers, without agitation. Fluorescence was measured following binding of thioflavin-T.
  • Fig. 12 is a graph demonstrating the in vitro activity of glucagon depsi - peptides 5 [G4:T5] (SEQ ID NO: 1265), 8 [D15:T16(Ac)] (SEQ ID NO: 1266), 9 [G4:T5(Ac)] (SEQ ID NO: 1277), 10 [G4:T5(KZ)] (SEQ ID NO: 1267) and 15
  • [F6:T7(EP)] (SEQ ID NO: 1268) relative to native glucagon.
  • the measured activity is an indirect function of cAMP synthesis as assessed in an engineered cell assay where the human glucagon receptor is over-expressed and coupled to a luminescence reporter.
  • Fig. 13 provides data demonstrating second trigger kinetics in vivo in rats administered four different depsi-glucagon analogs, three of which are further modified by attachment of a second trigger to the alpha amine of the depsi amino acid. 5 [G4:T5] (SEQ ID NO: 1265), 10 [G4:T5(KZ)] (SEQ ID NO: 1267) , 16
  • Fig. 14 presents data representing the administration of native glucagon (1), depsi-peptide 5 [G4:T5] (SEQ ID NO: 1265) [formulated in 0.01N HC1] and 10
  • Fig. 15 presents data representing the administration of native glucagon (1), depsi-peptides 13 [F6:T7(KZ)] (SEQ ID NO: 1273), 15 [F6:T7(EP)] (SEQ ID NO: 1268) and 4 [F6:T7] (SEQ ID NO: 1264) [all formulated in 0.01N HC1; dose: 10 nmol/kg]. Vehicle was 0.01N HC1.
  • Fig. 16 presents data representing the administration of depsi-peptide 15
  • Fig. 17A & 17B are graphs demonstrating blood glucose level (Fig. 17A) and change in blood glucose level (Fig. 17B) in normal rats after administration of GLP-1 analogs with and without Sitagliptin challenge.
  • the dose of Sitagliptin was 30 mg/kg (oral) and GLP-1 & GLP-l(Aib2) 10 nmol/kg (i.v.) DETAILED DESCRIPTION DEFINITIONS
  • the term "pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable salt refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases.
  • Salts derived from inorganic bases include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.
  • Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • treating diabetes will refer in general to altering glucose blood levels in the direction of normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.
  • an "effective" amount or a “therapeutically effective amount” of a glucagon peptide refers to a nontoxic but sufficient amount of the peptide to provide the desired effect.
  • one desired effect would be the prevention or treatment of hypoglycemia, as measured, for example, by an increase in blood glucose level.
  • the amount that is "effective” will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • parenteral means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.
  • purified and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment.
  • purified does not require absolute purity; rather, it is intended as a relative definition.
  • purified polypeptide is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.
  • isolated requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • native glucagon refers to a peptide consisting of the sequence of SEQ ID NO: 1
  • native GLP-1 is a generic term that designates GLP-l(7-36)amide, GLP-1 (7-37)acid or a mixture of those two
  • glucagon or “GLP-1” in the absence of any further designation is intended to mean native glucagon or native GLP-1, respectively.
  • a "glucagon peptide” as used herein includes any peptide comprising, either the amino acid sequence of SEQ ID NO: 1, or any analog of the amino acid sequence of SEQ ID NO: 1, including amino acid substitutions, additions, or deletions, or post translational modifications (e.g. methylation, acylation, ubiquitination and the like) of the peptide, wherein the analog stimulates glucagon or GLP-1 receptor activity, e.g., as measured by cAMP production using the assay described in Example 3.
  • glucagon agonist refers to a complex comprising a glucagon peptide that stimulates glucagon receptor activity, e.g., as measured by cAMP production using the assay described in Example 3.
  • a "glucagon agonist analog” may include a glucagon peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 or analog of such a sequence that has been modified to include one or more conservative amino acid substitutions at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29.
  • an amino acid “modification” refers to a substitution, addition or deletion of an amino acid, and includes substitution with or addition of any of the 20 amino acids commonly found in human proteins, as well as atypical or non- naturally occurring amino acids.
  • all references to a particular amino acid position by number refer to the amino acid at that position in native glucagon (SEQ ID NO: l) or the corresponding amino acid position in any analogs thereof.
  • a reference herein to "position 28" would mean the corresponding position 27 for a glucagon analog in which the first amino acid of SEQ ID NO: 1 has been deleted.
  • a reference herein to "position 28” would mean the corresponding position 29 for a glucagon analog in which one amino acid has been added before the N-terminus of SEQ ID NO: 1.
  • substitution refers to the replacement of one amino acid residue by a different amino acid residue.
  • conservative amino acid substitution is defined herein as exchanges within one of the following five groups:
  • polyethylene glycol refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH 2 CH 2 ) n OH, wherein n is at least 9. Absent any further characterization, the term is intended to include polymers of ethylene glycol with an average total molecular weight selected from the range of 500 to 40,000 Daltons. "polyethylene glycol” or “PEG” is used in combination with a numeric suffix to indicate the approximate average molecular weight thereof. For example, PEG-5,000 refers to polyethylene glycol having a total molecular weight average of about 5,000.
  • pegylated and like terms refers to a compound that has been modified from its native state by linking a polyethylene glycol polymer to the compound.
  • a "pegylated glucagon peptide” is a glucagon peptide that has a PEG chain covalently bound to the glucagon peptide.
  • a general reference to a peptide is intended to encompass peptides that have modified amino and carboxy termini.
  • an amino acid chain comprising an amide group in place of the terminal carboxylic acid is intended to be encompassed by an amino acid sequence designating the standard amino acids.
  • Linker is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups.
  • a "dimer” is a complex comprising two subunits covalently bound to one another via a linker.
  • dimer when used absent any qualifying language, encompasses both homodimers and heterodimers.
  • a homodimer comprises two identical subunits, whereas a heterodimer comprises two subunits that differ, although the two subunits are substantially similar to one another.
  • pH stabilized glucagon peptide refers to a glucagon agonist analog that exhibits superior stability and solubility, relative to native glucagon, in aqueous buffers in the broadest pH range used for pharmacological purposes.
  • charged amino acid refers to an amino acid that comprises a side chain that is negatively charged (i.e., de-protonated) or positively charged (i.e., protonated) in aqueous solution at physiological pH.
  • negatively charged amino acids include aspartic acid, glutamic acid, cysteic acid, homocysteic acid, and homoglutamic acid
  • positively charged amino acids include arginine, lysine and histidine.
  • Charged amino acids include the charged amino acids among the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.
  • Non-naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.
  • Commercial sources of atypical amino acids include Sigma- Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA).
  • Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from other amino acids.
  • acidic amino acid refers to an amino acid that comprises a second acidic moiety, including for example, a carboxylic acid or sulfonic acid group.
  • depsi-peptide defines a peptide having an ester linked amino acid.
  • a “depsi-glucagon” is a glucagon peptide that has been modified to comprise an ester linked amino acid within the peptide sequence, including for example at a position selected from positions 5, 7, 8, 11, 13, 16, 17, 18, 19, 20, 21, 25 or 28 according to the numbering of native glucagon.
  • alkyl refers to a linear or branched hydrocarbon containing the indicated number of carbon atoms.
  • exemplary alkyls include methyl, ethyl, and linear propyl groups.
  • heteroalkyl refers to a linear or branched hydrocarbon containing the indicated number of carbon atoms and at least one heteroatom in the backbone of the structure. Suitable heteroatoms for purposes herein include but are not limited to N, S, and O.
  • glucagon agonists are provided that have pharmacokinetic and pharmacodynamics profiles comparable to native hormone but with solubility at physiological pH and chemical stability, once commercially formulated, to constitute a ready-to-use medicine.
  • glucagon analogs having activity at the GLP-1 and/or GIP receptors are also modified in accordance with the present disclosure to comprise one or more ester linked amino acids both as a means of enhancing their solubility and as a means of delaying their time of action and increasing their therapeutic index.
  • the enhanced solubility of the glucagon analogs is derived from the substitution of one or more native amide bonds with an ester bond. Applicants have discovered that single amide bonds within the glucagon amino acid sequence can be replaced by ester bonds to enhance solubility and that those ester bonds will spontaneously rearrange after administration to a patient and exposure to
  • one embodiment of the present invention is directed to a glucagon agonist that has been modified relative to the wild type peptide of His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp- Ser- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1) to comprise one, two, three or more ester bonds replacing the native amide bonds (forming a "depsi-glucagon" analog) to improve the peptide's solubility in aqueous solutions, particularly at a pH ranging from about 5.5 to about 8.0.
  • the inactive depsi-glucagon analog is administered to patients, wherein the ester bonds are converted to amide bonds in vivo thus restoring activity to the glucagon analog.
  • the ester linked amino acids are present at one or more of positions 5, 7, 8, 11, 13, 16, 17, 18, 19, 20, 21, 25 or 28 according to the numbering of native glucagon.
  • an enhanced soluble analog of glucagon wherein the analog comprising one, two, three or more amino acids selected from positions 13, 17, 18, 19, 20, 21, 25 or 28 (according to the numbering of native glucagon) linked to the immediately preceding amino acid (i.e., positions 12, 16, 17, 18, 19, 20, 24 or 27 respectively) via an ester bond.
  • an enhanced soluble analog of glucagon is provided wherein the analog comprising one, two, three or more amino acids selected from positions 5, 7, 8, 11 or 16 (according to the numbering of native glucagon) linked to the immediately preceding amino acid (i.e., positions 4, 6, 7, 10 or 15 respectively ) via an ester bond.
  • a single amino acid selected from amino acids at positions 7, 8, 11 or 16 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 13, 17, 18, 19, or 25 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 7, 8, or 11 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 8, 11 or 16 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 7, 11 or 16 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 11 or 16 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at positions 11 or 16 is linked to the immediate preceding amino acid via an ester bond. In one embodiment a single amino acid selected from amino acids at position 16 is linked to the immediate preceding amino acid via
  • the side chain of a serine or threonine is linked via an ester bond with the alpha carboxy group of the immediate preceding amino acid in the glucagon amino acid sequence.
  • a glucagon analog is provided comprising a serine or threonine at one or more of positions 7, 8, 11 or 16 wherein the serine or threonine is linked to the immediate preceding amino acid via an ester bond.
  • a serine or threonine at a single position selected from positions 8, 11 or 16 is linked to the immediate preceding amino acid via an ester bond.
  • a serine or threonine at a single position selected from positions 11 or 16 is linked to the immediate preceding amino acid via an ester bond.
  • a serine or threonine at position 16 is linked to the immediate preceding amino acid via an ester bond.
  • the glucagon peptide modified to comprise the ester linked amino acid can be selected from any known glucagon peptide as disclosed herein or known to those skilled in the art.
  • glucagon peptide analog comprising one, two, three or more ester linked amino acids comprising the structure of Formula IV:
  • Ri5 is H or CH 3 .
  • the one, two, three or more ester linked amino acids comprising the structure of Formula IV are located at position 5, 7, 8, 11, 13, 16, 17, 18, 19, 20, 21, 25 or 28; or at position 7, 8, 11, 13, 16, 17, 18, 19, 20, 21 or 25; or at position 7, 8, 11, 13, 17, 18, 19, or 25; or at position 13, 17, 18, 19, 20, 21, 25 or 28; or at positions 5, 7, 8, 11 or 16, or at position 8, 11 or 16, or at position 7 or 16, or at position 16, relative to native glucagon.
  • the enhanced soluble glucagon peptide analogs of the present invention can be further structurally modified to stabilize the ester bond configuration at
  • the alpha amine of the ester linked amino acid of the stabilized glucagon peptide analog is covalently bound to a compound that is metastable or cleavable upon contact with mammalian serum.
  • the alpha amine of the ester linked amino acid is covalently bound to an amino acid or a dipeptide that is susceptible to cleavage by a peptidase present in mammalian serum, including for example, an
  • DPP IV Dipeptidyl Peptidase IV
  • the improved soluble glucagon peptide analog of the present disclosure comprises one, two, three or more ester linked amino acids comprising the structure of
  • Ri5 is H or CH 3 and
  • Ri6 is an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • R 1 ⁇ 2 is a dipeptide that is susceptible to cleavage by Dipeptidyl Peptidase IV.
  • Ri 6 is a dipeptide of the general formula X-Pro, wherein X is any amino acid and the proline is linked to the primary amine of the ester linked amino acid.
  • R 1 ⁇ 2 is selected from the group consisting of Gly-Pro, Lys-Pro and Lys- Azetidine-2-carboxylic acid wherein the proline or Azetidine-2-carboxylic acid (Azetidine) is linked to the primary amine of the ester linked amino acid.
  • R 1 ⁇ 2 is a dipeptide having the general structure of Formula I:
  • Ri is H or Ci-Cis alkyl
  • R 2 R 4 and R 8 are independently selected from the group consisting of H, Q- Ci8 alkyl, C 2 -Q 8 alkenyl, (Ci-Cis alkyl)OH, (Ci-Cis alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C1-C4 alkyl)CONH 2 , (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4
  • R 3 is selected from the group consisting of Ci-Ci 8 alkyl, (Ci-Ci 8 alkyl)OH, (C1-C18 alkyl)NH 2 , (Ci-Cis 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 -Ci 0 aryl)R 7 , and (C1-C4 alkyl)(C 3 -C 9 heteroaryl) or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NHR 6 or OH
  • R 6 is H, Ci-C 8 alkyl
  • R 7 is selected from the group consisting of hydrogen and OH.
  • Ri 6 is a dipeptide having the general structure of Formula
  • Ri is selected from the group consisting of H and C 1 -C 4 alkyl
  • R 2 is selected from the group consisting of H, Ci-C 6 alkyl, C 2 -C 8 alkenyl, (Ci- C 4 alkyl)OH, (d-C 4 alkyl)NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 6 -Ci 0 aryl)R 7 , and C3 ⁇ 4(C5-C9 heteroaryl);
  • R 3 is Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H and C 1 -C 4 alkyl or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 7 is selected from the group consisting of hydrogen and OH
  • Ri 6 is a dipeptide having the general structure of Formula
  • Ri is C1-C4 alkyl or (C1-C4 alkyl)NH 2;
  • R 2 is H
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and Ci-C 4 alkyl
  • R 8 is H
  • R5 is NH 2 or OH, optionally wherein R5 is NH 2 .
  • R 1 ⁇ 2 is a dipeptide having the general structure of
  • Ri is H, C 1 -C4 alkyl or (C 1 -C4 alkyl)NH 2;
  • R 2 is H or is Ci-C 4 alkyl
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and Q-C4 alkyl
  • R 8 is H
  • R5 is NH 2 or OH, optionally with the proviso that Ri and R 2 are not both H.
  • Ri 6 is a dipeptide having the general structure of Formula
  • Ri is C1-C4 alkyl
  • R 2 is Cl-C4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and Ci-C 4 alkyl
  • R 8 is H
  • R 5 is NH 2 .
  • the glucagon peptides of the current disclosure can be further modified to enhance their solubility by introducing charge at its carboxy terminus.
  • charge is added to the peptide by the substitution of native non-charged amino acids with charged amino acids selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid, or by the addition of charged amino acids to the amino or carboxy terminus of the peptide.
  • glucagon or any known glucagon analog can be enhanced by the introduction of an ester bond for the native amide bond at any of positions 5, 7, 8, 11 or 16 relative to native glucagon.
  • the glucagon related peptide is a Class 1 glucagon related peptide, which is described herein and in International Patent Publication No. WO 2009/155257 (published on December 23, 2009), International Patent
  • Class 1 glucagon peptides retain glucagon receptor activity relative to the native glucagon peptide (SEQ ID NO: 801).
  • the glucagon peptide can retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% activity, 80% activity,
  • the Class 1 glucagon related peptides have the same or greater activity (used synonymously with the term "potency" herein) than glucagon.
  • a Class 1 glucagon related peptide has been modified relative to the wild type peptide of His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 801) to improve the peptide's solubility in aqueous solutions, particularly at a pH ranging from about 5.5 to about 8.0, while retaining the native peptide's biological activity.
  • the solubility of any of the Class 1 glucagon related peptides described herein can be further improved by attaching a hydrophilic moiety to the peptide.
  • a hydrophilic moiety to the peptide.
  • Introduction of such groups also increases duration of action, e.g. as measured by a prolonged half-life in circulation.
  • Hydrophilic moieties are further described herein.
  • solubility is improved by adding charge to the Class 1 glucagon related peptide by the substitution of native non-charged amino acids with charged amino acids selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid, or by the addition of charged amino acids to the amino or carboxy terminus of the peptide.
  • the Class 1 glucagon related peptide is modified by amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide, and in some embodiments at a position C-terminal to position 27 of SEQ ID NO: 801.
  • one, two or three charged amino acids may be introduced within the C-terminal portion, and in some embodiments C-terminal to position 27.
  • the native amino acid(s) at positions 28 and/or 29 are substituted with a charged amino acid, and/or one to three charged amino acids are added to the C-terminus of the peptide, e.g. after position 27, 28 or 29.
  • one, two, three or all of the charged amino acids are negatively charged.
  • one, two, three or all of the charged amino acids are positively charged.
  • the Class 1 glucagon related peptide may comprise any one or two of the following modifications: substitution of N28 with E; substitution of N28 with D; substitution of T29 with D; substitution of T29 with E; insertion of E after position 27, 28 or 29; insertion of D after position 27, 28 or 29.
  • substitution of N28 with E substitution of N28 with D
  • substitution of T29 with D substitution of T29 with E
  • insertion of E after position 27, 28 or 29 substitution of D after position 27, 28 or 29.
  • Any of the Class 1 glucagon peptides may additionally exhibit improved stability and/or reduced degradation, for example, retaining at least 95% of the original peptide after 24 hours at 25 ° C.
  • Any of the Class 1 glucagon related peptides disclosed herein may additionally exhibit improved stability at a pH within the range of 5.5 to 8, for example, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the original peptide after 24 hours at 25 °C.
  • any of the foregoing Class 1 glucagon related peptides can be further modified to improve stability by modifying the amino acid at position 15 of SEQ ID NO: 801 to reduce degradation of the peptide over time, especially in acidic or alkaline buffers.
  • Asp at position 15 is substituted with a Glu, homo-Glu, cysteic acid, or homo-cysteic acid.
  • any of the Class 1 glucagon related peptides described herein can be further modified to improve stability by modifying the amino acid at position 16 of SEQ ID NO: 801.
  • Ser at position 16 is substituted with Thr or Aib, or any of the amino acids substitutions described herein with regard to Class 1 glucagon related peptides which enhance potency at the glucagon receptor.
  • Such modifications reduce cleavage of the peptide bond between Aspl5-Serl6.
  • any of the Class 1 glucagon related peptides described herein can be further modified to reduce degradation at various amino acid positions by modifying any one, two, three, or all four of positions 20, 21, 24, or 27.
  • Exemplary embodiments include substitution of Gin at position 20 with Ser, Thr, Ala or Aib, substitution of Asp at position 21 with Glu, substitution of Gin at position 24 with Ala or Aib, substitution of Met at position 27 with Leu or Nle. Removal or substitution of methionine reduces degradation due to oxidation of the methionine.
  • Class 1 glucagon related peptides are provided that have enhanced potency at the glucagon receptor, wherein the peptides comprise an amino acid modification at position 16 of native glucagon (SEQ ID NO: 801).
  • enhanced potency can be provided by substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, or a charged amino acid having a side chain containing at least one heteroatom, (e.g. N, O, S, P) and with a side chain length of about 4 (or 3-5) atoms.
  • heteroatom e.g. N, O, S, P
  • Substitution of serine at position 16 with glutamic acid enhances glucagon activity at least 2-fold, 4- fold, 5-fold and up to 10-fold greater at the glucagon receptor.
  • the Class 1 glucagon related peptide retains selectivity for the glucagon receptor relative to the GLP-1 receptors, e.g., at least 5-fold, 10-fold, or 15-fold selectivity.
  • the Class 1 glucagon peptides disclosed herein are further modified at position 1 or 2 to reduce susceptibility to cleavage by dipeptidyl peptidase IV. More particularly, in some embodiments, position 1 and/or position 2 of the Class 1 glucagon related peptide is substituted with the DPP-IV resistant amino acid(s) described herein. In some embodiments, position 2 of the analog peptide is substituted with an amino isobutyric acid. In some embodiments, position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, D-alanine, glycine, N-methyl serine, and ⁇ -amino butyric acid. In another embodiment, position 2 of the Class 1 glucagon related peptide is substituted with an amino acid selected from the group consisting of D-serine, glycine, and
  • aminoisobutyric acid In some embodiments, the amino acid at position 2 is not D- serine.
  • Reduction in glucagon activity upon modification of the amino acids at position 1 and/or position 2 of the glucagon peptide can be restored by stabilization of the alpha-helix structure in the C-terminal portion of the glucagon peptide (around amino acids 12-29).
  • the alpha helix structure can be stabilized by, e.g., formation of a covalent or non-covalent intramolecular bridge (e.g., a lactam bridge between side chains of amino acids at positions "i" and "i+4", wherein i is an integer from 12 to 25), substitution and/or insertion of amino acids around positions 12-29 with an alpha helix- stabilizing amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid), as further described herein.
  • a covalent or non-covalent intramolecular bridge e.g., a lactam bridge between side chains of amino acids at positions "i" and "i+4", wherein i is an integer from 12 to 25
  • substitution and/or insertion of amino acids around positions 12-29 with an alpha helix- stabilizing amino acid e.g., an ⁇ , ⁇ -disubstituted amino acid
  • Glucagon receptor activity can be reduced by an amino acid modification at position 3 (according to the amino acid numbering of wild type glucagon), e.g.
  • substitution of the naturally occurring glutamine at position 3, with an acidic, basic, or a hydrophobic amino acid substantially reduces or destroys glucagon receptor activity.
  • glucagon agonists can comprise the amino acid sequence of SEQ ID NO: 863, SEQ ID NO: 869, SEQ ID NO: 870, SEQ ID NO: 871, SEQ ID NO: 872, SEQ ID NO: 873, and SEQ ID NO: 874.
  • Enhanced activity at the GLP-1 receptor is provided by replacing the carboxylic acid of the C-terminal amino acid with a charge-neutral group, such as an amide or ester. Conversely, retaining the native carboxylic acid at the C-terminus of the peptide maintains the relatively greater selectivity of the Class 1 glucagon related peptide for the glucagon receptor vs. the GLP-1 receptor (e.g., greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-fold).
  • Class 1 glucagon related peptide may further increase solubility and/or stability and/or glucagon activity.
  • the Class 1 glucagon related peptide may alternatively comprise other modifications that do not substantially affect solubility or stability, and that do not substantially decrease glucagon activity.
  • the Class 1 glucagon related peptide may comprise a total of up to 11, or up to 12, or up to 13, or up to 14 amino acid modifications relative to the native glucagon sequence. For example, conservative or non-conservative substitutions, additions or deletions may be carried out at any of positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29.
  • Exemplary modifications of the Class 1 glucagon related peptide include but are not limited to:
  • non-conservative substitutions conservative substitutions, additions or deletions while retaining at least partial glucagon agonist activity, for example, conservative substitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29, substitution of Tyr at position 10 with Val or Phe, substitution of Lys at position 12 with Arg, substitution of one or more of these positions with Ala;
  • substitution with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid which may reduce degradation; or modification of the serine at position 16, for example, by substitution of threonine, Aib, glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, which likewise may reduce degradation due to cleavage of the Aspl5-Serl6 bond;
  • hydrophilic moiety such as the water soluble polymer polyethylene glycol, as described herein, e.g. at position 16, 17, 20, 21, 24, 29, 40 or at the C-terminal amino acid, which may increase solubility and/or half-life;
  • acylating or alkylating the glucagon peptide as described herein which may increase the activity at the glucagon receptor and/or the GLP-1 receptor, increase half- life in circulation and/or extending the duration of action and/or delaying the onset of action, optionally combined with addition of a hydrophilic moiety, additionally or alternatively, optionally combined with a modification which selectively reduces activity at the GLP-1 peptide, e.g., a modification of the Thr at position 7, such as a substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He; deleting amino acids C-terminal to the amino acid at position 27 (e.g., deleting one or both of the amino acids at positions 28 and 29, yielding a peptide 27 or 28 amino acids in length);
  • Lys at position 12 is substituted with Arg.
  • amino acids at positions 29 and/or 28, and optionally at position 27, are deleted.
  • the glucagon peptide comprises (a) an amino acid modification at position 1 and/or 2 that confers DPP-IV resistance, e.g., substitution with DMIA at position 1, or Aib at position 2, (b) an intramolecular bridge within positions 12-29, e.g.
  • the amino acid at position 29 in certain embodiments is Thr or Gly.
  • the glucagon peptide comprises (a) Asp28Glu29, or
  • the glucagon peptide is T16, A20, E21, A24, Nle27, D28, and E29.
  • the Class 1 glucagon related peptide comprises the amino acid sequence:
  • XI and/or X2 is a non-native amino acid that reduces susceptibility of (or increases resistance of) the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
  • Z is selected from the group consisting of -COOH (the naturally occurring C-terminal carboxylate), -Asn-COOH, Asn-Thr-COOH, and Y-COOH, wherein Y is 1 to 2 amino acids, and
  • an intramolecular bridge preferably a covalent bond, 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.
  • the intramolecular bridge is a lactam bridge.
  • the amino acids at positions i and i+4 of SEQ ID NO: 839 are Lys and Glu, e.g., Glul6 and Lys20.
  • XI is selected from the group consisting of: D-His, N-methyl-His, alpha-methyl-His, imidazole acetic acid, des- amino-His, hydroxyl-His, acetyl-His, homo-His, and alpha, alpha-dimethyl imidiazole acetic acid (DMIA).
  • X2 is selected from the group consisting of: D-Ser, D-Ala, Gly, N-methyl-Ser, Val, and alpha, amino isobutyric acid (Aib).
  • the glucagon peptide is covalently linked to a hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, within a C-terminal extension, or at the C-terminal amino acid.
  • this hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl- phenylalanine residue at any of these positions.
  • Exemplary hydrophilic moieties include polyethylene glycol (PEG), for example, of a molecular weight of about 1,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons.
  • the Class I glucagon related peptide comprises the amino acid sequence:
  • Xl-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: 940), wherein XI and/or X2 is a non-native amino acid that reduces susceptibility of (or increases resistance of) the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), wherein one, two, three, four or more of positions 16, 20, 21, and 24 of the glucagon peptide is substituted with an a, a-disubstituted amino acid, and
  • DPP-IV dipeptidyl peptidase IV
  • Z is selected from the group consisting of -COOH (the naturally occurring C-terminal carboxylate), -Asn-COOH, Asn-Thr-COOH, and Y-COOH, wherein Y is 1 to 2 amino acids.
  • Exemplary further amino acid modifications to the foregoing Class 1 glucagon related peptides or analogs include substitution of Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., aminobutyric acid (Abu), He, optionally, in combination with substitution or addition of an amino acid comprising a side chain covalently attached (optionally, through a spacer) to an acyl or alkyl group, which acyl or alkyl group is non-native to a naturally-occurring amino acid, substitution of Lys at position 12 with Arg; substitution of Asp at position 15 with Glu; substitution of Ser at position 16 with Thr or Aib; substitution of Gin at position 20 with Ser, Thr, Ala or Aib; substitution of Asp at position 21 with Glu; substitution of Gin at position 24 with Ser, Thr, Ala or Aib; substitution of Met at position 27 with Leu or Nle; substitution of Asn at position 28 with a charged amino acid; substitution of Asn at position 28 with a charged
  • Class 1 glucagon related peptides can be prepared that retain at least 20% of the activity of native glucagon at the glucagon receptor, and which are soluble at a concentration of at least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, (e.g. pH 7), and optionally retain at least 95% of the original peptide (e.g. 5% or less of the original peptide is degraded or cleaved) after 24 hours at 25°C.
  • high potency Class 1 glucagon peptides can be prepared that exhibit at least about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 10-fold or more of the activity of native glucagon at the glucagon receptor, and optionally are soluble at a concentration of at least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, (e.g. pH 7), and optionally retains at least 95% of the original peptide (e.g. 5% or less of the original peptide is degraded or cleaved) after 24 hours at 25°C.
  • the Class 1 glucagon peptides described herein may exhibit at least any of the above indicated relative levels of activity at the glucagon receptor but no more than 1,000%, 5,000% or 10,000% of the activity of native glucagon at the glucagon receptor.
  • a glucagon analog of SEQ ID NO: 833 is provided wherein 1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from the corresponding amino acid of
  • a glucagon analog of SEQ ID NO: 833 is provided wherein 1 to 3 amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from the corresponding amino acid of SEQ ID NO: 801.
  • a glucagon analog of SEQ ID NO: 807, SEQ ID NO: 808 or SEQ ID NO: 834 is provided wherein 1 to 2 amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from the corresponding amino acid of SEQ ID NO: 801, and in a further embodiment those one to two differing amino acids represent conservative amino acid substitutions relative to the amino acid present in the native sequence (SEQ ID NO: 801).
  • a glucagon peptide of SEQ ID NO: 811 or SEQ ID NO: 813 wherein the glucagon peptide further comprises one, two or three amino acid substitutions at positions selected from positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27 or 29. In some embodiments the substitutions at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27 or 29 are conservative amino acid substitutions.
  • a glucagon agonist comprising an analog peptide of SEQ ID NO: 801 wherein the analog differs from SEQ ID NO: 801 by having an amino acid other than serine at position 2 and by having an acidic amino acid substituted for the native amino acid at position 28 or 29 or an acidic amino acid added to the carboxy terminus of the peptide of SEQ ID NO: 801.
  • the acidic amino acid is aspartic acid or glutamic acid.
  • a glucagon analog of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832 is provided wherein the analog differs from the parent molecule by a substitution at position 2. More particularly, position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of D- serine, alanine, D-alanine, glycine, n-methyl serine and amino isobutyric acid.
  • a glucagon agonist comprising an analog peptide of SEQ ID NO: 801 wherein the analog differs from SEQ ID NO: 801 by having an amino acid other than histidine at position 1 and by having an acidic amino acid substituted for the native amino acid at position 28 or 29 or an acidic amino acid added to the carboxy terminus of the peptide of SEQ ID NO: 801.
  • the acidic amino acid is aspartic acid or glutamic acid.
  • a glucagon analog of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832 is provided wherein the analog differs from the parent molecule by a substitution at position 1.
  • position 1 of the analog peptide is substituted with an amino acid selected from the group consisting of DMIA, D-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine and homo- histidine.
  • the modified glucagon peptide comprises a sequence selected from the group consisting of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 and SEQ ID NO: 832.
  • a glucagon peptide is provided comprising a sequence of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832 further comprising one to two amino acids, added to the C-terminus of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832, wherein the additional amino acids are independently selected from the group consisting of Lys, Arg, His, Asp Glu, cysteic acid or homocysteic acid.
  • the additional amino acids added to the carboxy terminus are selected from the group consisting of Lys, Arg, His, Asp or Glu or in a further embodiment the additional amino acids are Asp or Glu.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 807 or a glucagon agonist analog thereof.
  • the peptide comprising a sequence selected from the group consisting of SEQ ID NO: 808, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812 and SEQ ID NO: 813.
  • the peptide comprising a sequence selected from the group consisting of SEQ ID NO: 808, SEQ ID NO: 810 and SEQ ID NO: 811.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 808, SEQ ID NO: 810 and SEQ ID NO: 811 further comprising an additional amino acid, selected from the group consisting of Asp and Glu, added to the C-terminus of the glucagon peptide.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 811 or SEQ ID NO: 813, and in a further embodiment the glucagon peptide comprises the sequence of SEQ ID NO: 811.
  • Xaa at position 15 is Asp, Glu, cysteic acid, homoglutamic acid or homocysteic acid
  • the Xaa at position 28 is Asn or an acidic amino acid
  • the Xaa at position 29 is Thr or an acidic amino acid
  • R is an acidic amino acid, COOH or CONH 2 , with the proviso that an acidic acid residue is present at one of positions 28, 29 or 30.
  • R is COOH, and in another embodiment R is CONH 2 .
  • the present disclosure also encompasses glucagon fusion peptides wherein a second peptide has been fused to the C-terminus of the glucagon peptide to enhance the stability and solubility of the glucagon peptide.
  • the fusion glucagon peptide may comprise a glucagon agonist analog comprising a glucagon peptide NH 2 -His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R (SEQ ID NO: 834), wherein R is an acidic amino acid or a bond and an amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or
  • the glucagon peptide is selected from the group consisting of SEQ ID NO: 833, SEQ ID NO: 807 or SEQ ID NO: 808 further comprising an amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to the carboxy terminal amino acid of the glucagon peptide.
  • the glucagon fusion peptide comprises SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805 and SEQ ID NO: 806 or a glucagon agonist analog thereof, further comprising an amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to amino acid 29 of the glucagon peptide.
  • the fusion peptide further comprises a PEG chain linked to an amino acid at position 16, 17, 21, 24, 29, within a C-terminal extension, or at the C-terminal amino acid, wherein the PEG chain is selected from the range of 500 to 40,000 Daltons.
  • the amino acid sequence of SEQ ID NO: 820 is selected from the range of 500 to 40,000 Daltons.
  • the glucagon peptide portion of the glucagon fusion peptide comprises a sequence selected from the group consisting of SEQ ID NO: 810, SEQ ID NO: 811 and SEQ ID NO: 813.
  • the glucagon peptide portion of the glucagon fusion peptide comprises the sequence of SEQ ID NO: 811 or SEQ ID NO: 813, wherein a PEG chain is linked at position 21, 24, 29, within a C-terminal extension or at the C-terminal amino acid, respectively.
  • the glucagon peptide sequence of the fusion peptide comprises the sequence of SEQ ID NO: 811, further comprising an amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to amino acid 29 of the glucagon peptide.
  • the glucagon fusion peptide comprises a sequence selected from the group consisting of SEQ ID NO: 824, SEQ ID NO: 825 and SEQ ID NO: 826.
  • the fusion glucagon peptide comprises a glucagon agonist analog selected from the group consisting of SEQ ID NO: 810, SEQ ID NO: 811 and SEQ ID NO: 813, further comprising an amino acid sequence of SEQ ID NO: 823 (GPSSGAPPPS-CONH 2 ) linked to amino acid 29 of the glucagon peptide.
  • the glucagon peptide of SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 813, or SEQ ID NO: 832 is modified to comprise one or more hydrophilic groups covalently linked to the side chains of amino acids present at positions 21 and 24 of the glucagon peptide.
  • the glucagon peptide of SEQ ID NO: 811 is modified to contain one or more amino acid substitution at positions 16, 17, 20, 21, 24 and/or 29, wherein the native amino acid is substituted with an amino acid having a side chain suitable for crosslinking with hydrophilic moieties, including for example, PEG.
  • the native peptide can be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non- naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.
  • the polyethylene glycol chain may be in the form of a straight chain or it may be branched. In accordance with some embodiments the polyethylene glycol chain has an average molecular weight selected from the range of about 500 to about 40,000 Daltons. In some embodiments the polyethylene glycol chain has a molecular weight selected from the range of about 500 to about 5,000 Daltons. In another embodiment the polyethylene glycol chain has a molecular weight of about 20,000 to about 40,000 Daltons.
  • any of the glucagon peptides described above may be further modified to include a covalent or non-covalent intramolecular bridge or an alpha helix-stabilizing amino acid within the C-terminal portion of the glucagon peptide (amino acid positions 12-29).
  • the glucagon peptide comprises any one or more of the modifications discussed above in addition to an amino acid substitution at positions 16, 20, 21, or 24 (or a combination thereof) with an ⁇ , ⁇ -disubstituted amino acid, e.g., Aib.
  • the glucagon peptide comprises any one or more modifications discussed above in addition to an intramolecular bridge, e.g., a lactam, between the side chains of the amino acids at positions 16 and 20 of the glucagon peptide.
  • an intramolecular bridge e.g., a lactam
  • the glucagon peptide comprises the amino acid sequence of SEQ ID NO: 877, wherein the Xaa at position 3 is an amino acid comprising a side chain of Structure I, II, or III:
  • R 1 is C 0 - 3 alkyl or C 0 - 3 heteroalkyl
  • R 2 is NHR 4 or Ci_ 3 alkyl
  • R 3 is Ci_ 3 alkyl
  • R 4 is H or Ci_ 3 alkyl
  • X is NH, O, or S
  • Y is NHR 4 , SR 3 , or OR 3 .
  • X is NH or Y is NHR 4 .
  • R 1 is Co- 2 alkyl or Ci heteroalkyl.
  • R 2 is NHR 4 or Ci alkyl.
  • R 4 is H or C 1 alkyl.
  • an amino acid comprising a side chain of Structure I wherein, R 1 is CH 2 -S, X is NH, and R 2 is CH 3 (acetamidomethyl-cysteine, C(Acm)); R 1 is CH 2 , X is NH, and R 2 is CH 3
  • R 1 is C 0 alkyl
  • X is NH
  • R 2 is NHR 4
  • R 4 is H
  • R 1 is CH 2 -CH 2
  • X is NH
  • R is CH 3 (acetylornithine, Orn(Ac)).
  • an amino acid comprising a side chain of Structure II is provided, wherein R 1 is CH 2 , Y is NHR 4 , and R 4 is CH 3 (methylglutamine, Q(Me));
  • an amino acid comprising a side chain of Structure III is provided wherein, R 1 is CH 2 and R 4 is H (methionine- sulfoxide, M(O));
  • the amino acid at position 3 is substituted with Dab(Ac).
  • glucagon agonists can comprise the amino acid sequence of SEQ ID NO: 863, SEQ ID NO: 869, SEQ ID NO: 871, SEQ ID NO: 872, SEQ ID NO: 873, and SEQ ID NO: 874.
  • the glucagon peptide is an analog of the glucagon peptide of SEQ ID NO: 877.
  • the analog comprises any of the amino acid modifications described herein, including, but not limited to: a substitution of Asn at position 28 with a charged amino acid; a 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; a substitution at position 28 with Asn, Asp, or Glu; a substitution at position 28 with Asp; a substitution at position 28 with Glu; a substitution of Thr at position 29 with a charged amino acid; a 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; a substitution at position 29 with Asp, Glu, or Lys; a substitution at position 29 with Glu; a substitution at position 29 with Glu
  • the analog of the glucagon peptide of SEQ ID NO: 877 comprises an ⁇ , ⁇ -disubstituted amino acid, such as Aib, at one, two, three, or all of positions 16, 20, 21, and 24.
  • the analog of the glucagon peptide of SEQ ID NO: 877 comprises one or more of the following: 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 Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He; substitution of Tyr at position 10 with Phe or Val; substitution of Lys at position 12 with Arg; substitution of Asp at position 15 with Glu, substitution of Ser at position 16 with Thr or Aib; substitution of Gin at position 20 with Ala or Aib; substitution of Asp at position 21 with Glu; substitution of Gin at position 24 with Ala or Aib; substitution of Met at
  • the glucagon peptide comprises the amino acid sequence of any of SEQ ID NOs: 862-867 and 869-874.
  • the analog of the glucagon peptide comprising SEQ ID NO: 877 comprises a hydrophilic moiety, e.g., PEG, covalently linked to the amino acid at any of positions 16, 17, 20, 21, 24, and 29 or at the C-terminal amino acid.
  • analog of the glucagon peptide comprising SEQ ID NO: 1
  • ID NO: 877 comprises an amino acid comprising a side chain covalently attached, optionally, through a spacer, to an acyl group or an alkyl group, which acyl group or alkyl group is non-native to a naturally-occurring amino acid.
  • the acyl group in some embodiments is a C4 to C30 fatty acyl group.
  • the alkyl group is a C4 to C30 alkyl.
  • the acyl group or alkyl group is covalently attached to the side chain of the amino acid at position 10.
  • the amino acid at position 7 is He or Abu.
  • the glucagon agonist may be a peptide comprising the amino acid sequence of any of the SEQ ID NOs: 801-919, optionally with up to 1, 2, 3, 4, or 5 further modifications that retain glucagon agonist activity.
  • the glucagon agonist comprises the amino acids of any of SEQ ID NOs: 859-919.
  • the glucagon related peptide is a Class 2 glucagon related peptide, which is described herein and in International Patent Publication No.
  • SEQ ID NOs: 1-262 in International Patent Publication No. WO 2010/011439.
  • SEQ ID NOs: 1284 to 1296 relating to Class 2 glucagon related peptides correspond to SEQ ID NOs: 657 to 669 in U.S. Application No. 61/187,578.
  • Native glucagon does not activate the GIP receptor, and normally has about 1% of the activity of native-GLP-1 at the GLP-1 receptor. Modifications to the native glucagon sequence described herein produce Class 2 glucagon related peptides that can exhibit potent glucagon activity equivalent to or better than the activity of native glucagon (SEQ ID NO: 1001), potent GIP activity equivalent to or better than the activity of native GIP (SEQ ID NO: 1004), and/or potent GLP-1 activity equivalent to or better than the activity of native GLP-1.
  • the Class 2 glucagon related peptide may be one of a glucagon/GIP co-agonist, glucagon/GIP/GLP-1 tri- agonist, GIP/GLP-1 co-agonist, or a GIP agonist glucagon peptide, as further described herein.
  • the Class 2 glucagon related peptides described herein exhibit an EC 50 for GIP receptor activation activity of about 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less. In some embodiments, the Class 2 glucagon related peptides exhibit an EC 50 for glucagon receptor activation of about 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less. In some embodiments, the Class 2 glucagon related peptides exhibit an EC 50 for GLP-1 receptor activation of about 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less. Receptor activation can be measured by in vitro assays measuring cAMP induction in HEK293 cells over-expressing the receptor, e.g. assaying
  • HEK293 cells co-transfected with DNA encoding the receptor and a luciferase gene linked to cAMP responsive element as described in Example 2.
  • Class 2 glucagon related peptides exhibit at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175% or 200% or higher activity at the GIP receptor relative to native GIP (GIP potency).
  • the glucagon peptides described herein exhibit no more than 1000%, 10,000%,
  • Class 2 glucagon related peptides exhibit at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or higher activity at the glucagon receptor relative to native glucagon (glucagon potency).
  • the glucagon peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the glucagon receptor relative to native glucagon.
  • Class 2 glucagon related peptides exhibit at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%,
  • the glucagon peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GLP-1 receptor relative to native GLP-1.
  • a Class 2 glucagon related peptide's activity at a receptor relative to a native ligand of the receptor is calculated as the inverse ratio of EC50S for the Class 2 glucagon related peptide vs. the native ligand.
  • Class 2 glucagon related peptides exhibit activity at both the glucagon receptor and the GIP receptor ("glucagon/GIP co-agonists"). These Class 2 glucagon related peptides have lost native glucagon's selectivity for glucagon receptor compared to GIP receptor.
  • the EC 50 of the Class 2 glucagon related peptide at the GIP receptor is less than about 50-fold, 40-fold, 30- fold or 20-fold different (higher or lower) from its EC 50 at the glucagon receptor.
  • the GIP potency of the Class 2 glucagon related peptide is less than about 500-, 450-, 400-, 350-, 300-, 250-, 200-, 150-, 100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its glucagon potency.
  • the ratio of the EC 50 of the Class 2 glucagon related peptide at the GIP receptor divided by the EC 50 of the Class 2 glucagon related peptide at the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the EC 50 at the GIP receptor divided by the EC 50 at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the Class 2 glucagon related peptide compared to the glucagon potency of the Class 2 glucagon related peptide is less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the potency at the GIP receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2).
  • GLP-1 activity have been significantly reduced or destroyed, e.g., by an amino acid modification at position 7, a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28, yielding a 27- or 28-amino acid peptide, or a combination thereof.
  • Class 2 glucagon related peptides exhibit activity at the glucagon, GIP and GLP-1 receptors ("glucagon/GIP/GLP-1 tri-agonists"). These Class 2 glucagon related peptides have lost native glucagon's selectivity for the glucagon receptor compared to both the GLP-1 and GIP receptors.
  • the EC 50 of the Class 2 glucagon related peptide at the GIP receptor is less than about 50-fold, 40-fold, 30-fold or 20-fold different (higher or lower) from its respective EC50S at the glucagon and GLP-1 receptors.
  • the GIP potency of the Class 2 glucagon related peptide is less than about 500-, 450-, 400-, 350-, 300-, 250-, 200-, 150-, 100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its glucagon and GLP-1 potencies.
  • the ratio of the EC 50 of the tri-agonist at the GIP receptor divided by the EC 50 of the tri- agonist at the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the EC 50 at the GIP receptor divided by the EC 50 at the GLP-1 receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the tri-agonist compared to the GLP-1 potency of the tri-agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the potency at the GIP receptor divided by the potency at the GLP-1 receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In related embodiments, the ratio of the EC 50 of the tri-agonist at the GIP receptor divided by the EC 50 of the tri-agonist at the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the EC 50 at the GIP receptor divided by the EC 50 at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the tri-agonist compared to the glucagon potency of the tri-agonist is less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the potency at the GIP receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the EC 50 of the tri- agonist at the GLP-1 receptor divided by the EC 50 of the tri-agonist at the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the EC 50 at the GLP-1 receptor divided by the EC 50 at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GLP-1 potency of the tri-agonist compared to the glucagon potency of the tri-agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.
  • the ratio of the potency at the GLP-1 receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2).
  • Class 2 glucagon related peptides exhibit activity at the GLP-1 and GIP receptors, but in which the glucagon activity has been significantly reduced or destroyed (“GIP/GLP-1 co-agonists"), e.g., by an amino acid modification at position 3. For example, substitution at this position with an acidic, basic, or a hydrophobic amino acid (glutamic acid, ornithine, norleucine) reduces glucagon activity.
  • the EC 50 of the glucagon peptide at the GIP receptor is less than about 50-fold, 40-fold, 30-fold or 20-fold different (higher or lower) from its EC 50 at the GLP-1 receptor.
  • the GIP potency of the Class 2 glucagon related peptide is less than about 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its GLP-1 potency. In some embodiments these Class 2 glucagon related peptides have about 10% or less of the activity of native glucagon at the glucagon receptor, e.g. about 1-10%, or about 0.1-10%, or greater than about 0.1% but less than about 10%.
  • the ratio of the EC 50 of the Class 2 glucagon related peptide at the GIP receptor divided by the EC 50 of the Class 2 glucagon related peptide at the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the GIP potency of the Class 2 glucagon related peptide compared to the GLP-1 potency of the Class 2 glucagon related peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • Class 2 glucagon related peptides exhibit activity at the GIP receptor, in which the glucagon and GLP-1 activity have been significantly reduced or destroyed ("GIP agonist glucagon peptides"), e.g., by amino acid modifications at positions 3 with Glu and 7 with He.
  • GIP agonist glucagon peptides e.g., by amino acid modifications at positions 3 with Glu and 7 with He.
  • these Class 2 glucagon related peptides have about 10% or less of the activity of native glucagon at the glucagon receptor, e.g. about 1-10%, or about 0.1-10%, or greater than about 0.1%, 0.5%, or 1% but less than about 1%, 5%, or 10%.
  • these Class 2 glucagon related peptides also have about 10% or less of the activity of native GLP-1 at the GLP-1 receptor, e.g. about 1-10%, or about 0.1- 10%, or greater than about 0.1%, 0.5%, or 1% but less than about 1%, 5%, or 10%.
  • the EC 50 of the Class 2 glucagon related peptide for GIP receptor activation is about 4, 2, 1 nM or less, or the analog has at least about 1%, 2%, 3%, 4% or 5% of the activity of native GIP at the GIP receptor.
  • the EC 50 of the unpegylated Class 2 glucagon related peptide for GLP-1 receptor activation is about 4, 2, 1 nM or less or has at least about 1%, 2%, 3%, 4% or 5% of the activity of native GLP-1 at the GLP-1 receptor.
  • the EC 50 of the unpegylated Class 2 glucagon related peptide for glucagon receptor activation is about 4, 2, 1 nM or less, or at least about 5%, 10%, 15% or 20% of the activity of native glucagon at the glucagon receptor.
  • the unpegylated Class 2 glucagon related peptide has less than about 1% of the activity of native glucagon at the glucagon receptor. In other embodiments, the unpegylated Class 2 glucagon related peptide has less than about 10%, 5% or 1% of the activity of native GLP-1 at the GLP-1 receptor.
  • the relative EC 50s at one or more receptors may be higher e.g., about 10-fold higher.
  • GIP receptor activation is about 10 nM or less, or the Class 2 glucagon related peptide has at least about 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the activity of native GIP at the GIP receptor.
  • the EC 50 of a pegylated Class 2 glucagon related peptide for GLP-1 receptor activation is about 10 nM or less or has at least about 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the activity of native GLP- 1 at the GLP- 1 receptor.
  • the EC 50 of a pegylated Class 2 glucagon related peptide for glucagon receptor activation is about 10 nM or less, or at least about 0.5%, 1%, 1.5% or 2% of the activity of native glucagon at the glucagon receptor. In some embodiments, the Class 2 glucagon related peptide has less than about 1% of the activity of native glucagon at the glucagon receptor. In other embodiments, the Class 2 glucagon related peptide has less than about 10%, 5% or
  • the modifications disclosed herein in reference to a Class 2 glucagon related peptide permit the manipulation of glucagon (SEQ ID NO: 1001) to create glucagon peptides that exhibit increased GIP activity, glucagon activity, and/or GLP-1 activity.
  • Other modifications disclosed herein in reference to a Class 2 glucagon related peptide prolong the half-life, increase solubility, or increase stability of the resulting peptide.
  • Yet other modifications disclosed herein in reference to a Class 2 glucagon related peptide have no effect on activity, or can be made without destroying the desired activity or activities. Any of the combinations in reference to a Class 2 glucagon related peptide that serve the same purpose (e.g. increasing GIP activity) can be applied individually or in combination.
  • any of the single or sets of combinations in reference to a Class 2 glucagon related peptide that confer enhanced properties can be applied individually or in combination, e.g. increased GIP and/or GLP-1 activity can be combined with increased half-life.
  • 1, 2, 3, 4, 5, 6 or more of the amino acid modifications may be non-conservative substitutions, additions or deletions.
  • 1, 2, 3, 4, 5, 6 or more of the amino acid modifications may be conservative substitutions.
  • Enhanced activity at the GIP receptor is provided by an amino acid modification at position 1.
  • His at position 1 is substituted with a large, aromatic amino acid, optionally Tyr, Phe, Trp, amino-Phe, nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3 -amino Tyr.
  • the combination of Tyr at position 1 with stabilization of the alpha helix within the region corresponding to amino acids 12-29 provided a Class 2 glucagon related peptide that activates the GIP receptor as well as the GLP-1 receptor and the glucagon receptor.
  • the alpha helix structure can be stabilized by, e.g., formation of a covalent or non-covalent intramolecular bridge, or substitution and/or insertion of amino acids around positions 12-29 with an alpha helix-stabilizing amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid).
  • an alpha helix-stabilizing amino acid e.g., an ⁇ , ⁇ -disubstituted amino acid
  • Enhanced activity at the GIP receptor is also provided by amino acid modifications at positions 27 and/or 28, and optionally at position 29.
  • the Met at position 27 is substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 is substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 is substituted with a small aliphatic amino acid, optionally Gly.
  • Substitution with LAG at positions 27-29 provides increased GIP activity relative to the native MNT sequence at those positions.
  • Enhanced activity at the GIP receptor is also provided by an amino acid modification at position 12.
  • position 12 is substituted with a large, aliphatic, nonpolar amino acid, optionally He.
  • Enhanced activity at the GIP receptor is also provided by an amino acid modification at positions 17 and/or 18.
  • position 17 is substituted with a polar residue, optionally Gin
  • position 18 is substituted with a small aliphatic amino acid, optionally Ala.
  • a substitution with QA at positions 17 and 18 provides increased GIP activity relative to the native RR sequence at those positions.
  • an intramolecular bridge can be formed by a covalent bond between the side chains of two amino acids at positions i and i+4 or between positions j and j+3, or between positions k and k+7.
  • the bridge is between positions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20.
  • non-covalent interactions such as salt bridges can be formed between positively and negatively charged amino acids at these positions.
  • enhanced glucagon potency is provided by an amino acid modification at position 16 of native glucagon (SEQ ID NO: 1001).
  • such enhanced potency can be provided by substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, or a charged amino acid having a side chain containing at least one heteroatom, (e.g. N, O, S, P) and with a side chain length of about 4 (or 3-5) atoms.
  • the glucagon peptide retains its original selectivity for the glucagon receptor relative to the GLP-1 receptors.
  • Glucagon receptor activity can be reduced by an amino acid modification at position 3, e.g. substitution of the naturally occurring glutamine at position 3, with an acidic, basic, or a hydrophobic amino acid.
  • substitution at position 3 with glutamic acid, ornithine, or norleucine substantially reduces or destroys glucagon receptor activity.
  • Maintained or enhanced activity at the glucagon receptor may be achieved by modifying the Gin at position 3 with a glutamine analog, as described herein.
  • glucagon agonists can comprise the amino acid sequence of any of SEQ ID NOs: 1243-1248, 1250, 1251, and 1253-1256.
  • glucagon activity which has been reduced by amino acid modifications at positions 1 and 2 is provided by modifications that that stabilize the alpha helix structure of the C-terminal portion (amino acids 12-29) of the glucagon peptide or analog thereof.
  • an intramolecular bridge can be formed by a covalent bond between the side chains of two amino acids at positions i and i+4 or between positions j and j+3, or between positions k and k+7.
  • non-covalent interactions such as salt bridges can be formed between positively and negatively charged amino acids at these positions.
  • one or more a, a-disubstituted amino acids are inserted or substituted into this C-terminal portion (amino acids 12-29) at positions that retain the desired activity.
  • amino acids 12-29 amino acids 12-29
  • one, two, three or all of positions 16, 20, 21 or 24 are substituted with an a, a- disubstituted amino acid, e.g., Aib.
  • Enhanced activity at the GLP-1 receptor is provided by replacing the carboxylic acid of the C-terminal amino acid with a charge-neutral group, such as an amide or ester.
  • Enhanced activity at the GLP-1 receptor is also provided by stabilizing the alpha-helix structure in the C-terminal portion of glucagon (around amino acids 12- 29), e.g., through formation of an intramolecular bridge between the side chains of two amino acids, or substitution and/or insertion of amino acids around positions 12- 29 with an alpha helix- stabilizing amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid), as further described herein.
  • an alpha helix- stabilizing amino acid e.g., an ⁇ , ⁇ -disubstituted amino acid
  • the side chains of the amino acid pairs 12 and 16, 13 and 17, 16 and 20 , 17 and 21, 20 and 24 or 24 and 28 are linked to one another and thus stabilize the glucagon alpha helix.
  • the bridge or linker is about 8 (or about 7-9) atoms in length, particularly when the bridge is between positions i and i+4. In some embodiments, the bridge or linker is about 6 (or about 5-7) atoms in length, particularly when the bridge is between positions j and j+3.
  • intramolecular bridges are formed by (a) substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, or a charged amino acid having a side chain containing at least one heteroatom, (e.g.
  • the side chains of such amino acids at positions 16 and 20 can form a salt bridge or can be covalently linked.
  • the two amino acids are bound to one another to form a lactam ring.
  • stabilization of the alpha helix structure in the C- terminal portion of the glucagon peptide is achieved through the formation of an intramolecular bridge other than a lactam bridge.
  • suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, the use of a, co-diaminoalkane tethers, the formation of metal-atom bridges, and other means of peptide cyclization are used to stabilize the alpha helix.
  • one or more a, a-disubstituted amino acids are inserted or substituted into this C-terminal portion (amino acids 12-29) at positions that retain the desired activity.
  • amino acids 12-29 amino acids 12-29
  • one, two, three or all of positions 16, 20, 21 or 24 are substituted with an a, a-disubstituted amino acid, e.g., Aib.
  • Increased activity at the GLP-1 receptor is provided by an amino acid modification at position 20 as described herein.
  • Increased activity at the GLP-1 receptor is provided by adding GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096) to the C-terminus, wherein X is any amino acid.
  • GLP-1 activity in such analogs can be further increased by modifying the amino acid at position 18, 28 or 29, or at position 18 and 29, as described herein.
  • a further modest increase in GLP-1 potency is provided by modifying the amino acid at position 10 to be a large, aromatic amino acid residue, optionally Trp. Reduced activity at the GLP-1 receptor is provided, e.g., by an amino acid modification at position 7 as described herein.
  • Potency at the GLP-1 receptor can be further enhanced by an alanine substitution for the native arginine at position 18.
  • the invention provides glucagon peptides that comprise modifications at position 16, at position 20, and at the C-terminal carboxylic acid group, optionally with a covalent bond between the amino acids at positions 16 and 20; glucagon peptides that comprise modifications at position 16 and at the C-terminal carboxylic acid group; glucagon peptides that comprise modifications at positions 16 and 20, optionally with a covalent bond between the amino acids at positions 16 and 20; and glucagon peptides that comprise modifications at position 20 and at the C- terminal carboxylic acid group.
  • Modifications at position 1 and/or 2 can increase the peptide's resistance to dipeptidyl peptidase IV (DPP IV) cleavage.
  • DPP IV dipeptidyl peptidase IV
  • position 1 and/or position 2 may be substituted with a DPP-IV resistant amino acid as described herein.
  • the amino acid at position 2 is substituted with N-methyl alanine.
  • modifications at position 2 may reduce glucagon activity, sometimes significantly; surprisingly, this reduction in glucagon activity can be restored by stabilizing the alpha-helix structure in the C-terminal portion of glucagon (around amino acids 12-29), e.g., through formation of a covalent bond between the side chains of two amino acids, as described herein.
  • the covalent bond is between amino acids at positions "i" and "i+4", or positions "j" and "j+3", e.g., between positions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20.
  • this covalent bond is a lactam bridge between a glutamic acid at position 16 and a lysine at position 20. In some embodiments, this covalent bond is an intramolecular bridge other than a lactam bridge, as described herein. Modifications that reduce degradation
  • any of the Class 2 glucagon related peptides can be further modified to improve stability by modifying the amino acid at position 15 and/or 16 of SEQ ID NO: 1001 to reduce degradation of the peptide over time, especially in acidic or alkaline buffers. Such modifications reduce cleavage of the Aspl5-Serl6 peptide bond.
  • the amino acid modification at position 15 is a deletion or substitution of Asp with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid. In other exemplary
  • the amino acid modification at position 16 is a deletion or substitution of Ser with Thr or Aib.
  • Ser at position 16 is substituted with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid.
  • the methionine residue present at position 27 of the native peptide is modified, e.g. by deletion or substitution. Such modifications may prevent oxidative degradation of the peptide.
  • the Met at position 27 is substituted with leucine, isoleucine or norleucine. In some specific embodiments, Met at position 27 is substituted with leucine or norleucine.
  • the Gin at position 20 and/or 24 is modified, e.g. by deletion or substitution. Such modifications can reduce degradation that occurs through deamidation of Gin.
  • the Gin at position 20 and/or 24 is substituted with Ser, Thr, Ala or Aib.
  • the Gin at position 20 and/or 24 is substituted with Lys, Arg, Orn, or Citrulline.
  • the Asp at position 21 is modified, e.g. by deletion or substitution. Such modifications can reduce degradation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by
  • position 21 is substituted with Glu, homoglutamic acid or homocysteic acid. In some specific embodiments, position 21 is substituted with Glu.
  • Stabilization of the alpha-helix structure in the C-terminal portion of the Class 2 glucagon related peptide provides enhanced GLP-1 and/or GIP activity and restores glucagon activity which has been reduced by amino acid modifications at positions 1 and/or 2.
  • the alpha helix structure can be stabilized by, e.g., formation of a covalent or non-covalent intramolecular bridge, or substitution and/or insertion of amino acids around positions 12-29 with an alpha helix-stabilizing amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid).
  • Stabilization of the alpha-helix structure of a GIP agonist may be accomplished as described herein.
  • the glucagon peptides disclosed herein are modified to comprise an acyl group or alkyl group, e.g., an acyl or alkyl group which is non-native to a naturally-occurring amino acid as described herein.
  • Acylation or alkylation can increase the half-life of the glucagon peptides in circulation. Acylation or alkylation can advantageously delay the onset of action and/or extend the duration of action at the glucagon and/or GLP-1 receptors and/or improve resistance to proteases such as DPP-IV and/or improve solubility. Activity at the glucagon and/or GLP-1 and/or GIP receptors of the glucagon peptide may be maintained after acylation. In some embodiments, the potency of the acylated glucagon peptides is comparable to the unacylated versions of the glucagon peptides. Class 2 glucagon related peptides may be acylated or alkylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position, as described herein.
  • the invention provides a glucagon peptide modified to comprise an acyl group or alkyl group covalently linked to the amino acid at position 10 of the glucagon peptide.
  • the glucagon peptide may further comprise a spacer between the amino acid at position 10 of the glucagon peptide and the acyl group or alkyl group.
  • the acyl group is a fatty acid or bile acid, or salt thereof, e.g.
  • the spacer is any moiety with suitable reactive groups for attaching acyl or alkyl groups.
  • the spacer comprises an amino acid, a dipeptide, a tripeptide, a hydrophilic bifunctional, or a hydrophobic bifunctional spacer.
  • the spacer is selected from the group consisting of: Trp, Glu, Asp, Cys and a spacer comprising NH 2 (CH 2 CH 2 0) n (CH 2 ) m COOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12.
  • acylated or alkylated glucagon peptides may also further comprise a hydrophilic moiety, optionally a polyethylene glycol. Any of the foregoing glucagon peptides may comprise two acyl groups or two alkyl groups, or a combination thereof.
  • the GIP agonist can be linked, optionally via covalent bonding and optionally via a linker, to a conjugate moiety as described herein.
  • the second peptide is XGPSSGAPPPS (SEQ ID NO: 1096), wherein X is selected from one of the 20 common amino acids, e.g., glutamic acid, aspartic acid or glycine.
  • X represents an amino acid, for example Cys, that further comprises a hydrophilic moiety covalently linked to the side chain of that amino acid.
  • Such C-terminal extensions improve solubility and also can improve GIP or GLP-1 activity.
  • the glucagon peptide further comprises a carboxy terminal extension, the carboxy terminal amino acid of the extension ends in an amide group or an ester group rather than a carboxylic acid.
  • the threonine at position 29 of the native glucagon peptide is replaced with a glycine.
  • a glucagon peptide having a glycine substitution for threonine at position 29 and comprising the C-terminal extension of GPSSGAPPPS (SEQ ID NO: 1095) is four times as potent at the GLP-1 receptor as native glucagon modified to comprise the same C-terminal extension.
  • This T29G substitution can be used in conjunction with other modifications disclosed herein to enhance the affinity of the glucagon peptides for the GLP-1 receptor.
  • the T29G substitution can be combined with the S 16E and N20K amino acid substitutions, optionally with a lactam bridge between amino acids 16 and 20, and optionally with addition of a PEG chain as described herein.
  • an amino acid is added to the C-terminus, and the additional amino acid is selected from the group consisting of glutamic acid, aspartic acid and glycine.
  • the solubility of any of the glucagon peptides can be improved by amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide, preferably at a position C-terminal to position 27 of SEQ ID NO: 1001.
  • one, two or three charged amino acids may be introduced within the C-terminal portion, preferably C-terminal to position 27.
  • the native amino acid(s) at positions 28 and/or 29 are substituted with one or two charged amino acids, and/or in a further embodiment one to three charged amino acids are also added to the C-terminus of the peptide.
  • one, two or all of the charged amino acids are negatively charged.
  • the negatively charged (acidic amino acid) is aspartic acid or glutamic acid.
  • glucagon peptide may be made to the glucagon peptide that still allow it to retain GIP activity (and optionally GLP-1 activity and/or glucagon activity).
  • any of the modifications described above in reference to a Class 2 peptide which increase or decrease GIP activity, which increase or decrease glucagon receptor activity, and which increase GLP-1 receptor activity can be applied individually or in combination. Any of the modifications described above in reference to a Class 2 glucagon related peptide can also be combined with other modifications that confer other desirable properties, such as increased solubility and/or stability and/or duration of action, as described herein with regard to Class 2 glucagon related peptides.
  • any of the modifications described above in reference to Class 2 glucagon related peptides can be combined with other modifications described herein in reference to Class 2 glucagon related peptides that do not substantially affect solubility or stability or activity.
  • Exemplary modifications include but are not limited to:
  • (A) Improving solubility for example, by introducing one, two, three or more charged amino acid(s) to the C-terminal portion of native glucagon, preferably at a position C- terminal to position 27.
  • a charged amino acid can be introduced by substituting a native amino acid with a charged amino acid, e.g. at positions 28 or 29, or alternatively by adding a charged amino acid, e.g. after position 27, 28 or 29.
  • one, two, three or all of the charged amino acids are negatively charged.
  • one, two, three or all of the charged amino acids are positively charged.
  • Such modifications increase solubility, e.g.
  • solubility and duration of action or half-life in circulation by addition of a hydrophilic moiety such as a polyethylene glycol chain, as described herein, e.g. at position 16, 17, 20, 21, 24 or 29, within a C-terminal extension, or at the C-terminal amino acid of the peptide,
  • D Increasing duration of action or half-life in circulation through introducing resistance to dipeptidyl peptidase IV (DPP IV) cleavage by modification of the amino acid at position 1 or 2 as described herein.
  • (E) Increasing stability by modification of the Asp at position 15, for example, by deletion or substitution with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid.
  • Such modifications can reduce degradation or cleavage at a pH within the range of 5.5 to 8, for example, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, up to 100% of the original peptide after 24 hours at 25°C.
  • Such modifications reduce cleavage of the peptide bond between Aspl5-Serl6.
  • (G) Increasing stability by modification of the methionine at position 27, for example, by substitution with leucine or norleucine. Such modifications can reduce oxidative degradation. Stability can also be increased by modification of the Gin at position 20 or 24, e.g. by substitution with Ser, Thr, Ala or Aib. Such modifications can reduce degradation that occurs through deamidation of Gin. Stability can be increased by modification of Asp at position 21, e.g. by substitution with Glu. Such modifications can reduce degradation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to iso-aspartate.
  • GPSSGAPPPS SEQ ID NO: 1095
  • Some positions of the native glucagon peptide can be modified while retaining at least some of the activities of the parent peptide. Accordingly, applicants anticipate that one or more of the amino acids located at positions at positions 2, 5, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 can be substituted with an amino acid different from that present in the native glucagon peptide, and still retain activity at the glucagon receptor.
  • position 18 is substituted with an amino acid selected from the group consisting of Ala, Ser, or Thr.
  • amino acid at position 20 is substituted with Ser, Thr, Lys, Arg, Orn, Citrulline or Aib.
  • position 21 is substituted with Glu, homoglutamic acid or homocysteic acid.
  • the glucagon peptide comprises 1 to 10 amino acid modifications selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28 and 29.
  • the modifications are one or more amino acid substitutions selected from the group consisting of Glnl7, Alal8, Glu21, Ile23, Ala24, Val27 and Gly29.
  • 1 to 2 amino acids selected from positions 17-26 differ from the parent peptide. In other embodiments, 1 to 2 amino acids selected from positions 17-22 differ from the parent peptide. In yet other embodiments, the modifications are Glnl7, Alal8, Glu21, Ile23 and Ala24.
  • one or more amino acids are added to the carboxy terminus of the glucagon peptide.
  • the amino acid is typically selected from one of the 20 common amino acids, and in some embodiments the amino acid has an amide group in place of the carboxylic acid of the native amino acid.
  • the added amino acid is selected from the group consisting of glutamic acid and aspartic acid and glycine.
  • the Class 2 glucagon related peptides disclosed herein are modified by truncation of the C-terminus by one or two amino acid residues yet retain similar activity and potency at the glucagon, GLP-1 and/or GIP receptors.
  • the amino acid at position 29 and/or 28 can be deleted.
  • the analog of glucagon (SEQ ID NO: 1001) having GIP agonist activity comprises SEQ ID NO: 1001 with (a) an amino acid modification at position 1 that confers GIP agonist activity, (b) a modification which stabilizes the alpha helix structure of the C-terminal portion (amino acids 12-29) of the analog, and (c) optionally, 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) further amino acid modifications.
  • the analog exhibits at least about 1% activity of native GIP at the GIP receptor or any other activity level at the GIP receptor described herein.
  • the modification which stabilizes the alpha helix structure is one which provides or introduces an intramolecular bridge, including, for example, a covalent intramolecular bridge, such as any of those described herein.
  • the covalent intramolecular bridge in some embodiments is a lactam bridge.
  • the lactam bridge of the analog of these embodiments can be a lactam bridge as described herein.
  • the lactam bridge may be one which is 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.
  • the lactam bridge can be between the amino acids at positions 16 and 20, wherein one of the amino acids at positions 16 and 20 is substituted with Glu and the other of the amino acids at positions 16 and 20 is substituted with Lys.
  • the modification which stabilizes the alpha helix structure is the introduction of one, two, three, or four ⁇ , ⁇ -disubstituted amino acids at position(s) 16, 20, 21, and 24 of the analog.
  • the ⁇ , ⁇ - disubstituted amino acid is Aib.
  • the ⁇ , ⁇ -disubstituted amino acid (e.g., Aib) is at position 20 and the amino acid at position 16 is substituted with a positive-charged amino acid, such as, for example, an amino acid of Formula IV, which is described herein.
  • the amino acid of Formula rV may be homoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab).
  • the amino acid modification at position 1 is a substitution of His with an amino acid lacking an imidazole side chain, e.g. a large, aromatic amino acid (e.g., Tyr).
  • the analog of glucagon comprises amino acid modifications at one, two or all of positions 27, 28 and 29.
  • the Met at position 27 can be substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 can be substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 can be substituted with a small aliphatic amino acid, optionally Gly, or a combination of two or three of the foregoing.
  • the analog of glucagon comprises Leu at position 27, Ala at position 28, and Gly or Thr at position 29.
  • the analog of glucagon comprises an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29.
  • the extension can comprise the amino acid sequence of SEQ ID NO: 1095 or 1096, for instance.
  • the analog of glucagon can comprise an extension of which 1-6 amino acids of the extension are positive-charged amino acids.
  • the positive-charged amino acids may be amino acids of Formula IV, including, but not limited to Lys, homoLys, Orn, and Dab.
  • the analog of glucagon in some embodiments is acylated or alkylated as described herein.
  • the acyl or alkyl group may be attached to the analog of glucagon, with or without a spacer, at position 10 or 40 of the analog, as further described herein.
  • the analog may additionally or alternatively be modified to comprise a hydrophilic moiety as further described herein.
  • the analog comprises any one or a combination of the following modifications:
  • the analog of glucagon (SEQ ID NO: 1001) having GIP agonist activity comprises the following modifications:
  • the EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • lactam bridge of the analog of these embodiments can be a lactam bridge as described herein. See, e.g., the teachings of lactam bridges under the section
  • the lactam bridge can be between the amino acids at positions 16 and 20, wherein one of the amino acids at positions 16 and 20 is substituted with Glu and the other of the amino acids at positions 16 and 20 is substituted with Lys.
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID NOs: 1005-1094.
  • the analog of glucagon (SEQ ID NO: 1001) having GIP agonist activity comprises the following modifications:
  • amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an ⁇ , ⁇ -disubstituted amino acid, amino acid modifications at one, two or all of positions 27, 28 and 29, e.g., amino acid modifications at position 27 and/or 28, and
  • the EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • the ⁇ , ⁇ -disubstituted amino acid of the analog of these embodiments can be any ⁇ , ⁇ -disubstituted amino acid, including, but not limited to, amino iso-butyric acid (Aib), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g., 1- aminocyclooctane-l-carboxylic acid).
  • the ⁇ , ⁇ -disubstituted amino acid is Aib.
  • the amino acid at position 20 is substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., Aib.
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID Nos: 1099-1141, 1144-1164, 1166-1169, and 1173-1178.
  • the analog of glucagon (SEQ ID NO: 1001) having GIP agonist activity comprises the following modifications:
  • n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, each of Rl and R2 is independently selected from the group consisting of H, CI -CI 8 alkyl, (CI -CI 8 alkyl)OH, (C1-C18 alkyl)NH2, (C1-C18 alkyl)SH, (C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl), wherein R7 is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group, (c) an amino acid substitution of the Gin at position 20 with an alpha, alpha-disubstituted amino acid,
  • the EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • the amino acid of Formula IV of the analog of these embodiments may be any amino acid, such as, for example, the amino acid of Formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4, or 5, in which case, the amino acid is Dab, Orn, Lys, or homoLys respectively.
  • the alpha, alpha-disubstituted amino acid of the analog of these embodiments may be any alpha, alpha-disubstituted amino acid, including, but not limited to, amino iso-butyric acid (Aib), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or
  • cycloheptane e.g., 1-aminocyclooctane-l-carboxylic acid.
  • the alpha, alpha-disubstituted amino acid is Aib.
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID NOs: 1099-1165.
  • the analog of glucagon (SEQ ID NO:
  • GIP agonist activity comprises:
  • EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • the acylated or alkylated amino acid is an amino acid of Formula I, II, or III.
  • the amino acid of Formula I is Dab, Orn, Lys, or homoLys.
  • the extension of about 1 to about 21 amino acids comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSSGAPPPK (SEQ ID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small, aliphatic or non- polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to SEQ ID NO: 1095, 1096, 1170, 1171 or 1172.
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally- extended analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminally extended analog.
  • the analog having GIP agonist activity further comprises amino acid modifications at one, two or all of positions 27, 28 and 29, e.g., amino acid modifications at position 27 and/or 28.
  • the amino acid modification at position 1 that confers GIP agonist activity can be a substitution of His with an amino acid lacking an imidazole side chain.
  • the amino acid modification at position 1 can, for example, be a substitution of His with a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • the analog does not comprise an amino acid modification at position 1 which modification confers GIP agonist activity.
  • the amino acid at position 1 is not a large, aromatic amino acid, e.g., Tyr.
  • the amino acid at position 1 is an amino acid comprising an imidazole ring, e.g., His, analogs of His.
  • the analog is not any of the compounds disclosed in International Patent Application Publication No. WO
  • the analog comprises the amino acid sequence of any of SEQ ID Nos: 1284-1296.
  • amino acid modifications at one, two, or all of positions 27, 28, and 29 can be any of the modifications at these positions described herein.
  • the Met at position 27 can be substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 can be substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 can be substituted with a small aliphatic amino acid, optionally Gly.
  • the analog can comprise such amino acid modifications at position 27 and/or 28.
  • the analog of the above exemplary embodiments can further comprise 1-9 or 1-6 further, additional amino acid modifications, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half- life in circulation, delay the onset of action, or increase stability.
  • the analog can further comprise, for example, an amino acid modification at position 12, optionally, a substitution with lie, and/or amino acid modifications at positions 17 and 18, optionally substitution with Q at position 17 and A at position 18, and/or an addition of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), or sequences containing one or more conservative substitutions relative to SEQ ID NO: 1095 or 1096, to the C- terminus, wherein X is any amino acid.
  • the analog can comprise one or more of the following modifications:
  • the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gin with Glu), wherein the analog has less than 1% of the activity of glucagon at the glucagon receptor.
  • the analog can comprise an amino acid modification at position 7 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), wherein the analog has less than about 10% of the activity of GLP- 1 at the GLP- 1 receptor.
  • the analog can be covalently linked to a hydrophilic moiety.
  • the analog is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus.
  • the analog comprises a C-terminal extension (e.g., an amino acid sequence of SEQ ID NO: 1095) and an addition of an amino acid comprising the hydrophilic moiety, such that the hydrophilic moiety is covalently linked to the analog at position 40.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to the glucagon sequence (SEQ ID NO: 1001) or it may be an amino acid which is replacing a native amino acid of SEQ ID NO: 1001.
  • the linkage to the hydrophilic moiety can com rise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the analog can comprise a modified amino acid in which the side chain is covalently linked to an acyl or alkyl group (e.g., an acyl or alkyl group which is non-native to a naturally-occurring amino acid).
  • the acylated or alkylated analog can be in accordance with acylated or alkylated peptides described in the section "Acylation and alkylation.”
  • the acyl group is a C4 to a C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.
  • the acyl or alkyl group may be covalently attached to any amino acid of the analog, including, but not limited to the amino acid at position 10 or 40, or the C-terminal amino acid.
  • the analog comprises a C-terminal extension (e.g., an amino acid sequence of SEQ ID NO: 1095) and an addition of an amino acid comprising the acyl or alkyl group, such that the acyl or alkyl group is covalently linked to the analog at position 40.
  • the acyl or alkyl group is covalently linked to the side chain of an amino acid of Formula I, II, or III, e.g., a Lys residue.
  • the acyl or alkyl group may be covalently linked to an amino acid which is native to the glucagon sequence (SEQ ID NO: 1001) or may be linked to an amino acid which is added to the sequence of SEQ ID NO: 1001 or to the sequence of SEQ ID NO: 1001 followed by SEQ ID NO: 1095 (at the N- or C-terminus) or may be linked to an amino acid which replaces a native amino acid, e.g., the Tyr at position 10 of SEQ ID NO: 1001.
  • the analog may be attached to the acyl or alkyl group via a spacer, as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-PAla, Leu-Leu, Pro-Pro, y-Glu-y-Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • the amino acid spacer is not ⁇ -Glu.
  • the dipeptide spacer is not ⁇ -Glu- ⁇ -Glu.
  • the analog of glucagon having GIP agonist activity comprises the amino acid sequence according to any one of SEQ ID NOs: 1227, 1228, 1229 or 1230 that further comprises the following modifications: (a) optionally, an amino acid modification at position 1 that confers GIP agonist activity,
  • EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • the acylated or alkylated amino acid is an amino acid of Formula I, II, or III.
  • the amino acid of Formula I is Dab, Orn, Lys, or homoLys.
  • the about 1 to about 21 amino acids comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSSGAPPPK (SEQ ID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small, aliphatic or non- polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to SEQ ID NO: 1095, 1096, 1170, 1171 or 1172.
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally- extended analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminally extended analog.
  • the amino acid at position 1 that confers GIP agonist activity can be an amino acid lacking an imidazole side chain.
  • the amino acid at position 1 can, for example, be a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • the analog of the above exemplary embodiments can further comprise 1-6 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half-life in circulation, delay the onset of action, or increase stability.
  • glucagon analogs described in the above exemplary embodiment comprise further amino acid modifications at one, two or all of positions 27, 28 and 29. Modifications at these positions can be any of the modifications described herein relative to these positions.
  • position 27 can be substituted with a large aliphatic amino acid (e.g., Leu, He or norleucine) or Met
  • position 28 can be substituted with another small aliphatic amino acid (e.g., Gly or Ala) or Asn
  • position 29 can be substituted with another small aliphatic amino acid (e.g., Ala or Gly) or Thr.
  • the analog can comprise such amino acid modifications at position 27 and/or 28.
  • the analog can further comprise one or more of the following additional modifications:
  • amino acid at position 2 is any one of D-Ser, Ala, D-Ala, Gly, N-methyl- Ser, Aib, Val, or a-amino-N-butyric acid;
  • amino acid at position 10 is Tyr, Trp, Lys, Orn, Glu, Phe, or Val;
  • amino acid at position 12 is lie, Lys or Arg;
  • amino acid at position 16 is any one of Ser, Glu, Gin, homoglutamic acid, homocysteic acid, Thr, Gly, or Aib;
  • amino acid at position 17 is Gin or Arg
  • amino acid at position 18 is any one of Ala, Arg, Ser, Thr, or Gly;
  • the amino acid at position 20 is any one of Ala, Ser, Thr, Lys, Citrulline, Arg, Orn, or Aib or another alpha, alpha-disubstituted amino acid;
  • amino acid at position 21 is any one of Glu, Asp, homoglutamic acid, homocysteic acid;
  • amino acid at position 24 is any one of Gin, Asn, Ala, Ser, Thr, or Aib; and (xii) one or more conservative substitutions at any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.
  • the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gin with Glu), wherein the analog has less than 1% of the activity of glucagon at the glucagon receptor.
  • the analog can comprise an amino acid modification at position 7 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), wherein the analog has less than about 10% of the activity of GLP-1 at the GLP-1 receptor.
  • the analog can be covalently linked to a hydrophilic moiety.
  • the analog is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus.
  • the analog comprises a hydrophilic moiety covalently linked to the analog at position 24.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to SEQ ID NO: 1001, 1227, 1228, 1229 or 1230 or it may be a substituted amino acid.
  • the linkage may com rise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the analog can comprise a modified amino acid within the C-terminal extension in which the side chain is covalently linked to an acyl or alkyl group.
  • the acylated or alkylated analog can be in accordance with acylated or alkylated peptides described in the section "Acylation and alkylation.”
  • the acyl group is a C4 to a C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.
  • the acyl or alkyl group may be covalently attached to any amino acid of the analog, including, but not limited to the amino acid at position 10 or 40, or the C-terminal amino acid.
  • the acyl or alkyl group is covalently linked to the side chain of an amino acid of Formula I, II, or III, e.g., a Lys residue.
  • the acyl or alkyl group is covalently linked to an amino acid which is native to SEQ ID NO: 1001, 1227, 1228, 1229 or 1230 or it may be linked to a substituted amino acid.
  • the acyl or alkyl group is covalently linked to an amino acid which is native to SEQ ID NO: 1095, 1096, 1171 or 1172, or it may be linked to a substituted amino acid.
  • the analog may be attached to the acyl or alkyl group via a spacer, as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-PAla, Leu-Leu, Pro-Pro, y-Glu-y-Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • the amino acid spacer is not ⁇ -Glu.
  • the dipeptide spacer is not ⁇ -Glu- ⁇ -Glu.
  • an analog of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1099-1141, 1144-1164, 1166, 1192-1207, 1209-1221 and 1223 or selected from the group consisting of SEQ ID NOs: 1167-1169, 1173-1178 and 1225.
  • the analog of glucagon having GIP agonist activity comprises an acyl or alkyl group (e.g., an acyl or alkyl group which is non-native to a naturally occurring amino acid), wherein the acyl or alkyl group is attached to a spacer, wherein (i) the spacer is attached to the side chain of the amino acid at position 10 of the analog; or (ii) the analog comprises an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 and the spacer is attached to the side chain of an amino acid corresponding to one of positions 37-43 relative to SEQ ID NO: 1001, wherein the EC 50 of the analog for GIP receptor activation is about 10 nM or less.
  • an acyl or alkyl group e.g., an acyl or alkyl group which is non-native to a naturally occurring amino acid
  • the analog comprises an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 and the spacer is attached to the side chain of an
  • the analog may comprise an amino acid sequence of SEQ ID NO: 1001 with (i) an amino acid modification at position 1 that confers GIP agonist activity, (ii) amino acid modifications at one, two, or all of positions 27, 28, and 29, (iii) at least one of:
  • the analog comprises 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;
  • the analog comprises (i) an amino acid substitution of Ser at position 16 with an amino acid of Formula IV:
  • n 1 to 7, wherein each of Rl and R2 is independently selected from the group consisting of H, Ci-Cis alkyl, (Ci-Cis alkyl)OH, (Ci-Cis alkyl)NH 2 , (Ci-Cis 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)(C6-Cio aryl)R 7 , and (Ci-C 4 alkyl)(C 3 -C9 heteroaryl), wherein R 7 is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group; and (ii) an amino acid substitution of the Gin at position 20 with an alpha, alpha- disubstituted amino acid.
  • the alpha, alpha-disubstituted amino acid of the analog of these embodiments may be any alpha, alpha-disubstituted amino acid, including, but not limited to, amino iso-butyric acid (Aib), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or
  • cycloheptane e.g., 1-aminocyclooctane-l-carboxylic acid.
  • the alpha, alpha-disubstituted amino acid is Aib.
  • the amino acid of Formula IV of the analog of these embodiments may be any amino acid, such as, for example, the amino acid of Formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4, or 5, in which case, the amino acid is Dab, Orn, Lys, or homoLys respectively.
  • the amino acid modification at position 1 that confers GIP agonist activity can be a substitution of His with an amino acid lacking an imidazole side chain.
  • the amino acid modification at position 1 can, for example, be a substitution of His with a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • amino acid modifications at one, two, or all of positions 27, 28, and 29 can be any of the modifications at these positions described herein.
  • the Met at position 27 can be substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 can be substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 can be substituted with a small aliphatic amino acid, optionally Gly.
  • the analog can comprise such amino acid
  • analog of the above exemplary embodiments can further comprise 1-9 or
  • additional amino acid modifications e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half- life in circulation, delay the onset of action, or increase stability.
  • the analog can further comprise, for example, an amino acid modification at position 12, optionally, a substitution with lie, and/or amino acid modifications at positions 17 and 18, optionally substitution with Q at position 17 and A at position 18, and/or an addition of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), or sequences containing one or more conservative substitutions relative to SEQ ID NO: 1095 or 1096, to the C- terminus, wherein X is any amino acid.
  • the analog can comprise one or more of the following modifications:
  • the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gin with Glu), wherein the analog has less than 1% of the activity of glucagon at the glucagon receptor.
  • the analog can comprise an amino acid modification at position 7 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28, yielding a 27- or 28-amino acid peptide, or a combination thereof, wherein the analog has less than about 10% of the activity of GLP-1 at the GLP-1 receptor.
  • the analog can be covalently linked to a hydrophilic moiety.
  • the analog is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus.
  • the analog comprises a C-terminal extension (e.g., an amino acid sequence of SEQ ID NO: 1095) and an addition of an amino acid comprising the hydrophilic moiety, such that the hydrophilic moiety is covalently linked to the analog at position 40.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to the glucagon sequence (SEQ ID NO: 1001) or it may be an amino acid which is replacing a native amino acid of SEQ ID NO: 1001.
  • the linkage to the hydrophilic moiety can com rise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the spacer can be any spacer as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-PAla, Leu-Leu, Pro-Pro, y-Glu-y-Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • the amino acid spacer is not ⁇ -Glu.
  • the dipeptide spacer is not ⁇ -Glu- ⁇ -Glu.
  • the acyl or alkyl group is any acyl or alkyl group as described herein, such as an acyl or alkyl group which is non-native to a naturally occurring amino acid.
  • the acyl or alkyl group in some embodiments is a C4 to C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group, or a C4 to C30 alkyl group.
  • the acyl group is a C12 to C18 fatty acyl group (e.g., a C14 or C16 fatty acyl group).
  • the extension of about 1 to about 21 amino acids C- terminal to the amino acid at position 29 of the analog comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSSGAPPPK (SEQ ID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small, aliphatic or non-polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to SEQ ID NO: 1095, 1096, 1170, 1171 or 1172.
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extended analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminally extended analog.
  • the GIP agonist may be a peptide comprising the amino acid sequence of any of the amino acid sequences, e.g., SEQ ID NOs: 1005-1094, optionally with up to 1, 2, 3, 4, or 5 further modifications that retain GIP agonist activity.
  • the GIP agonist comprises the amino acids of any of SEQ ID NOs: 1099-1275.
  • the glucagon related peptide is a Class 3 glucagon related peptide, which is described herein and in International Patent Application Publication Nos. WO 2009/155258, WO 2008/101017, and U.S. Provisional
  • the Class 3 glucagon related peptide can be a peptide that exhibits increased activity at the glucagon receptor, and in further embodiments exhibits enhanced biophysical stability and/or aqueous solubility.
  • the Class 3 glucagon related peptide has lost native glucagon's selectivity for the glucagon receptor verses the GLP-1 receptor, and thus represents co-agonists of those two receptors. Selected amino acid modifications within the Class 3 glucagon related peptide can control the relative activity of the peptide at the GLP-1 receptor verses the glucagon receptor.
  • the Class 3 glucagon related peptide can be a peptide that exhibits increased activity at the glucagon receptor, and in further embodiments exhibits enhanced biophysical stability and/or aqueous solubility.
  • the Class 3 glucagon related peptide has lost native glucagon's selectivity for the glucagon receptor verses the GLP-1 receptor, and thus represents co-agonists of those two receptors.
  • glucagon/GLP-1 co-agonist that has higher activity at the glucagon receptor versus the GLP-1 receptor
  • a glucagon/GLP-1 co-agonist that has approximately equivalent activity at both receptors or a glucagon/GLP-1 co-agonist that has higher activity at the GLP-1 receptor versus the glucagon receptor.
  • the latter category of co-agonist can be engineered to exhibit little or no activity at the glucagon receptor, and yet retain ability to activate the GLP-1 receptor with the same or better potency than native GLP-1. Any of these co-agonists may also include modifications that confer enhanced biophysical stability and/or aqueous solubility.
  • Modifications of the Class 3 glucagon related peptide can be made to produce a glucagon peptide having anywhere from at least about 1% (including at least about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%) to about 200% or higher activity at the GLP-1 receptor relative to native GLP- 1 and anywhere from at least about 1% (including about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%) to about 500% or higher activity at the glucagon receptor relative to native glucagon.
  • a Class 3 glucagon related peptide may exhibit at least 10% of the activity of native glucagon at the glucagon receptor and at least 50% of the activity of native GLP-1 at the GLP-1 receptor, or at least 40% of the activity of native glucagon at the glucagon receptor and at least 40% of the activity of native GLP-1 at the GLP- 1 receptor, or at least 60% of the activity of native glucagon at the glucagon receptor and at least 60% of the activity of native GLP-1 at the GLP-1 receptor.
  • Selectivity of a Class 3 glucagon related peptide for the glucagon receptor versus the GLP-1 receptor can be described as the relative ratio of glucagon/GLP-1 activity (the peptide's activity at the glucagon receptor relative to native glucagon, divided by the peptide's activity at the GLP-1 receptor relative to native GLP-1).
  • a Class 3 glucagon related peptide that exhibits 60% of the activity of native glucagon at the glucagon receptor and 60% of the activity of native GLP-1 at the GLP-1 receptor has a 1: 1 ratio of glucagon/GLP-1 activity.
  • Exemplary ratios of glucagon/GLP-1 activity include about 1: 1, 1.5: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1, or about 1: 10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1: 1.5.
  • a glucagon/GLP-1 activity ratio of 10: 1 indicates a 10-fold selectivity for the glucagon receptor versus the GLP-1 receptor.
  • a GLP-1 /glue agon activity ratio of 10: 1 indicates a 10-fold selectivity for the GLP-1 receptor versus the glucagon receptor.
  • the Class 3 glucagon related peptides have about 10% or less of the activity of native glucagon at the glucagon receptor, e.g. about 1-10%, or about 0.1-10%, or greater than about 0.1% but less than about 10%, while exhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.
  • exemplary Class 3 glucagon related peptides described herein have about 0.5%, about 1% or about 7% of the activity of native glucagon, while exhibiting at least 20% of the activity of GLP- 1 at the GLP- 1 receptor.
  • the Class 3 glucagon related peptide can be a glucagon peptide with increased or decreased activity at the glucagon receptor, or GLP-1 receptor, or both.
  • the Class 3 glucagon related peptide can be a glucagon peptide with altered selectivity for the glucagon receptor versus the GLP-1 receptor.
  • high potency Class 3 glucagon related peptides are provided that also exhibit improved solubility and/or stability.
  • An exemplary high potency Class 3 glucagon related peptide exhibits at least about 200% of the activity of native glucagon at the glucagon receptor, and optionally is soluble at a
  • an exemplary Class 3 glucagon related peptide exhibits greater than about 40% or greater than about 60% activity at both the glucagon and the GLP- 1 receptors (at a ratio between about 1:3 and 3: 1, or between about 1:2 and 2: 1), is optionally soluble at a concentration of at least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7 and 9 (e.g.
  • Another exemplary Class 3 glucagon related peptide exhibits about 175% or more of the activity of native glucagon at the glucagon receptor and about 20% or less of the activity of native GLP-1 at the GLP-1 receptor, is optionally soluble at a concentration of at least 1 mg/niL at a pH between 6 and 8 or between 6 and 9, or between 7 and 9 (e.g. pH 7), and optionally retains at least 95% of the original peptide after 24 hours at 25°C.
  • Yet another exemplary Class 3 glucagon related peptide exhibits about 10% or less of the activity of native glucagon at the glucagon receptor and at least about 20% of the activity of native GLP-1 at the GLP-1 receptor, is optionally soluble at a concentration of at least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7 and 9 (e.g. pH 7), and optionally retains at least 95% of the original peptide after 24 hours at 25°C.
  • Yet another exemplary Class 3 glucagon related peptide exhibits about 10% or less but above 0.1% , 0.5% or 1% of the activity of native glucagon at the glucagon receptor and at least about 50%, 60%, 70%, 80%, 90% or 100% or more of the activity of native GLP-1 at the GLP-1 receptor, is optionally soluble at a concentration of at least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7 and 9 (e.g. pH 7), and optionally retains at least 95% of the original peptide after 24 hours at 25°C.
  • Class 3 glucagon related peptides retain at least 22, 23, 24, 25, 26, 27 or 28 of the naturally occurring amino acids at the corresponding positions in native glucagon (e.g. have 1-7, 1-5 or 1-3 modifications relative to naturally occurring glucagon).
  • Increased activity at the glucagon receptor is provided by an amino acid modification at position 16 of native glucagon (SEQ ID NO: 1). In some
  • the Class 3 glucagon related peptide is a glucagon agonist that has been modified relative to the wild type peptide of His-Ser-Gln-Gly-Thr-Phe- Thr-Ser-Asp- Tyr-Ser-Lys-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met- Asn-Thr (SEQ ID NO: 1) to enhance the peptide's potency at the glucagon receptor.
  • the normally occurring serine at position 16 of native glucagon can be substituted with select acidic amino acids to enhance the potency of glucagon, in terms of its ability to stimulate cAMP synthesis in a validated in vitro model assay (see Example 2). More particularly, this substitution enhances the potency of the analog at least 2-fold, 4-fold, 5-fold, and up to 10-fold greater at the glucagon receptor. This substitution also enhances the analog's activity at the GLP-1 receptor at least 5-fold, 10-fold, or 15-fold relative to native glucagon, but selectivity is maintained for the glucagon receptor over the GLP-1 receptor.
  • such enhanced potency can be provided by substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, or a charged amino acid having a side chain containing at least one heteroatom, (e.g. N, O, S, P) and with a side chain length of about 4 (or 3-5) atoms.
  • heteroatom e.g. N, O, S, P
  • the serine residue at position 16 of native glucagon is substituted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid, homocysteic acid, threonine, or glycine.
  • the serine residue at position 16 of native glucagon is substituted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid and homocysteic acid, and in some embodiments the serine residue is substituted with glutamic acid.
  • the enhanced potency Class 3 glucagon related peptide comprises a peptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or a glucagon agonist analog of SEQ ID NO: 5.
  • a Class 3 glucagon related peptide having enhanced potency at the glucagon receptor relative to wild type glucagon is provided wherein the peptide comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, wherein the glucagon peptide retains its selectivity for the glucagon receptor relative to the GLP-1 receptors.
  • the Class 3 glucagon related peptide having enhanced specificity for the glucagon receptor comprises the peptide of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a glucagon agonist analog thereof, wherein the carboxy terminal amino acid retains its native carboxylic acid group.
  • a Class 3 glucagon related peptide comprises the sequence of NH 2 -His-Ser-Gln-Gly-Thr-Phe- Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln- Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 10), wherein the peptide exhibits approximately fivefold enhanced potency at the glucagon receptor, relative to native glucagon as measured by the in vitro cAMP assay of Example 2.
  • Glucagon receptor activity can be reduced, maintained, or enhanced by an amino acid modification at position 3, e.g. substitution of the naturally occurring glutamine at position 3.
  • substitution of the amino acid at position 3 with an acidic, basic, or hydrophobic amino acid has been shown to substantially reduce or destroy glucagon receptor activity.
  • the analogs that are substituted with, for example, glutamic acid, ornithine, or norleucine have about 10% or less of the activity of native glucagon at the glucagon receptor, e.g.
  • exemplary analogs described herein have about 0.5%, about 1% or about 7% of the activity of native glucagon, while exhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.
  • any of the Class 3 glucagon related peptides including glucagon analogs, glucagon agonist analogs, glucagon co-agonists, and glucagon/GLP-1 co-agonist molecules, described herein may be modified to contain a modification at position 3, e.g., Gin substituted with Glu, to produce a peptide with high selectivity, e.g., tenfold selectivity, for the GLP-1 receptor as compared to the selectivity for the glucagon receptor.
  • a modification at position 3 e.g., Gin substituted with Glu
  • the naturally occurring glutamine at position 3 of any of the Class 3 glucagon peptides can be substituted with a glutamine analog without a substantial loss of activity at the glucagon receptor, and in some cases, with an enhancement of glucagon receptor activity, as described herein.
  • the amino acid at position 3 is substituted with Dab(Ac).
  • glucagon agonists can comprise the amino acid sequence of SEQ ID NO: 595, SEQ ID NO: 601 SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, and SEQ ID NO: 606.
  • modifications at position 2 may reduce glucagon activity.
  • This reduction in glucagon activity can be restored by stabilizing the alpha-helix in the C-terminal portion of glucagon, e.g. through means described herein, for example, through a covalent bond between the side chains of the amino acids at positions "i" and "i+4", e.g., 12 and 16, 16 and 20, or 20 and 24.
  • this covalent bond is a lactam bridge between a glutamic acid at position 16 and a lysine at position 20.
  • this covalent bond is an intramolecular bridge other than a lactam bridge.
  • suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur- containing bridge formation, the use of a, co-diaminoalkane tethers, the formation of metal-atom bridges, and other means of peptide cyclization.
  • Enhanced activity at the GLP-1 receptor is provided by replacing the carboxylic acid of the C-terminal amino acid with a charge-neutral group, such as an amide or ester.
  • these Class 3 glucagon related peptides comprise a sequence of SEQ ID NO: 20, wherein the carboxy terminal amino acid has an amide group in place of the carboxylic acid group found on the native amino acid.
  • These Class 3 glucagon related peptides have strong activity at both the glucagon and GLP-1 receptors and thus act as co-agonists at both receptors.
  • the Class 3 glucagon related peptide is a glucagon and GLP-1 receptor co-agonist, wherein the peptide comprises the sequence of SEQ ID NO: 20, wherein the amino acid at position 28 is Asn or Lys and the amino acid at position 29 is Thr-amide.
  • Increased activity at the GLP-1 receptor is provided by modifications that stabilize the alpha helix in the C-terminal portion of glucagon (e.g. around residues 12-29).
  • intramolecular bridge between the side chains of two amino acids that are separated by three intervening amino acids i.e., an amino acid at position "i” and an amino acid at position "i+4", wherein i is any integer between 12 and 25
  • two intervening amino acids i.e., an amino acid at position "j” and an amino acid at position "j+3,” wherein j is any integer between 12 and 27, or by six intervening amino acids, i.e., an amino acid at position "k” and an amino acid at position "k+7,” wherein k is any integer between 12 and 22.
  • the bridge or linker is about 8 (or about 7-9) atoms in length and forms between side chains of amino acids at positions 12 and 16, or at positions 16 and 20, or at positions 20 and 24, or at positions 24 and 28.
  • the two amino acid side chains can be linked to one another through non-covalent bonds, e.g., hydrogen-bonding, ionic interactions, such as the formation of salt bridges, or by covalent bonds.
  • the Class 3 glucagon related peptide exhibits glucagon/GLP-1 receptor co-agonist activity and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 47, 48 and 49.
  • the side chains are covalently bound to one another, and in some embodiments the two amino acids are bound to one another to form a lactam ring.
  • the Class 3 glucagon related peptide comprises SEQ ID NO: 45, wherein at least one lactam ring is formed between the side chains of an amino acid pair selected from the group consisting of amino acid pairs 12 and 16, 16 and 20, 20 and 24 or 24 and 28.
  • the Class 3 glucagon related peptide comprises a glucagon peptide analog of SEQ ID NO: 20, wherein the peptide comprises an intramolecular lactam bridge formed between amino acid positions 12 and 16 or between amino acid positions 16 and 20.
  • the Class 3 glucagon related peptide comprises the sequence of SEQ ID NO: 20, wherein an intramolecular lactam bridge is formed between amino acid positions 12 and 16, between amino acid positions 16 and 20, or between amino acid positions 20 and 24 and the amino acid at position 29 is glycine, wherein the sequence of SEQ ID NO: 29 is linked to the C-terminal amino acid of SEQ ID NO: 20.
  • the amino acid at position 28 is aspartic acid.
  • stabilization of the alpha helix structure in the C-terminal portion of the Class 3 glucagon related peptide is achieved through the formation of an intramolecular bridge other than a lactam bridge.
  • suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, the use of a, co-diaminoalkane tethers, the formation of metal-atom bridges, and other means of peptide cyclization are used to stabilize the alpha helix.
  • enhanced activity at the GLP-1 receptor may be achieved by stabilizing the alpha-helix structure in the C-terminal portion of the glucagon peptide (around amino acids 12-29) through purposeful introduction of one or more a, a- disubstituted amino acids at positions that retain the desired activity.
  • Such peptides may be considered herein as a peptide lacking an intramolecular bridge.
  • stabilization of the alpha-helix is accomplished in this manner without introduction of an intramolecular bridge such as a salt bridge or covalent bond.
  • one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of a glucagon peptide is substituted with an a, a-disubstituted amino acid.
  • substitution of position 16 of the Class 3 glucagon related peptide with amino iso-butyric acid (Aib) enhances GLP-1 activity, in the absence of a salt bridge or lactam.
  • one, two, three or more of positions 16, 20, 21 or 24 are substituted with Aib.
  • Enhanced activity at the GLP-1 receptor may be achieved by an amino acid modification at position 20.
  • the glutamine at position 20 is replaced with another hydrophilic amino acid having a side chain that is either charged or has an ability to hydrogen-bond, and is at least about 5 (or about 4-6) atoms in length, for example, lysine, citrulline, arginine, or ornithine.
  • GLP-1 activity at the GLP-1 receptor is demonstrated in Class 3 glucagon related peptides comprising the C-terminal extension of SEQ ID NO: 26.
  • GLP-1 activity in such Class 3 glucagon related peptides comprising SEQ ID NO: 26 can be further increased by modifying the amino acid at position 18, 28 or 29, or at position 18 and 29, as described herein.
  • a further modest increase in GLP-1 potency may be achieved by modifying the amino acid at position 10 to be Trp.
  • the Class 3 glucagon related peptides can comprise modifications at position 16, at position 20, and at the C-terminal carboxylic acid group, optionally with a covalent bond between the amino acids at positions 16 and 20; can comprise modifications at position 16 and at the C-terminal carboxylic acid group; can comprise modifications at positions 16 and 20, optionally with a covalent bond between the amino acids at positions 16 and 20; or can comprise modifications at position 20 and at the C-terminal carboxylic acid group; optionally with the proviso that the amino acid at position 12 is not Arg; or optionally with the proviso that the amino acid at position 9 is not Glu.
  • the Class 3 glucagon related peptides can be further modified to improve the peptide's solubility and stability in aqueous solutions at physiological pH, while retaining the high biological activity relative to native glucagon.
  • Hydrophilic moieties as discussed herein can be attached to the Class 3 glucagon related peptide as further discussed herein.
  • introduction of hydrophilic groups at positions 17, 21, and 24 of the Class 3 glucagon related peptide comprising SEQ ID NO: 9 or SEQ ID NO: 10 are anticipated to improve the solubility and stability of the high potency glucagon analog in solutions having a physiological pH. Introduction of such groups also increases duration of action, e.g. as measured by a prolonged half- life in circulation.
  • the Class 3 glucagon related peptide comprises a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, wherein the side chain of an amino acid residue at one of position 16, 17, 21 or 24 of said Class 3 glucagon related peptide further comprises a polyethylene glycol chain, having a molecular weight selected from the range of about 500 to about 40,000 Daltons.
  • the polyethylene glycol chain has a molecular weight selected from the range of about 500 to about 5,000 Daltons.
  • the polyethylene glycol chain has a molecular weight of about 10,000 to about 20,000 Daltons.
  • the polyethylene glycol chain has a molecular weight of about 20,000 to about 40,000 Daltons.
  • Suitable hydrophilic moieties include any water soluble polymers known in the art, including the hydrophilic moieties described herein, homo- or co-polymers of PEG, and a monomethyl-substituted polymer of PEG (mPEG).
  • the hydrophilic group comprises a polyethylene (PEG) chain.
  • the Class 3 glucagon related peptide comprises the sequence of SEQ ID NO: 6 or SEQ ID NO: 7 wherein a PEG chain is covalently linked to the side chains of amino acids present at positions 21 and 24 of the Class 3 glucagon related peptide and the carboxy terminal amino acid of the Class 3 glucagon related peptide has the carboxylic acid group.
  • the polyethylene glycol chain has an average molecular weight selected from the range of about 500 to about 10,000 Daltons.
  • the pegylated Class 3 glucagon related peptide comprises two or more polyethylene glycol chains covalently bound to the Class 3 glucagon related peptide wherein the total molecular weight of the glucagon chains is about 1,000 to about 5,000 Daltons.
  • the pegylated glucagon agonist comprises a peptide consisting of SEQ ID NO: 5 or a glucagon agonist analog of SEQ ID NO: 5, wherein a PEG chain is covalently linked to the amino acid residue at position 21 and at position 24, and wherein the combined molecular weight of the two PEG chains is about 1,000 to about 5,000 Daltons.
  • the solubility of the Class 3 glucagon related peptide comprising SEQ ID NO: 20 can be further improved, for example, by introducing one, two, three or more charged amino acid(s) to the C-terminal portion of glucagon peptide of SEQ ID NO: 20, preferably at a position C-terminal to position 27.
  • a charged amino acid can be introduced by substituting a native amino acid with a charged amino acid, e.g. at positions 28 or 29, or alternatively by adding a charged amino acid, e.g. after position 27, 28 or 29.
  • one, two, three or all of the charged amino acids are negatively charged. Additional modifications, e.g. conservative
  • an analog of the Class 3 glucagon related peptide of SEQ ID NO: 20 is provided wherein the analog differs from SEQ ID NO: 20 by 1 to 2 amino acid substitutions at positions 17-26, and, in some embodiments, the analog differs from the peptide of SEQ ID NO: 20 by an amino acid substitution at position 20.
  • the glucagon peptide is modified to comprise an acyl or alkyl group, e.g., a C4 to C30 acyl or alkyl group.
  • the acyl group or alkyl group is not naturally occurring on an amino acid.
  • the acyl or alkyl group is non-native to any naturally-occurring amino acid.
  • Acylation or alkylation can increase the half-life in circulation and/or delay the onset of and/or extend the duration of action and/or improve resistance to proteases such as DPP-IV.
  • the activity at the glucagon receptor and GLP-1 receptor of the Class 3 glucagon related peptides is maintained, if not substantially enhanced after acylation. Further, the potency of the acylated analogs were comparable to the unacylated versions of the Class 3 glucagon related peptides, if not substantially enhanced.
  • the invention provides a Class 3 glucagon related peptide modified to comprise an acyl group or alkyl group covalently linked to the amino acid at position 10 of the glucagon peptide.
  • the glucagon peptide may further comprise a spacer between the amino acid at position 10 of the Class 3 glucagon related peptide and the acyl group or alkyl group.
  • Any of the foregoing Class 3 glucagon related peptides may comprise two acyl groups or two alkyl groups, or a combination thereof.
  • the acylated Class 3 glucagon related peptide comprises the amino acid sequence of any of SEQ ID NOs: 534-544 and 546- 549.
  • the Class 3 glucagon related peptides described herein are further modified by truncation or deletion of one or two amino acids of the C- terminus of the glucagon peptide (i.e., position 29 and/or 28) without affecting activity and/or potency at the glucagon and GLP-1 receptors.
  • the Class 3 glucagon related peptide can comprise amino acids 1-27 or 1-28 of the native glucagon peptide (SEQ ID NO: 1), optionally with one or more modifications described herein.
  • the truncated Class 3 glucagon related peptide comprises SEQ ID NO: 550 or SEQ ID NO: 551.
  • the truncated glucagon agonist peptide comprises SEQ ID NO: 552 or SEQ ID NO: 553.
  • the Class 3 glucagon related peptides disclosed herein are modified by the addition of a second peptide to the carboxy terminus of the glucagon peptide, for example, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69 is covalently bound through a peptide bond to a second peptide, wherein the second peptide comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the threonine at position 29 of the native glucagon peptide is replaced with a glycine.
  • a Class 3 glucagon related peptide having a glycine substitution for threonine at position 29 and comprising the carboxy terminal extension of SEQ ID NO: 26 is four times as potent at the GLP-1 receptor as native glucagon modified to comprise the carboxy terminal extension of SEQ ID NO: 26. Potency at the GLP-1 receptor can be further enhanced by an alanine substitution for the native arginine at position 18.
  • Class 3 glucagon related peptide can have a carboxy terminal extension of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28.
  • Class 3 glucagon related peptide comprising SEQ ID NO: 33 or SEQ ID NO: 20, further comprises the amino acid sequence of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked to amino acid 29 of the glucagon peptide.
  • the Class 3 glucagon related peptide comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14 and SEQ ID NO: 15, further comprising the amino acid sequence of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked to amino acid 29 of the glucagon peptide.
  • the glucagon peptide comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 55 and SEQ ID NO: 56 further comprising the amino acid sequence of SEQ ID NO: 820
  • the Class 3 glucagon related peptide comprises the sequence of SEQ ID NO: 64.
  • (A) Improving solubility for example, by introducing one, two, three or more charged amino acid(s) to the C-terminal portion of native glucagon, preferably at a position C- terminal to position 27.
  • a charged amino acid can be introduced by substituting a native amino acid with a charged amino acid, e.g. at positions 28 or 29, or alternatively by adding a charged amino acid, e.g. after position 27, 28 or 29.
  • one, two, three or all of the charged amino acids are negatively charged.
  • one, two, three or all of the charged amino acids are positively charged.
  • Such modifications increase solubility, e.g. provide at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-fold or greater solubility relative to native glucagon at a given pH between about 5.5 and 8, e.g., pH 7, when measured after 24 hours at 25°C.
  • (C) Increasing stability by modification of the aspartic acid at position 15, for example, by deletion or substitution with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid. Such modifications can reduce degradation or cleavage at a pH within the range of 5.5 to 8, especially in acidic or alkaline buffers, for example, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the original peptide after 24 hours at 25 °C.
  • (D) Increasing stability by modification of the methionine at position 27, for example, by substitution with leucine or norleucine. Such modifications can reduce oxidative degradation. Stability can also be increased by modification of the Gin at position 20 or 24, e.g. by substitution with Ser, Thr, Ala or Aib. Such modifications can reduce degradation that occurs through deamidation of Gin. Stability can be increased by modification of Asp at position 21, e.g. by substitution with Glu. Such modifications can reduce degradation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to iso-aspartate.
  • substitution of His at position 1 with a large, aromatic amino acid e.g., Tyr, Phe, Trp or amino-Phe
  • Ser at position 2 with Ala substitution of Tyr at position 10 with Val or Phe
  • substitution of Lys at position 12 with Arg substitution of Asp at position 15 with Glu
  • substitution of Ser at position 16 with Thr or Aib substitution of His at position 1 with a large, aromatic amino acid (e.g., Tyr, Phe, Trp or amino-Phe); Ser at position 2 with Ala; substitution of Tyr at position 10 with Val or Phe; substitution of Lys at position 12 with Arg; substitution of Asp at position 15 with Glu; substitution of Ser at position 16 with Thr or Aib.
  • Class 3 glucagon related peptides with GLP-1 activity that contain a non- conservative substitution of His at position 1 with a large, aromatic amino acid (e.g., Tyr) can retain GLP- 1 activity provided that the alpha-helix is stabilized via an intramolecular bridge, e.g., such as any of those described herein.
  • a large, aromatic amino acid e.g., Tyr
  • the Class 3 glucagon related peptide can be linked, optionally via covalent bonding and optionally via a linker, to a conjugate moiety.
  • the Class 3 glucagon related peptide also can be part of a fusion peptide or protein wherein a second peptide or polypeptide has been fused to a terminus, e.g., the carboxy terminus of the Class 3 glucagon related peptide.
  • the fusion Class 3 glucagon related peptide may comprise a glucagon agonist of SEQ ID NO: 55, SEQ ID NO: 9 or SEQ ID NO: 10 further comprising an amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to amino acid 29 of the glucagon peptide.
  • the amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) is bound to amino acid 29 of the Class 3 glucagon related peptide through a peptide bond.
  • glucagon related peptide fusion peptides comprising the C-terminal extension peptide of Exendin-4 (e.g., SEQ ID NO: 820 or SEQ ID NO: 821)
  • substitution of the native threonine residue at position 29 with glycine dramatically increases GLP-1 receptor activity.
  • This amino acid substitution can be used in conjunction with other modifications disclosed herein with regard to Class 3 glucagon related peptides to enhance the affinity of the glucagon analogs for the GLP-1 receptor.
  • the T29G substitution can be combined with the S 16E and N20K amino acid substitutions, optionally with a lactam bridge between amino acids 16 and 20, and optionally with addition of a PEG chain as described herein.
  • a Class 3 glucagon related peptide comprises the sequence of SEQ ID NO: 64.
  • the Class 3 glucagon related peptide portion of the glucagon fusion peptide is selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 wherein a PEG chain, when present at positions 17, 21, 24, or the C-terminal amino acid, or at both 21 and 24, is selected from the range of 500 to 40,000 Daltons.
  • the Class 3 glucagon related peptide segment is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 63, wherein the PEG chain is selected from the range of 500 to 5,000.
  • the Class 3 glucagon related peptide is a fusion peptide comprising the sequence of SEQ ID NO: 55 and SEQ ID NO: 65 wherein the peptide of SEQ ID NO: 65 is linked to the carboxy terminus of SEQ ID NO: 55.
  • an additional chemical modification of the Class 3 glucagon related peptide of SEQ ID NO: 10 bestows increased GLP-1 receptor potency to a point where the relative activity at the glucagon and GLP-1 receptors is virtually equivalent.
  • a Class 3 glucagon related peptide comprises a terminal amino acid comprising an amide group in place of the carboxylic acid group that is present on the native amino acid.
  • the relative activity of the Class 3 glucagon related peptide at the respective glucagon and GLP-1 receptors can be adjusted by further modifications to the Class 3 glucagon related peptide to produce analogs demonstrating about 40% to about 500% or more of the activity of native glucagon at the glucagon receptor and about 20% to about 200% or more of the activity of native GLP-1 at the GLP-1 receptor, e.g. 50-fold, 100-fold or more increase relative to the normal activity of glucagon at the GLP-1 receptor.
  • the glucagon peptides described herein exhibit up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon at the glucagon receptor.
  • the glucagon peptides described herein exhibit up to about 100%, 1000%, 10,000%, 100,000%, or
  • a glucagon analog comprising the sequence of SEQ ID NO: 55, wherein said analog differs from SEQ ID NO: 55 by 1 to 3 amino acids, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 24, 27, 28, and 29, wherein said glucagon peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor.
  • glucagon/GLP-1 receptor co-agonist comprising the sequence:
  • Xaa at position 15 is selected from the group of amino acids consisting of Asp, Glu, cysteic acid, homoglutamic acid and homocysteic acid
  • Xaa at position 16 is selected from the group of amino acids consisting of Ser, Glu, Gin, homoglutamic acid and homocysteic acid
  • the Xaa at position 20 is Gin or Lys
  • the Xaa at position 24 is Gin or Glu
  • the Xaa at position 28 is Asn, Lys or an acidic amino acid
  • the Xaa at position 29 is Thr, G
  • the glucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO: 33 wherein the amino acid at position 28 is aspartic acid and the amino acid at position 29 is glutamic acid.
  • the amino acid at position 28 is the native asparagine
  • the amino acid at position 29 is glycine
  • the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 65 is covalently linked to the carboxy terminus of SEQ ID NO: 33.
  • the glucagon analog comprises a sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.
  • a glucagon peptide analog of SEQ ID NO: 33 wherein said analog differs from SEQ ID NO: 33 by 1 to 3 amino acids, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, with the proviso that when the amino acid at position 16 is serine, either position 20 is lysine, or a lactam bridge is formed between the amino acid at position 24 and either the amino acid at position 20 or position 28.
  • the analog differs from SEQ ID NO: 33 by 1 to 3 amino acids selected from positions 1, 2, 3, 21 and 27.
  • the glucagon peptide analog of SEQ ID NO: 33 differs from that sequence by 1 to 2 amino acids, or in some embodiments by a single amino acid, selected form positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, with the proviso that when the amino acid at position 16 is serine, either position 20 is lysine, or a lactam bridge is formed between the amino acid at position 24 and either the amino acid at position 20 or position 28.
  • a relatively selective GLP-1 receptor agonist comprising the sequence NH 2 -His-Ser-Xaa-Gly-Thr-Phe- Thr-Ser- Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu- Met-Xaa-Xaa-R (SEQ ID NO: 53) wherein the Xaa at position 3 is selected from the group of amino acids consisting of Glu, Orn or Nle, the Xaa at position 15 is selected from the group of amino acids consisting of Asp, Glu, cysteic acid, homoglutamic acid and homocysteic acid, Xaa at position 16 is selected from the group of amino acids consisting of Ser, Glu, Gin, homoglutamic acid and homocysteic acid, the Xaa at position 20 is Gin or Lys,
  • the amino acid at position 3 is glutamic acid.
  • the acidic amino acid substituted at position 28 and/or 29 is aspartic acid or glutamic acid.
  • the glucagon peptide, including a co- agonist peptide comprises the sequence of SEQ ID NO: 33 further comprising an additional acidic amino acid added to the carboxy terminus of the peptide.
  • the carboxy terminal amino acid of the glucagon analog has an amide in place of the carboxylic acid group of the natural amino acid.
  • glucagon/GLP-1 receptor co-agonist comprising a modified glucagon peptide selected from the group consisting of:
  • Xaa at position 15 is selected from the group of amino acids consisting of Asp, Glu, cysteic acid, homoglutamic acid and homocysteic acid
  • Xaa at position 16 is selected from the group of amino acids consisting of Ser, Glu, Gin, homoglutamic acid and homocysteic acid
  • the Xaa at position 20 is Gin or Lys
  • the Xaa at position 24 is Gin or Glu
  • the Xaa at position 27 is Met, Leu or Nle
  • the Xaa at position 28 is Asn, Asp
  • R is CONH 2
  • the Xaa at position 15 is Asp
  • the Xaa at position 16 is selected from the group of amino acids consisting of Glu, Gin, homoglutamic acid and homocysteic acid
  • the Xaas at positions 20 and 24 are each Gin the Xaa at position 28 is Asn or
  • Asp and the Xaa at position 29 is Thr.
  • the Xaas at positions 15 and 16 are each Glu
  • the Xaas at positions 20 and 24 are each Gin
  • the Xaa at position 28 is Asn or Asp
  • the Xaa at position 29 is Thr and R is CONH 2 .
  • amino acids located at positions at positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide of SEQ ID NO: 11 can be substituted with an amino acid different from that present in the native glucagon peptide, and still retain activity at the glucagon receptor.
  • methionine residue present at position 27 of the native peptide is changed to leucine or norleucine to prevent oxidative degradation of the peptide.
  • amino acid at position 20 is substituted with Lys, Arg, Orn or Citrullene and/or position 21 is substituted with Glu, homoglutamic acid or homocysteic acid.
  • a glucagon analog of SEQ ID NO: 20 wherein 1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 27, 28 or 29 of the analog differ from the corresponding amino acid of SEQ ID NO: 1, with the proviso that when the amino acid at position 16 is serine, position 20 is Lys, or alternatively when position 16 is serine the position 24 is Glu and either position 20 or position 28 is Lys.
  • a glucagon analog of SEQ ID NO: 20 is provided wherein 1 to 3 amino acids selected from positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27, 28 or 29 of the analog differ from the corresponding amino acid of SEQ ID NO: 1.
  • a glucagon analog of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 11 is provided wherein 1 to 2 amino acids selected from positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20 or 21 of the analog differ from the corresponding amino acid of SEQ ID NO: 1, and in a further embodiment the one to two differing amino acids represent conservative amino acid substitutions relative to the amino acid present in the native glucagon sequence (SEQ ID NO: 1).
  • glucagon peptide of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is provided wherein the glucagon peptide further comprises one, two or three amino acid substitutions at positions selected from positions 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27 or 29. In some embodiments the substitutions at positions 2, 5, 7, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 27 or 29 are conservative amino acid substitutions.
  • a glucagon/GLP-1 receptor co-agonist comprising a variant of the sequence of SEQ ID NO 33, wherein 1 to 10 amino acids selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28 and 29, respectively, of the variant differ from the corresponding amino acid of SEQ ID NO: 1.
  • a variant of the sequence of SEQ ID NO 33 is provided wherein the variant differs from SEQ ID NO: 33 by one or more amino acid substitutions selected from the group consisting of Glnl7, Alal8, Glu21, Ile23, Ala24, Val27 and Gly29.
  • a glucagon/GLP-1 receptor co-agonist comprising variants of the sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected from positions 17-26 of the variant differ from the corresponding amino acid of SEQ ID NO: 1.
  • a variant of the sequence of SEQ ID NO 33 is provided wherein the variant differs from SEQ ID NO: 33 by an amino acid substitution selected from the group consisting of Glnl7, Alal8, Glu21, Ile23 and Ala24.
  • a variant of the sequence of SEQ ID NO 33 wherein the variant differs from SEQ ID NO: 33 by an amino acid substitution at position 18 wherein the substituted amino acid is selected from the group consisting of Ala, Ser, Thr, and Gly.
  • a variant of the sequence of SEQ ID NO 33 is provided wherein the variant differs from SEQ ID NO: 33 by an amino acid substitution of Ala at position 18. Such variations are encompassed by SEQ ID NO: 55.
  • a glucagon/GLP-1 receptor co-agonist comprising variants of the sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected from positions 17-22 of the variant differ from the corresponding amino acid of SEQ ID NO: 1, and in a further embodiment a variant of SEQ ID NO 33 is provided wherein the variant differs from SEQ ID NO: 33 by lor 2 amino acid substitutions at positions 20 and 21.
  • a glucagon/GLP-1 receptor co-agonist comprising the sequence:
  • R is CONH2.
  • a glucagon/GLP-1 receptor co- agonist comprising a variant of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, wherein the variant differs from said sequence by an amino acid substitution at position 20.
  • the amino acid substitution is selected form the group consisting of Lys, Arg, Orn or citrulline for position 20.
  • a glucagon agonist comprising an analog peptide of SEQ ID NO: 34 wherein the analog differs from SEQ ID NO: 34 by having an amino acid other than serine at position 2.
  • the serine residue is substituted with aminoisobutyric acid, D-alanine, and in some embodiments the serine residue is substituted with aminoisobutyric acid.
  • Such modifications suppresses cleavage by dipeptidyl peptidase IV while retaining the inherent potency of the parent compound (e.g. at least 75, 80, 85, 90, 95% or more of the potency of the parent compound).
  • the solubility of the analog is increased, for example, by introducing one, two, three or more charged amino acid(s) to the C- terminal portion of native glucagon, preferably at a position C-terminal to position 27.
  • one, two, three or all of the charged amino acids are negatively charged.
  • the analog further comprises an acidic amino acid substituted for the native amino acid at position 28 or 29 or an acidic amino acid added to the carboxy terminus of the peptide of SEQ ID NO: 34.
  • the glucagon analogs disclosed herein are further modified at position 1 or 2 to reduce susceptibility to cleavage by dipeptidyl peptidase IV.
  • a glucagon analog of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is provided wherein the analog differs from the parent molecule by a substitution at position 2 and exhibits reduced susceptibility (i.e., resistance) to cleavage by dipeptidyl peptidase IV.
  • position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, D- alanine, valine, amino n-butyric acid, glycine, N-methyl serine and aminoisobutyric acid.
  • position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, D-alanine, glycine, N- methyl serine and aminoisobutyric acid.
  • position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, glycine, N-methyl serine and aminoisobutyric acid.
  • the amino acid at position 2 is not D-serine.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 21 or SEQ ID NO: 22.
  • a glucagon analog of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is provided wherein the analog differs from the parent molecule by a substitution at position 1 and exhibits reduced susceptibility (i.e., resistance) to cleavage by dipeptidyl peptidase IV.
  • position 1 of the analog peptide is substituted with an amino acid selected from the group consisting of D-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine and homo- histidine.
  • a glucagon agonist is provided comprising an analog peptide of SEQ ID NO: 34 wherein the analog differs from SEQ ID NO: 34 by having an amino acid other than histidine at position 1.
  • the solubility of the analog is increased, for example, by introducing one, two, three or more charged amino acid(s) to the C-terminal portion of native glucagon, preferably at a position C-terminal to position 27.
  • one, two, three or all of the charged amino acids are negatively charged.
  • the analog further comprises an acidic amino acid substituted for the native amino acid at position 28 or 29 or an acidic amino acid added to the carboxy terminus of the peptide of SEQ ID NO: 34.
  • the acidic amino acid is aspartic acid or glutamic acid.
  • the glucagon/GLP-1 receptor co-agonist comprises a sequence of SEQ ID NO: 20 further comprising an additional carboxy terminal extension of one amino acid or a peptide selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the amino acid is typically selected from one of the 20 common amino acids, and in some
  • the additional carboxy terminus amino acid has an amide group in place of the carboxylic acid of the native amino acid.
  • the additional amino acid is selected from the group consisting of glutamic acid, aspartic acid and glycine.
  • a glucagon/GLP-1 receptor co-agonist wherein the peptide comprises at least one lactam ring formed between the side chain of a glutamic acid residue and a lysine residue, wherein the glutamic acid residue and a lysine residue are separated by three amino acids.
  • the carboxy terminal amino acid of the lactam bearing glucagon peptide has an amide group in place of the carboxylic acid of the native amino acid. More particularly, in some embodiments a glucagon and GLP-1 co-agonist is provided comprising a modified glucagon peptide selected from the group consisting of:
  • Xaa at position 28 is Asp, or Asn
  • Xaa at position 29 is Thr or Gly
  • R is selected from the group consisting of COOH, CONH 2 , glutamic acid, aspartic acid, glycine, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, and a lactam bridge is formed between Lys at position 12 and Glu at position 16 for SEQ ID NO: 66, between Glu at position 16 and Lys at position 20 for SEQ ID NO: 67, between Lys at position 20 and Glu at position 24 for SEQ ID
  • R is selected from the group consisting of COOH, CONH 2 , glutamic acid, aspartic acid, glycine, the amino acid at position 28 is Asn, and the amino acid at position 29 is threonine.
  • R is CONH 2 , the amino acid at position 28 is Asn and the amino acid at position 29 is threonine.
  • R is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 65 and the amino acid at position 29 is glycine.
  • the glucagon/GLP-1 receptor co-agonist is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, wherein the peptide further comprises an additional carboxy terminal extension of one amino acid or a peptide selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the terminal extension comprises the sequence of SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 and the glucagon peptide comprises the sequence of SEQ ID NO: 55.
  • the glucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO: 33 wherein the amino acid at position 16 is glutamic acid, the amino acid at position 20 is lysine, the amino acid at position 28 is asparagine and the amino acid sequence of SEQ ID No: 26 or SEQ ID NO: 29 is linked to the carboxy terminus of SEQ ID NO: 33.
  • the amino acid is typically selected from one of the 20 common amino acids, and in some embodiments the amino acid has an amide group in place of the carboxylic acid of the native amino acid. In some embodiments the additional amino acid is selected from the group consisting of glutamic acid and aspartic acid and glycine.
  • the glucagon agonist analog further comprises a carboxy terminal extension, the carboxy terminal amino acid of the extension, in some embodiments, ends in an amide group or an ester group rather than a carboxylic acid.
  • the glucagon/GLP-1 receptor co-agonist comprises the sequence: NH 2 -His-Ser-Gln-Gly-Thr-Phe- Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Xaa-CONH 2 (SEQ ID NO: 19), wherein the Xaa at position 30 represents any amino acid.
  • Xaa is selected from one of the 20 common amino acids, and in some embodiments the amino acid is glutamic acid, aspartic acid or glycine.
  • the solubility of this peptide can be further improved by covalently linking a PEG chain to the side chain of amino acid at position 17, 21, 24 or 30 of SEQ ID NO: 19.
  • the peptide comprises an additional carboxy terminal extension of a peptide selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the glucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.
  • SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 64 can be made to yield a set of glucagon agonists that possess variable degrees of GLP-1 agonism. Accordingly, peptides that possess virtually identical in vitro potency at each receptor have been prepared and characterized. Similarly, peptides with tenfold selectively enhanced potency at each of the two receptors have been identified and characterized.
  • substitution of the serine residue at position 16 with glutamic acid enhances the potency of native glucagon at both the Glucagon and GLP-1 receptors, but maintains approximately a tenfold selectivity for the glucagon receptor.
  • substitution of the native glutamine at position 3 with glutamic acid SEQ ID NO: 22
  • substitution of the native glutamine at position 3 with glutamic acid SEQ ID NO: 22
  • the solubility of the glucagon/GLP-1 co-agonist peptides can be further enhanced in aqueous solutions at physiological pH, while retaining the high biological activity relative to native glucagon by the introduction of hydrophilic groups at positions 16, 17, 21, and 24 of the peptide, or by the addition of a single modified amino acid (i.e., an amino acid modified to comprise a hydrophilic group) at the carboxy terminus of the glucagon/GLP-1 co-agonist peptide.
  • the hydrophilic group comprises a polyethylene (PEG) chain.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 wherein a PEG chain is covalently linked to the side chain of an amino acids at position 16, 17, 21, 24, 29 or the C-terminal amino acid of the glucagon peptide, with the proviso that when the peptide comprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 the polyethylene glycol chain is covalently bound to an amino acid residue at position 17, 21 or 24, when the peptide comprises SEQ ID NO: 14 or SEQ ID NO: 15 the polyethylene glycol chain is covalently bound to an amino acid residue at position 16, 17 or 21, and when the peptide comprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 the polyethylene glycol chain is covalently
  • the glucagon peptide comprises the sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein a PEG chain is covalently linked to the side chain of an amino acids at position 17, 21, 24, or the C-terminal amino acid of the glucagon peptide, and the carboxy terminal amino acid of the peptide has an amide group in place of the carboxylic acid group of the native amino acid.
  • the glucagon/GLP-1 receptor co-agonist peptide comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, wherein a PEG chain is covalently linked to the side chain of an amino acid at position 17, 21 or 24 of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 19, or at position 16, 17 or 21 of SEQ ID NO: 14 and SEQ ID NO: 15 or at position 17 or 21 of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 of the glucagon peptide.
  • the glucagon/GLP-1 receptor co- agonist peptide comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 19, wherein a PEG chain is covalently linked to the side chain of an amino acids at position 17, 21 or 24 or the C-terminal amino acid of the glucagon peptide.
  • the glucagon co-agonist peptide is modified to contain one or more amino acid substitution at positions 16, 17, 21, 24, or 29 or the C- terminal amino acid, wherein the native amino acid is substituted with an amino acid having a side chain suitable for crosslinking with hydrophilic moieties, including for example, PEG.
  • the native peptide can be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non- naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.
  • the amino acid having a side chain suitable for crosslinking with hydrophilic moieties can be added to the carboxy terminus of any of the glucagon analogs disclosed herein.
  • an amino acid substitution is made in the glucagon/GLP-1 receptor co- agonist peptide at a position selected from the group consisting of 16, 17, 21, 24, or 29 replacing the native amino acid with an amino acid selected from the group consisting of lysine, cysteine, ornithine, homocysteine and acetyl phenylalanine, wherein the substituting amino acid further comprises a PEG chain covalently bound to the side chain of the amino acid.
  • a glucagon peptide selected form the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 is further modified to comprise a PEG chain is covalently linked to the side chain of an amino acid at position 17 or 21 of the glucagon peptide.
  • the pegylated glucagon/GLP-1 receptor co-agonist further comprises the sequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 29.
  • SEQ ID NO: 55 or SEQ ID NO: 56 further comprising a C-terminal extension of SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 linked to the C-terminal amino acid of SEQ ID NO: 55 or SEQ ID NO: 56, and optionally further comprising a PEG chain covalently linked to the side chain of an amino acids at position 17, 18, 21, 24 or 29 or the C-terminal amino acid of the peptide.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 55 or SEQ ID NO: 56, wherein a PEG chain is covalently linked to the side chain of an amino acids at position 21 or 24 of the glucagon peptide and the peptide further comprises a C-terminal extension of SEQ ID NO: 26, or SEQ ID NO: 29.
  • the pegylated glucagon analog further comprises a C-terminal extension of SEQ ID NO: 26 or SEQ ID NO: 29 linked to the C-terminal amino acid of SEQ ID NO: 33 or SEQ ID NO: 34.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 19, wherein a PEG chain is covalently linked to the side chain of the amino acid at position 30 of the glucagon peptide and the peptide further comprises a C-terminal extension of SEQ ID NO: 26 or SEQ ID NO: 29 linked to the C-terminal amino acid of SEQ ID NO: 19.
  • the polyethylene glycol chain may be in the form of a straight chain or it may be branched. In accordance with some embodiments the polyethylene glycol chain has an average molecular weight selected from the range of about 500 to about 10,000 Daltons. In some embodiments the polyethylene glycol chain has an average molecular weight selected from the range of about 1,000 to about 5,000 Daltons. In an alternative embodiment the polyethylene glycol chain has an average molecular weight selected from the range of about 10,000 to about 20,000 Daltons. In accordance with some embodiments the pegylated glucagon peptide comprises two or more polyethylene glycol chains covalently bound to the glucagon peptide wherein the total molecular weight of the glucagon chains is about 1,000 to about 5,000 Daltons.
  • the pegylated glucagon agonist comprises a peptide consisting of SEQ ID NO: 5 or a glucagon agonist analog of SEQ ID NO: 5, wherein a PEG chain is covalently linked to the amino acid residue at position 21 and at position 24, and wherein the combined molecular weight of the two PEG chains is about 1,000 to about 5,000 Daltons.
  • the glucagon peptide comprises the amino acid sequence of SEQ ID NO: 1 with up to ten amino acid modifications and comprises an amino acid at position 10 which is acylated or alkylated.
  • the amino acid at position 10 is acylated or alkylated with a C4 to C30 fatty acid.
  • the amino acid at position 10 comprises an acyl group or an alkyl group which is non-native to a naturally-occurring amino acid.
  • the glucagon peptide comprising an amino acid at position 10 which is acylated or alkylated comprises a stabilized alpha helix.
  • the glucagon peptide comprises an acyl or alkyl group as described herein and an intramolecular bridge, e.g., a covalent intramolecular bridge (e.g., a lactam bridge) between 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.
  • the glucagon peptide comprises an acyl or alkyl group as described herein and one, two, three or more of positions 16, 20, 21 and/or 24 of the glucagon peptide are substituted with an a, a-disubstituted amino acid, e.g., Aib.
  • the non-native glucagon peptide comprises Glu at position 16 and Lys at position 20, wherein optionally a lactam bridge links the Glu and the Lys, and, optionally, the glucagon peptide further comprises one or more modifications selected from the group consisting of: Gin at position 17, Ala at position 18, Glu at position 21, lie at position 23, and Ala at position 24.
  • the glucagon peptide comprises an amino acid at position 10 which is acylated or alkylated
  • the glucagon peptide can further comprise a C-terminal amide in lieu of the C-terminal alpha carboxylate.
  • the glucagon peptide comprising an acyl or alkyl group as described herein further comprises an amino acid substitution at position 1, at position 2, or at positions 1 and 2, wherein the amino acid substitution(s) achieve DPP-IV protease resistance.
  • the His at position 1 may be substituted with an amino acid selected from the group consisting of: D-histidine, alpha, alpha- dimethyl imidiazole acetic acid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine and homo-histidine.
  • the Ser at position 2 may be substituted with an amino acid selected from the group consisting of: D-serine, alanine, D- alanine, valine, glycine, N-methyl serine, N-methyl alanine, and amino isobutyric acid.
  • the amino acid at position 2 is not D-serine.
  • the glucagon peptide comprising the amino acid at position 10 which is acylated or alkylated as described herein can comprise any amino acid sequence which is substantially related to SEQ ID NO: 1.
  • the glucagon peptide comprises SEQ ID NO: 1 with up to 10 amino acid modifications (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 modifications).
  • the amino acid sequence of the acylated or alkylated glucagon peptide is greater than 25% identical to SEQ ID NO: 1 (e.g., greater than 30%, 35%, 40%, 50%, 60%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or nearly 100% identical to SEQ ID NO: 1).
  • the glucagon peptide is one which comprises SEQ ID NOs: 55 with an amino acid at position 10 acylated or alkylated as described herein.
  • the glucagon peptide can be any of SEQ ID NOs: 55, 55 with 1 or 2 amino acid modifications, 2-4, 9-18, 20, 23-25, 33, 40-44, 53, 56, 61, 62, 64, 66-514, and 534.
  • the acyl or alkyl group of these embodiments may be any acyl or alkyl group described herein.
  • the acyl group may be a C4 to C30 (e.g., C8 to C24) fatty acyl group and the alkyl group may be a C4 to C30 (e.g., C8 to C24) alkyl group.
  • the amino acid to which the acyl or alkyl group is attached may be any of the amino acids described herein, e.g., an amino acid of any of Formula I (e.g., Lys), Formula II, and Formula III.
  • the acyl group or alkyl group is directly attached to the amino acid at position 10.
  • the acyl or alkyl group is attached to the amino acid at position 10 via a spacer, such as, for example, a spacer which is 3 to 10 atoms in length, e.g., an amino acid or dipeptide. Suitable spacers for purposes of attaching an acyl or alkyl group are described herein.
  • the Class 3 glucagon related peptide may be an analog of any of the foregoing Class 3 glucagon related peptides as described herein, which analog exhibits agonist activity at the GIP receptor.
  • the activity level of the analog at the glucagon receptor, the GLP-1 receptor, and the GIP receptor, the potency at each of these receptors, and the selectivity for each of these receptors may be in accordance with the teachings of Class 2 glucagon related peptides described herein. See, the teachings under the subsection of the Class 2 glucagon related peptide section entitled "Activity.”
  • an analog of a glucagon peptide which analog exhibits agonist activity at the GIP receptor, is provided.
  • the analog in certain embodiments comprises the amino acid sequence of SEQ ID NO: 1 with at least one amino acid modification (optionally, up to 15 amino acid modifications), and an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 of the analog.
  • the analogs comprise at least one amino acid modification and up to 15 amino acid modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid modifications, up to 10 amino acid modifications). In certain embodiments, the analogs comprise at least one amino acid modification at up to 10 amino acid modifications and additional conservative amino acid modifications. Conservative amino acid modifications are described herein.
  • At least one of the amino acid modifications confers a stabilized alpha helix structure in the C-terminal portion of the analog. Modifications which achieve a stabilized alpha helix structure are described herein. See, for example, the teachings under the section entitled Stabilization of the alpha helix/Intramolecular bridges.
  • the analog comprises an
  • intramolecular bridge (e.g., a covalent intramolecular bridge, a non-covalent intramolecular bridge) between the side chains of two amino acids of the analog.
  • an intramolecular bridge links the side chains of the amino acids at positions i and i+4, wherein i is 12, 13, 16, 17, 20, or 24.
  • an intramolecular bridge connects the side chains of the amino acids at positions j and j+3, wherein j is 17, or at positions k and k+7" wherein k is any integer between 12 and 22.
  • the intramolecular bridge is a covalent
  • the lactam bridge connects the side chains of the amino acids at positions 16 and 20.
  • one of the amino acids at positions 16 and 20 is a positive-charged amino -I l l- acid and the other is a negative-charged amino acid.
  • the analog can comprise a lactam bridge connecting the side chains of a Glu at position 16 and a Lys at position 20.
  • the negative-charged amino acid and the positive- charged amino acid form a salt bridge.
  • the intramolecular bridge is a non-covalent intramolecular bridge.
  • the amino acid modification which confers a stabilized alpha helix is an insertion or substitution of an amino acid of SEQ ID NO: 1 with an ⁇ , ⁇ -disubstituted amino acid.
  • Suitable ⁇ , ⁇ -disubstituted amino acids for purposes of stabilizing the alpha helix are described herein and include, for example, Aib.
  • one, two, three, or more of the amino acids at positions 16, 20, 21, and 24 of SEQ ID NO: 1 are substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., Aib.
  • the amino acid at position 16 is Aib.
  • the analog which exhibits agonist activity at the GIP receptor can comprise additional modifications, such as any of those described herein.
  • the amino acid modifications may increase or decrease activity at one or both of the GLP- 1 receptor and glucagon receptor.
  • the amino acid modifications may increase stability of the peptide, e.g., increase resistance to DPP-IV protease degradation, stabilize the bond between amino acids 15 and 16.
  • the amino acid modifications may increase the solubility of the peptide and/or alter the time of action of the analog at any of the GIP, glucagon, and GLP-1 receptors. A combination of any of these types of modifications may be present in the analogs which exhibit agonist activity at the GIP receptor.
  • the analog comprises the amino acid sequence of SEQ ID NO: 1 with one or more of: Gin at position 17, Ala at position 18, Glu at position 21, He at position 23, and Ala or Cys at position 24, or conservative amino acid substitutions thereof.
  • the analog comprises a C-terminal amide in place of the C-terminal alpha carboxylate.
  • the analog comprises an amino acid substitution at position 1, position 2, or positions 1 and 2, which substitution(s) achieve DPP-IV protease resistance. Suitable amino acid substitutions are described herein. For example, DMIA at position 1 and/or d-Ser or Aib at position 2.
  • the amino acid at position 2 is not D-serine.
  • the analog may comprise one or a combination of: (a) Ser at position 2 substituted with Ala; (b) Gin at position 3 substituted with Glu or a glutamine analog; (c) Thr at position 7 substituted with a lie; (d) Tyr at position 10 substituted with Trp or an amino acid comprising an acyl or alkyl group which is non-native to a naturally- occurring amino acid; (e) Lys at position 12 substituted with He; (f) Asp at position 15 substituted with Glu; (g) Ser at position 16 substituted with Glu; (h) Gin at position 20 substituted with Ser, Thr, Ala, Aib; (i) Gin at position 24 substituted with Ser, Thr, Ala, Aib; j) Met at position 27 substituted with Leu or Nle; (k) Asn at position 29 substituted with a charged amino acid, optionally, Asp or Glu; and (1) Thr at position 29 substituted with Gly or a charged amino acid, optionally, Asp or Glu;
  • the analog comprises an extension of 1-21 amino acids (e.g., 5-19, 7-15, 9-12 amino acids).
  • the extension of the analog may comprise any amino acid sequence, provided that the extension is 1 to 21 amino acids. In some aspects, the extension is 7 to 15 amino acids and in other aspects, the extension is 9 to 12 amino acids.
  • the extension comprises (i) the amino acid sequence of SEQ ID NO: 26 or 674, (ii) an amino acid sequence which has high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 98%, 99%) with the amino acid sequence of SEQ ID NO: 26 or 674, or (iii) the amino acid sequence of (i) or (ii) with one or more conservative amino acid modifications.
  • At least one of the amino acids of the extension is acylated or alkylated.
  • the amino acid comprising the acyl or alkyl group may be located at any position of extension of the analog.
  • the acylated or alkylated amino acid of the extension is located at one of positions 37, 38, 39, 40, 41, or 42 (according to the numbering of SEQ ID NO: 1) of the analog.
  • the acylated or alkylated amino acid is located at position 40 of the analog.
  • the acyl or alkyl group is an acyl or alkyl group which is non-native to a naturally-occurring amino acid.
  • the acyl or alkyl group may be a C4 to C30 (e.g., C12 to C18) fatty acyl group or C4 to C30 (e.g., C12 to CI 8) alkyl.
  • the acyl or alkyl group may be any of those discussed herein.
  • the acyl or alkyl group is attached directly to the amino acid, e.g., via the side chain of the amino acid.
  • the acyl or alkyl group is attached to the amino acid via a spacer (e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilic bifunctional spacer, a hydrophobic bifunctional spacer).
  • the spacer is 3 to 10 atoms in length.
  • the amino acid spacer is not ⁇ -Glu.
  • the dipeptide spacer is not ⁇ - Glu- ⁇ -Glu.
  • the amino acid to which the acyl or alkyl group is attached may be any of those described herein, including, for example, an amino acid of Formula I, II, or III.
  • the amino acid which is acylated or alkylated may be a Lys, for example.
  • Suitable amino acids comprising an acyl or alkyl group, as well as suitable acyl groups and alkyl groups, are described herein. See, for example, the teachings under the sections entitled Acylation and Alkylation.
  • 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 amino acids) of the extension are positive-charged amino acids, e.g., amino acids of Formula IV, such as, for example, Lys.
  • positive-charged amino acid refers to any amino acid, naturally-occurring or non-naturally occurring, comprising a positive charge on an atom of its side chain at a physiological pH.
  • the positive-charged amino acids are located at any of positions 37, 38, 39, 40, 41, 42, and 43.
  • a positive-charged amino acid is located at position 40.
  • the extension is acylated or alkylated as described herein and comprises 1-6 positive charged amino acids as described herein.
  • the analogs which exhibit agonist activity at the GIP receptor comprises (i) SEQ ID NO: 1 with at least one amino acid modification, (ii) an extension of 1 to 21 amino acids (e.g., 5 to 18, 7 to 15, 9 to 12 amino acids) C- terminal to the amino acid at position 29 of the analog, and (iii) an amino acid comprising an acyl or alkyl group which is non-native to a naturally-occurring amino acid which is located outside of the C-terminal extension (e.g., at any of positions 1- 29).
  • the analog comprises an acylated or alkylated amino acid at position 10.
  • the acyl or alkyl group is a C4 to C30 fatty acyl or C4 to C30 alkyl group.
  • the acyl or alkyl group is attached via a spacer, e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic bifunctional spacer).
  • the analog comprises an amino acid modification which stabilizes the alpha helix, such as a salt bridge between a Glu at position 16 and a Lys at position 20, or an alpha, alpha-disubstituted amino acid at any one, two, three, or more of positions 16, 20, 21, and 24.
  • the analog additionally comprises amino acid modifications which confer DPP-IV protease resistance, e.g., DMIA at position 1, Aib at position 2. Analogs comprising further amino acid modifications are contemplated herein.
  • the analogs having GIP receptor activity exhibit at least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%) activity of native GIP at the GIP receptor. In some embodiments, the analogs exhibit more than 20% (e.g., more than 50%, more than 75%, more than 100%, more than 200%, more than 300%, more than 500%) activity of native GIP at the GIP receptor. In some embodiments, the analog exhibits appreciable agonist activity at one or both of the GLP-1 and glucagon receptors. In some aspects, the selectivity for these receptors (GIP receptor and GLP-1 receptor and/or glucagon receptor) are within 1000-fold.
  • the selectivity for the GLP-1 receptor of the analogs having GIP receptor activity can be less than 500-fold, 100-fold, within 50-fold, within 25 fold, within 15 fold, within 10 fold) the selectivity for the GIP receptor and/or the glucagon receptor.
  • the Class 3 glucagon related peptide comprises the amino acid sequence of native glucagon (SEQ ID NO: 1) comprising the following modifications: Aib at position 2, Glu at position 3, Lys at position 10, Glu at position 16, Gin at position 17, Ala at position 18, Lys at position 20, Glu at position 21, He at position 23, Ala at position 24; wherein Lys at position 10 is acylated with a C 14 or C 16 fatty acid, and wherein the C-terminal carboxylate is replaced with an amide.
  • this Class 3 glucagon related peptide is attached via a linker (L) to a NHR ligand (Y).
  • the Class 3 glucagon related peptide comprises, consists essentially of, or consists of an amino acid sequence of any of SEQ ID NOs: 70-514, 517-534, or 554, optionally with up to 1, 2, 3, 4, or 5 further modifications that retain GLP-1 agonist and/or glucagon agonist activity.
  • the Class 3 glucagon related peptide comprises the amino acids of any of SEQ ID NOs: 562-760.
  • the Class 3 glucagon related peptide comprises the amino acid sequences of any of SEQ ID NOs: 1301-1421.
  • glucagon analog having enhanced solubility relative to native glucagon
  • said analog comprises one or more amino acids selected from positions 5, 7, 8, 11 or 16 linked via an ester bond and said glucagon analog comprises the sequence
  • Xi is selected from the group consisting of His, D-His, N-methyl-His, alpha-methyl- His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo-His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA);
  • X2 IS selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Gly, N-methyl- Ser, Aib, Val, or a-amino-N-butyric acid;
  • X 3 is an amino acid comprising a side chain of Structure I, II, or III:
  • R 1 is C 0 -3 alkyl or C 0 -3 heteroalkyl
  • R 2 is NHR 4 or Ci_ 3 alkyl
  • R 3 is Ci_ 3 alkyl
  • R 4 is H or Ci_ 3 alkyl
  • X is NH, O, or S
  • Y is NHR 4 , SR 3 , or OR 3 ;
  • Xio is selected from the group consisting of Tyr, Lys;
  • X12 is Lys, He or Arg
  • Xi5 is Asp, Glu, cysteic acid, homoglutamic acid or homocysteic acid;
  • Xi 6 is Glu, Lys, Asp, Ser, glutamine, homoglutamic acid, homocysteic acid, Thr or Aib;
  • Xi7 is Arg or Gin
  • Xi8 is Ala, Arg
  • X20 is Gin, Glu, Ser, Thr, Ala, Lys, Citrulline, Arg, Orn, or Aib;
  • X21 is Glu, Aib, Asp, Lys, Cys, Orn, homocysteine or acetyl phenylalanine;
  • X2 4 is Asn, Aib, Gin, Glu or Lys;
  • X27 is Met, Leu or Nle;
  • X 2 8 is Asn or Ala
  • X 29 is Thr or Gly
  • Z is selected from the group consisting of -COOH, -X 28 -COOH, X
  • sequence XiX 2 X 3 GTFTSDXioSXi 2 YLXi 5 Xi 6 Xi 7 Xi 8 AX 2 oX 2 iFVX 24 WLX 27 -Z (SEQ ID NO: 943 further comprises
  • glucagon analog having enhanced solubility relative to native glucagon
  • said analog comprises one or more amino acids selected from positions 5, 7, 8, 11 or 16 linked via an ester bond and said glucagon analog comprises the sequence
  • YAQGTFTSDX 1 0SKYLDERAAQDFVQWLLEGGPSSGAPPPS-NH 2 (SEQ ID NO: 945) or
  • X 2 is Ser, D-Ser or Aib
  • X 10 is Lys acylated with a C 16 to C18 carbon atom chain, optionally via a yGlu linker;
  • X 20 is Gin or Aib. In one embodiment the sequence
  • YX 2 EGTFTSDX 10 SIYLDKQAAX 20 EFVNWLLAGGPSSGAPPPS (SEQ ID NO: 944) comprise acid comprising the structure of
  • Ri 5 is H or CH 3 and
  • Ri 6 is an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • Ri 6 is a dipeptide that is susceptible to cleavage by Dipeptidyl Peptidase IV. In an alternative embodiment Ri 6 is a dipeptide having the general structure of Formula I:
  • Ri , R 2, R 4 and R 8 are independently selected from the group consisting of H,
  • R 3 is selected from the group consisting of Ci-Ci 8 alkyl, (Ci-Ci 8 alkyl)OH, (Ci-Cis alkyl)NH 2, (Ci-Cis 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 -Cio aryl)R 7 , and (C1-C4 alkyl)(C 3 -C 9 heteroaryl) or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NHR 6 or OH
  • R 6 is H, Ci-Cg alkyl
  • R 7 is selected from the group consisting of hydrogen and OH.
  • R 1 ⁇ 2 is a dipeptide having the general structure of Formula I: R 5
  • Ri is selected from the group consisting of H and Ci-C 4 alkyl
  • R 2 is selected from the group consisting of H, Ci-C 6 alkyl, C 2 -C 8 alkenyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , and CH 2 (Cs-C9 heteroaryl) or R 2 and R 6 together with the atoms to which they are attached form a 5 member heterocyclic ring;
  • R 3 is Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H and Ci-C 4 alkyl or R 3 and R 4 together with the atoms to which they are attached form a 5 member heterocyclic ring;
  • R 7 is selected from the group consisting of hydrogen and OH.
  • Ri 6 is a dipeptide having the general structure of Formula
  • Ri is C1-C4 alkyl or (C1-C4 alkyl)NH 2;
  • R 2 is H or is Ci-C 4 alkyl
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and C 1 -C 4 alkyl
  • R 8 is H
  • R 5 is NH 2 or OH.
  • a glucagon analog having enhanced solubility relative to native glucagon comprising one or more amino acids selected from positions 5, 7, 8, 11 or 16 linked via an ester bond and said glucagon analog comprises the sequence XiX 2 QGTFTSDYSKYLXi 5 Xi 6 RRAX 2 oDFVX 24 WLMX 2 8T (SEQ ID NO: 946) wherein
  • Xi is selected from the group consisting of His, D-His, N-methyl-His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo- His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA);
  • X 2 is selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Gly, N- methyl-Ser, Aib, Val, or a-amino-N-butyric acid;
  • Xi 5 is Asp, Glu, cysteic acid, homoglutamic acid or homocysteic acid;
  • X 16 is Glu, Lys, Asp, Ser, glutamine, homoglutamic acid, homocysteic acid, Thr or Aib;
  • X 2 o is Gin or Lys
  • X 24 is Gin or Glu
  • X 28 is Asn, Asp or Lys
  • SEQ ID NO: 946 that differs from SEQ ID NO: 946 by 1 to 2 amino acid modifications, selected from positions 1, 2, 7, 10, 11, 13, 14, 17, 18, 19, 21, 27 and 29, wherein the glucagon peptide exhibits enhanced activity at the GLP-1 receptor as compared to native glucagon, optionally wherein the side chains of the amino acids at positions 12 and 16, positions 16 and 20, positions 20 and 24, or positions 24 and 28 of the glucagon peptide are linked by covalent bonds to provide the enhanced activity at the GLP-1 receptor, with the proviso that when the amino acid at position 16 is serine either position 20 is lysine, or the side chain of the amino acid at position 24 and the side chain of either the amino acid at position 20 or position 28 are linked by covalent bonds.
  • the sequence is serine either position 20 is lysine, or the side chain of the amino acid at position 24 and the side chain of either the amino acid at position 20 or position 28 are linked by covalent bonds.
  • XiX 2 QGTFTSDYSKYLXi 5 Xi 6 RRAX 2 oDFVX 24 WLMX 28 T (SEQ ID NO: 946) comprises an ester linked amino acid comprising the structure of
  • Ri 5 is H or C3 ⁇ 4 and
  • Ri 6 is an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a diketopiperazine or diketomorpholine.
  • the ester linked amino acid is located at position 16.
  • Ri 6 is a dipeptide that is susceptible to cleavage by Dipeptidyl Peptidase IV.
  • R 1 ⁇ 2 is a dipeptide having the general structure of Formula I:
  • Ri , R 2i R 4 and R 8 are independently selected from the group consisting of H, Ci-Cis alkyl, C 2 -Ci 8 alkenyl, (Ci-Cis alkyl)OH, (Ci-Cis alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (Ci-C 4 alkyl)CONH 2 , (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4
  • R 3 is selected from the group consisting of Ci-Ci 8 alkyl, (Ci-Ci 8 alkyl)OH, (Ci-Cis alkyl)NH 2 , (Ci-Cis 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 -Cio aryl)R 7 , and (C1-C4 alkyl)(C 3 -C 9 heteroaryl) or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NHR 6 or OH
  • R 6 is H, Ci-C 8 alkyl
  • R 7 is selected from the group consisting of hydrogen and OH.
  • R 1 ⁇ 2 is a dipeptide having the general structure of Formula
  • Ri is selected from the group consisting of H and Ci-C 4 alkyl
  • R 2 is selected from the group consisting of H, Ci-C 6 alkyl, C 2 -C 8 alkenyl, (C 1 -C4 alkyl)OH, (C 1 -C4 alkyl)NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , and CH 2 (C5-C9 heteroaryl) or R 2 and R 6 together with the atoms to which they are attached form a 5 member heterocyclic ring;
  • R 3 is Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H and C 1 -C 4 alkyl or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 7 is selected from the group consisting of hydrogen and OH
  • Ri 6 is a dipeptide having the general structure of Formula I:
  • Ri is C1-C4 alkyl or (C1-C4 alkyl)NH 2;
  • R 2 is H or is Ci-C 4 alkyl
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 4 is selected from the group consisting of H, and Q-C4 alkyl
  • R 8 is H
  • R 5 is NH 2 or OH.
  • glucagon analog having enhanced solubility relative to native glucagon
  • said analog comprises one or more amino acids selected from positions 5, 7, 8, 11 or 16 linked via an ester bond and said glucagon analog comprises the sequence
  • Xi is selected from the group consisting of His, D-His, N-methyl-His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo- His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA);
  • X 2 is selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Gly, N- methyl-Ser, Aib, Val, or a-amino-N-butyric acid;
  • Xi6 is amino isobutyric acid (Aib);
  • X 20 is Gin, Glu, Ser, Thr, Ala, Lys, Citrulline, Arg, Orn, or Aib;
  • X 24 is Gin, Lys, Cys, Orn, homocysteine or acetyl phenylalanine;
  • X 2 8 is a Asn, Arg, His, Glu, Lys or Asp,
  • amino acid optionally, wherein the charged amino acid is selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid;
  • XiX 2 X 3 GTFTSDYSKYLDXi 6 RRAX 2 oDFVX 24 WLMX 28 T (SEQ ID NO: 942) comprises an ester linked amino acid comprising the structure of
  • Ri 5 is H or CH 3 and
  • Ri 6 is an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a diketopiperazine or diketomorpholine.
  • the ester linked amino acid is at position 16.
  • R 1 ⁇ 2 is a dipeptide that is susceptible to cleavage by Dipeptidyl Peptidase IV.
  • glucagon analog having enhanced solubility relative to native glucagon
  • said analog comprising one or more amino acids selected from positions 5, 7, 8, 11 or 16 linked via an ester bond and said glucagon analog comprises the sequence
  • X 2 is selected from the group consisting of Ser, D-Ser, Ala, dAla, Gly, N- methyl-Ser, Aib, Val, or a-amino-N-butyric acid.
  • X2 is Aib or D-Ser. In one embodiment X 2 is Aib.
  • the ester linked amino acid is at position 7, 8, 11 or 16. In one embodiment the ester linked amino acid is at position 8, 11 or 16. In one embodiment the ester linked amino acid is at position 11 or 16. In one embodiment the ester linked amino acid is at 16.
  • Ri 5 is H or CH 3 ;
  • Ri 6 is H, an amino acid or dipeptide that is susceptible to cleavage by a peptidase, or a dipeptide that will chemically cleave by formation of a diketopiperazine or diketomorpholine.
  • Ri 6 is a dipeptide of the general formula X-Pro, wherein X is any amino acid and the proline is linked to the primary amine of the ester linked amino acid.
  • R 1 ⁇ 2 is selected from the group consisting of Gly-Pro, Lys-Pro and Lys- Azetidine-2- carboxylic acid wherein the proline or Azetidine-2-carboxylic acid (Azetidine) is linked to the primary amine of the ester linked amino acid.
  • Ri5 is H or CH 3 ;
  • A is proline or azetidine
  • B is any amino acid, optionally selected from the group consisting of Lys, Glu, Asp, and Ala.
  • A is an N-alkylated amino acid, optionally alkylated with a Ci-C 4 alkyl and B is any amino acid, optionally lysine.
  • a glucagon analog having improved solubility relative to native glucagon comprising an amino acid sequence of NH 2 -Xaa-Xaa-Gln-Gly-Xaa-Phe- Xaa-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 1298) wherein
  • the Xaa at position 1 is Tyr, His, D-His, N-methyl-His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo-His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA),
  • the Xaa at position 2 is Ser, D-Ser or Aib,
  • Xaa at position 5 and 7 are independently Thr or an ester linked amino acid having the general structure of Formula V:
  • Ri5 is H or CH 3 with the proviso that at least one of the positions is an ester linked amino acid having the general structure of Formula V,
  • the Xaa at position 15 is selected from the group of amino acids consisting of Asp, Glu, cysteic acid, homoglutamic acid and homocysteic acid,
  • the Xaa at position 16 is selected from the group of amino acids consisting of Aib, Ser, Glu, Gin, homoglutamic acid, homocysteic acid and an amino acid of Formula V,
  • the Xaa at position 20 is Aib, Gin or Lys,
  • the Xaa at position 24 is Gin or Glu
  • the Xaa at position 28 is Asn, Lys or an acidic amino acid
  • the Xaa at position 29 is Thr, Gly or an acidic amino acid, and R is COOH or CONH 2 , with the proviso that when position 16 is serine, position 24 is Glu and either position 20 or position 28 is Lys.
  • the Xaa at position 28 is Asn and the Xaa at position 29 is Thr or Gly.
  • Xi is selected from the group consisting of His, D-His, N-methyl-His, alpha- methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo- His, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA);
  • X 2 is selected from the group consisting of Ser, D-Ser, and Aib; X5 and X 7 are independently Thr or an ester linked amino acid having the general structure of Formula V:
  • Ri5 is H or CH 3 with the proviso that at least one of X5 and X 7 is an ester linked amino acid having the general structure of Formula V;
  • Ri6 is H, an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • X 16 is an ester linked amino acid having the general structure of Formula V, wherein Ri5 is H or CH 3 ;
  • Ri6 is H, an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • X 2 is selected from the group consisting of Ser, D-Ser, and Aib;
  • X5 and X 7 are independently Thr or an ester linked amino acid having general structure of Formula V:
  • Ri5 is H or CH 3 with the proviso that at least one of X5 and X 7 is an ester linked amino acid having the general structure of Formula V;
  • Ri6 is H, an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • Xi6 is an ester linked amino acid having the general structure of Formula V, wherein
  • Ri5 is H or CH 3 ;
  • Ri6 is H, an amino acid or dipeptide that is susceptible to cleavage by a serum peptidase, or a dipeptide that will chemically cleave by formation of a
  • X 2 is selected from the group consisting of Ser, D-Ser, and Aib;
  • X5 and X 7 are independently Thr or an ester linked amino acid having general structure of Formula V:

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  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des peptides modifiés du glucagon présentant une solubilité améliorée. Les peptides du glucagon ont été modifiés par le remplacement d'une ou de plusieurs des liaisons amide natives par des liaisons ester. Les modifications augmentent la solubilité du peptide du glucagon dans lequel les liaisons amide natives sont reconstituées in vivo après administration à un patient.
PCT/US2017/034961 2016-06-02 2017-05-30 Agonistes hydrosolubles et chimiquement stables du depsi-glucagon Ceased WO2017210168A1 (fr)

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US201662344669P 2016-06-02 2016-06-02
US62/344,669 2016-06-02
US201662413082P 2016-10-26 2016-10-26
US62/413,082 2016-10-26

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10604555B2 (en) 2018-05-04 2020-03-31 Novo Nordisk A/S GIP derivatives and uses thereof
US11220534B2 (en) 2016-12-05 2022-01-11 Lancaster University Business Enterprises Limited Treatment of neurological diseases

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110098217A1 (en) * 2007-10-30 2011-04-28 Indiana University Research And Technology Corporation Compounds exhibiting glucagon antagonist and glp-1 agonist activity
US20110257076A1 (en) * 2008-12-19 2011-10-20 Dimarchi Richard D Amide based insulin prodrugs
WO2016049190A1 (fr) * 2014-09-24 2016-03-31 Indiana University Research And Technology Corporation Conjugués d'insuline-incrétines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110098217A1 (en) * 2007-10-30 2011-04-28 Indiana University Research And Technology Corporation Compounds exhibiting glucagon antagonist and glp-1 agonist activity
US20110257076A1 (en) * 2008-12-19 2011-10-20 Dimarchi Richard D Amide based insulin prodrugs
WO2016049190A1 (fr) * 2014-09-24 2016-03-31 Indiana University Research And Technology Corporation Conjugués d'insuline-incrétines

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11220534B2 (en) 2016-12-05 2022-01-11 Lancaster University Business Enterprises Limited Treatment of neurological diseases
US11851468B2 (en) 2016-12-05 2023-12-26 University Of Lancaster Treatment of neurological diseases
US10604555B2 (en) 2018-05-04 2020-03-31 Novo Nordisk A/S GIP derivatives and uses thereof
US11633459B2 (en) 2018-05-04 2023-04-25 Novo Nordisk A/S GIP derivatives and uses thereof

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