WO2011163473A1 - Analogues du glucagon présentant une solubilité et une stabilité améliorées dans des tampons à ph physiologique - Google Patents

Analogues du glucagon présentant une solubilité et une stabilité améliorées dans des tampons à ph physiologique Download PDF

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WO2011163473A1
WO2011163473A1 PCT/US2011/041623 US2011041623W WO2011163473A1 WO 2011163473 A1 WO2011163473 A1 WO 2011163473A1 US 2011041623 W US2011041623 W US 2011041623W WO 2011163473 A1 WO2011163473 A1 WO 2011163473A1
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glucagon
amino acid
peptide
seq
analog
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PCT/US2011/041623
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English (en)
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Richard D. Dimarchi
Maria Dimarchi
Joseph Chabenne
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Indiana University Research And Technology Corporation
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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

  • 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.
  • glucagon agonists exhibit enhanced biophysical stability and aqueous solubility at physiological pH in pharmaceutical compositions suitable for commercial use.
  • Native glucagon is neither soluble, nor stable in the physiological pH range and thus must be manufactured as a dry product that requires reconstitution and immediate use.
  • the glucagon analogs described herein have enhanced physical properties that render them superior for use in current medicinal settings where the native hormone is currently employed. These compounds can be used in accordance with one embodiment to prepare pre- formulated solutions ready for injection to treat hypoglycemia.
  • the glucagon agonists can be co-administered with insulin to buffer the effects of insulin to allow for a more stable maintenance of blood glucose levels.
  • other beneficial uses of compositions comprising the modified glucagon peptides disclosed herein are described in detail below.
  • One embodiment of the invention provides glucagon peptides 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 of one embodiment of the invention exhibit at least 2-fold, 5-fold, or even higher solubility compared to native glucagon at a pH between 6 and 8, or between 6 and 9, for example, at pH 7 after 24 hours at 25°C.
  • the glucagon peptides retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% activity, 80% activity, 85% activity, or 90% of the activity of native glucagon (calculated as the inverse ratio of EC50s for the glucagon peptide vs. glucagon, e.g., as measured by cAMP production using the assay generally described in Example 13).
  • the glucagon peptides of the present invention have the same or greater activity (used synonymously with the term
  • the glucagon peptides retain up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon.
  • Glucagon normally has about 1% of the activity of native GLP-1 at the GLP-1 receptor.
  • GLP-1 (7-36) amide (SEQ ID NO: 57) or GLP-1 (7-37)acid (SEQ ID NO: 58) are biologically potent forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor.
  • Glucagon is also 10- to 20- fold more selective for the glucagon receptor compared to GLP-1 receptor (selectivity calculated as the inverse ratio of EC50 of glucagon for the glucagon receptor vs. for the GLP-1 receptor).
  • the calculated selectivity is 17.5-fold.
  • Activity can be measured, e.g., by cAMP production using the assay generally described in Example 13.
  • the glucagon peptides of the present invention exhibit less than about 5%, 4%, 3%, 2% or 1% of the activity of native GLP-1 at the GLP-1 receptor and/or a greater than about 5-fold, 10-fold, or 15- fold selectivity for glucagon receptor compared to GLP-1 receptor.
  • the glucagon peptides of the present invention exhibit less than 5% of the activity of native GLP-1 at the GLP-1 receptor and exhibit a greater than 5- fold selectivity for glucagon receptor compared to GLP-1 receptor.
  • any of the glucagon peptides of the invention 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.
  • the glucagon peptides of the invention exhibit improved stability, such that at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, more than 95%, up to 100%) of a concentration of the peptide or less than about 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%, 4%, 3%, 2%, 1%, down to 0%) of degraded peptide is detectable at 280 nm by an ultraviolet (UV) detector after 1 or more weeks (e.g., 2 weeks, 4 weeks, 1 month, two months, four months, six months, eight months, ten months, twelve months) in solution at a temperature of at least 20 °C (e.g., 21 °C, 22 °C,
  • the glucagon analog comprises a modified amino acid sequence of native human glucagon (which native sequence is set forth herein as SEQ ID NO: 1) which glucagon analog exhibits increased solubility in an aqueous solution at physiological pH.
  • the glucagon analog comprises a modified amino acid sequence of native human glucagon (SEQ ID NO: 1) with the following amino acid modifications: (1) substitution of Ser at position 16 with an alpha, alpha-disubstituted amino acid, e.g. AIB or alpha-methyl serine; (2) substitution of Gin at position 3 with another amino acid which does not substantially reduce the peptide's ability to activate the glucagon receptor, e.g.
  • a glutamine analog such as described herein; (3) substitution of the Arg at position 17 with a negatively charged amino acid; (4) optionally, substitution of the Gin at position 20 with a nonpolar amino acid, e.g. Ala, Ser, Thr or AIB; (5) optionally, substitution of the Gin at position 24 with a nonpolar amino acid, e.g. Ala, Ser, Thr or AIB; (6) optionally, substitution or insertion of at least one charged, e.g. negatively charged, amino acid C-terminal to the amino acid at position 27; and optionally, up to 10 (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1) additional amino acid
  • the glucagon analog comprises a Gin analog at position 3, AIB at position 16, a negatively charged amino acid at position 17, Ala at position 20, Ala at position 24, and a negatively charged amino acid at position 28.
  • a glucagon peptide is provided with improved solubility, wherein the 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 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.
  • 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 glucagon peptide comprises an amino acid sequence of SEQ ID NO: 11, or an analog thereof that contains 1 to 3 further amino acid modifications relative to native glucagon, or a glucagon agonist analog thereof.
  • SEQ ID NO: 11 represents a modified glucagon peptide wherein the asparagine residue at position 28 of the native protein has been substituted with an aspartic acid.
  • the glucagon peptide comprises an amino acid sequence of SEQ ID NO: 38, wherein the asparagine residue at position 28 of the native protein has been substituted with glutamic acid.
  • Other exemplary embodiments include glucagon peptides of SEQ ID NOS: 24, 25, 26, 33, 35, 36 and 37.
  • glucagon 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: 1).
  • 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
  • glucagon 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.
  • any of the foregoing compounds can be further improved by attaching 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.
  • the hydrophilic moiety is a polyethylene glycol (PEG) chain or other water soluble polymer that is covalently linked to the side chain of an amino acid residue at one or more of positions 16, 17, 21, 24, 29, 40 of said glucagon peptide, within a C-terminal extension, or at the C-terminal amino acid.
  • the native amino acid at that position is substituted with an amino acid having a side chain suitable for crosslinking with hydrophilic moieties, to facilitate linkage of the hydrophilic moiety to the peptide.
  • exemplary amino acids include Cys, Lys, Orn, homo-Cys, or acetyl phenylalanine (Ac-Phe).
  • an amino acid modified to comprise a hydrophilic group is added to the peptide at the C- terminus.
  • the polyethylene glycol chain in accordance with one embodiment has a molecular weight selected from the range of about 500 to about 40,000 Daltons. In one embodiment 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 10,000 to about 20,000 Daltons. In yet other exemplary embodiments the polyethylene glycol chain has a molecular weight of about 20,000 to about 40,000 Daltons.
  • 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.
  • Acylation or alkylation can increase the half-life of the glucagon peptides in circulation, 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. Acylation or alkylation may also enhance solubility of the peptide at neutral pH. As shown herein, the activity at the glucagon receptor and GLP-1 receptor of the glucagon peptide is maintained if not enhanced after acylation.
  • the glucagon peptide is covalently attached to an acyl or alkyl group via a spacer, e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, or hydrophobic bifunctional spacer.
  • a spacer e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, or hydrophobic bifunctional spacer.
  • the spacer is an amino acid or dipeptide having an amino acid or peptide backbone structure that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length.
  • the total length of the spacer and acyl or alkyl group is 14 to 28 atoms, e.g., 17 to 28, 19 to 26 atoms, 19 to 21 atoms.
  • the acylated or alkylated peptides described herein further comprise a modification which selectively decreases activity at the GLP- 1 receptor, 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., aminobutyric acid (Abu) or He; deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28 (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).
  • a modification which selectively decreases activity at the GLP- 1 receptor 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., aminobutyric acid (Abu) or He; deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28 (e.g.,
  • Glucagon 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.
  • 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, e.g., an amino poly(alkyloxy)carboxylate, or a hydrophobic bifunctional spacer.
  • the spacer is selected from the group consisting of: Trp, Glu, Asp, Cys and a spacer comprising
  • 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 present invention further encompasses pharmaceutically acceptable salts of said glucagon agonists.
  • any of the foregoing compounds can be further modified to alter its pharmaceutical properties by the addition of a second peptide to the carboxy terminus of the glucagon peptide.
  • a glucagon peptide 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: 20, SEQ ID NO: 21 and SEQ ID NO: 22.
  • modifications at position 1 or 2 can increase the peptide's resistance to dipeptidyl peptidase IV (DPP r ) cleavage.
  • 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 e.g. AIB at position 2
  • modifications at position 1 e.g. DMIA at position 1
  • 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.
  • Such peptides are considered herein as a peptide lacking an intramolecular bridge.
  • 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.
  • the invention provides a glucagon peptide with glucagon agonist activity, 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),
  • 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: 39 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 invention provides a glucagon peptide with glucagon agonist activity, 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),
  • 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.
  • 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: 20 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
  • 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 AIB, 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
  • 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.
  • a glucagon peptide comprising a glutamine analog at position 3 may exhibit about 20%, about 50%, about 75%, about 100%, about 200% or about 500% or greater the activity of a corresponding glucagon peptide having the same amino acid sequence as the peptide comprising the glutamine analog, except for the modified amino acid at position 3 (e.g. SEQ ID NO: 69 or SEQ ID NO: 70) at the glucagon receptor.
  • the modified amino acid at position 3 e.g. SEQ ID NO: 69 or SEQ ID NO: 70
  • a glucagon peptide comprising a glutamine analog at position 3 exhibits enhanced activity at the glucagon receptor, but the enhanced activity is no more than 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon or of a corresponding glucagon peptide having the same amino acid sequence as the peptide comprising the glutamine analog, except for the modified amino acid at position 3.
  • 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 C 1-3 alkyl
  • R 3 is C 1-3 alkyl
  • R 4 is H or C 1-3 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 C alkyl.
  • R 4 is H or C 1 alkyl.
  • 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 R2 is CH 3 (acetyldiaminobutanoic acid, Dab(Ac)); R 1 is C 0 alkyl, X is NH, R 2 is NHR 4 , and R 4 is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R 1 is CH 2 -CH 2 , X is NH, and R is CH (acetylornithine, Orn(Ac)).
  • glucagon agonists can comprise the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74.
  • 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.
  • any of the glucagon peptides described herein can be modified without adversely affecting activity at the glucagon receptor, while retaining at least partial glucagon receptor activity.
  • 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.
  • Lys at position 12 is substituted with Arg.
  • amino acids at positions 29 and/or 28, and optionally at position 27, are deleted.
  • any of the glucagon peptides described herein may exhibit an EC50 at the human glucagon receptor of about 100 nM, 75 nM, 50 nM, 40nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM or less when tested for cAMP induction in HEK293 cells over- expressing glucagon receptor, e.g. using the assay of Example 13.
  • pegylated peptides will exhibit a higher EC50 compared to the unpegylated peptide.
  • the glucagon peptides described herein when unpegylated, may exhibit activity at the glucagon receptor which is at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90% at least 95%, at least 98%, at least 99%, 100%, 150%, 200%, 400%, 500% or more) of the activity of native glucagon (SEQ ID NO: 1) at the glucagon receptor.
  • SEQ ID NO: 1 native glucagon
  • the glucagon peptides described herein exhibit the indicated % activity of native glucagon at the glucagon receptor, when lacking a hydrophilic moiety, but exhibit a decreased % activity of native glucagon at the glucagon receptor, when comprising a hydrophilic moiety.
  • the glucagon peptides described herein, when pegylated may exhibit activity at the glucagon receptor which is at least 2% (e.g. at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the activity of native glucagon.
  • the glucagon peptides described herein may exhibit any of the above indicated activities but no more than 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon at the glucagon receptor.
  • 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 acids at positions 16, 20, 21, and 24 are substituted amino acids at positions 16, 20, 21, and 24 with an ⁇ , ⁇ disubstituted amino acid, optionally (c) linked to a hydrophilic moiety such as PEG, e.g., through Cys at position 24, 29 or at the C-terminal amino acid, optionally (d) an amino acid modification at position 27 that substitutes Met with, e.g., Nle, optionally (e) amino acid modifications at positions 20, 21 and 24 that reduce degradation, and optionally (f) linked to SEQ ID NO: 20.
  • the amino acid at position 29 in certain embodiments is Thr or Gly.
  • the glucagon peptide comprises (a) Asp28Glu29, or
  • glu28Glu29 or Glu29Glu30, or Glu28Glu30 or Asp28Glu30, and optionally (b) an amino acid modification at position 16 that substitutes Ser with, e.g. Thr or AIB, and optionally (c) an amino acid modification at position 27 that substitutes Met with, e.g., Nle, and optionally (d) amino acid modifications at positions 20, 21 and 24 that reduce degradation.
  • the glucagon peptide is
  • 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: 20 to 22 are also contemplated.
  • glucagon 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 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 glucagon peptides described herein exhibit the indicated % activity of native glucagon at the glucagon receptor, when lacking a hydrophilic moiety, but exhibit a decreased % activity of native glucagon at the glucagon receptor, when comprising a hydrophilic moiety.
  • the 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 10,000%, 100,000% or 1,000,000% of the activity of native glucagon at the glucagon receptor.
  • 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 glucagon peptide of the invention.
  • 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 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.
  • Exemplary glucagon peptides are selected from the group consisting 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, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 33, wherein amino acid 29 of the glucagon peptide is bound to a second peptide through a peptide bond, and said second peptide comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  • the glucagon peptide is pegylated.
  • the method comprises the step of administering a peptide comprising the sequence of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, wherein a polyethylene chain is covalently linked to amino acid position 21 or at position 24.
  • 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: 21 (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: 21), 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: 21.
  • 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 bar graph representing the stability of Glucagon
  • Fig. 2 represents data generated from HPLC analysis of Glucagon
  • Fig. 3 represents data showing the solubility of glucagon analogs (D28, E29, E30) relative to native glucagon after 60 hours at 25 °C at pH of 2, 4, 5.5, 7 and 8, respectively.
  • Fig. 4 represents data showing the solubility of glucagon analogs (E15D28, D28E29 and D28E30) relative to native glucagon after 24 hours at 25 °C and then 24 hours a 4 °C at pH of 2, 4, 5.5 and 7, respectively.
  • Fig. 5 represents the maximum solubility of glucagon analogs D28, D28E30 and E15,D28 after 24 hours, pH 7 at 4 °C.
  • Fig. 6 represents data showing a glucagon receptor mediated cAMP induction by glucagon analogs (K29 A . K30 T , and K29K30 ⁇ ) relative to native glucagon ⁇ .
  • Fig. 7 represents data showing a glucagon receptor mediated cAMP induction by glucagon analogs (D28 ⁇ , E29 ⁇ , E30 V, K30K31 O and K30, T) relative to native glucagon ⁇ .
  • Fig. 8 represents data showing a glucagon receptor mediated cAMP induction by glucagon analogs (D28 ⁇ , E28 ⁇ and K29, A ) relative to native glucagon ⁇ .
  • Fig. 9 represents data showing a glucagon receptor mediated cAMP induction by glucagon analogs (D28E29 +, D28E30 X , E15D28 * and E29 ⁇ ) relative to native glucagon ⁇ .
  • Fig. 10 represents data showing the change in serum glucose concentrations in beagle dogs after intramuscular administration of glucagon and glucagon analogs.
  • the animals were administered a 0.005 mg/kg dose of either glucagon, a glucagon analog comprising glucagon with the sequence of SEQ ID NO: 31 linked to the carboxy terminus of glucagon (glucagon-CEX) or a glucagon analog comprising an aspartic acid substitution at amino acid 28 (glucagon-Asp28) SEQ ID NO: 11.
  • Figs. 11 A and 1 IB respectively represent data showing glucagon receptor mediated cAMP induction, and GLP- 1 receptor mediated cAMP induction, by a glucagon analog having multiple substitutions: T16,A20,E21,A24,Nle27,D28,E29.
  • Fig. 12 is a graph of the area under the curve of the UV absorbtion at 280 nm of the formulation comprising the peptide of SEQ ID NO: 71 as a function of time (months).
  • Fig. 13 is a graph of the area under the curve of the UV absorbtion at 280 nm of the formulation comprising the peptide of SEQ ID NO: 76 as a function of time (months).
  • Fig. 14 is a graph of the area under the curve of the UV absorbtion at 280 nm of the formulation comprising the peptide of SEQ ID NO: 78 as a function of time (months).
  • Figure 15 represents a graph of the total change in body weight (%) of mice injected with vehicle control, Liraglutide, (CI 6) Glucagon Amide, YE-yE-C16 Glucagon Amide, AA-C16 Glucagon Amide, or ⁇ -06 Glucagon Amide at the indicated dose.
  • Figure 16 represents a graph of the fat mass (g) as measured on Day 7 of the study of mice injected with vehicle control, Liraglutide, (CI 6) Glucagon Amide, ⁇ - yE-C16 Glucagon Amide, AA-C16 Glucagon Amide, or ⁇ - ⁇ Glucagon Amide at the indicated dose.
  • Figure 17 represents a graph of the change in blood glucose (mg/dL) of mice injected with vehicle control, Liraglutide, (CI 6) Glucagon Amide, ⁇ - ⁇ -CI 6 Glucagon Amide, AA-C16 Glucagon Amide, or ⁇ -06 Glucagon Amide at the indicated dose.
  • Figure 18 represents a graph of the concentration of peptide (mg/ml) vs. pH of the buffer in which the peptide was assayed.
  • the black bars represent wildtype glucagon
  • the white bars represent Peptide AA
  • the hatched bars represent Peptide BB.
  • Figure 19 represents a graph of the concentration of peptide (mg/ml) vs. the peptide (Peptide AA or Peptide BB) and the temperature at which the peptide solution was assayed. Black bars represent PBS (pH 7.5), white bars represent PBS (pH 7.0), and hatched bars represent 0.1 AcO (pH 6.5).
  • Figure 20 represents the RP-HPLC spectral data of Peptide AA reconstituted in 50 mM Tris (pH 8.5), PBS (pH 7.4), or 50 mM sodium acetate (pH 6.5), as indicated, and maintained at 37 °C or kept frozen for 4 weeks. The samples were diluted as indicated and then assayed via RP-HPLC.
  • Figure 21 represents the RP-HPLC spectral data of Peptide BB reconstituted in 50 mM Tris (pH 8.5), PBS (pH 7.4), or 50 mM sodium acetate (pH 6.5), as indicated, and maintained at 37 °C or kept frozen for 4 weeks. The samples were diluted as indicated and then assayed via RP-HPLC.
  • Figure 22 represents the RP-HPLC spectral data of Peptide BB reconstituted in either a Tris buffer (pH 8.5) or PBS (pH 7.5).
  • 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).
  • the referenced material e.g., the natural environment if it is naturally occurring.
  • 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.
  • glucagon refers to a peptide consisting of the sequence of SEQ ID NO: 1
  • native GLP-1 is a generic term that designates GLP-1 (7-36)amide (consisting of the sequence of SEQ ID NO: 57), GLP- l(7-37)acid (consisting of the sequence of SEQ ID NO: 58) or a mixture of those two compounds.
  • 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 13.
  • the term "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 13.
  • glucagon agonist analog is 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: 1) 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: I. Small aliphatic, nonpolar or slightly polar residues:
  • 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.
  • 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.
  • native glucagon can be modified by introducing charge at its carboxy terminus to enhance the solubility of the peptide while retaining the agonist properties of the peptide.
  • the enhanced solubility allows for the preparation and storage of glucagon solutions at near neutral pH.
  • Formulating glucagon solutions at relatively neutral pHs e.g. pH of about 6.0 to about 8.0 improves the long term stability of the glucagon peptides.
  • 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 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.
  • 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 peptides of one embodiment of the invention retain glucagon activity and exhibit 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.
  • Any of the glucagon 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.
  • the glucagon peptides of the invention exhibit improved stability, such that at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, more than 95%, up to 100%) of a concentration of the peptide or less than about 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%, 4%, 3%, 2%, 1%, down to 0%) of degraded peptide is detectable at 280 nm by an ultraviolet (UV) detector after about 1 or more weeks (e.g., about 2 weeks, about 4 weeks, about 1 month, about two months, about four months, about six months, about eight months, about ten months, about twelve months) in solution at a temperature of at least 20 °C (e.g., 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, at least 27.5 °C, at least 30 °C, at least 35
  • UV ultraviolet
  • the glucagon peptides may include additional modifications that alter its pharmaceutical properties, e.g. increased potency, prolonged half-life in circulation, increased shelf-life, reduced precipitation or aggregation, and/or reduced degradation, e.g., reduced occurrence of cleavage or chemical modification after storage.
  • a glucagon peptide with improved solubility may be prepared, for example, by introducing one, two, three or more charged amino acid(s) to the C-terminal portion of native glucagon, and in one embodiment at a position C- terminal to position 27.
  • a charged amino acid can be introduced, for example 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.
  • the glucagon 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.
  • glucagon peptide may further increase solubility and/or stability and/or glucagon activity.
  • the glucagon peptide may alternatively comprise other modifications that do not substantially affect solubility or stability, and that do not substantially decrease glucagon activity.
  • the glucagon peptide may comprise a total of 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10, or up to 11, or up to 12, or up to 13, or up to 14 amino acid modifications relative to the native glucagon sequence.
  • such glucagon analogs 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).
  • 1, 2, 3, 4 or 5 non-conservative substitutions are carried out at any of positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 and up to 5 further conservative substitutions are carried out at any of these positions.
  • 1, 2, or 3 amino acid modifications are carried out within amino acids at positions 1-16, and 1, 2 or 3 amino acid modifications are carried out within amino acids at positions 17-26.
  • Exemplary modifications 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;
  • modification of the aspartic acid at position 15 for example, by 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;
  • Thr Ala or AIB, to reduce degradation that occurs through deamidation of Gin (g) modification of Asp at position 21, e.g. by substitution with Glu, to reduce degradation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to iso-aspartate;
  • acylating or alkylating the glucagon peptide as described herein which may increase the activity at the glucagon and GLP-1 receptors, 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
  • 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); or a combination thereof,
  • Exemplary modifications include at least one amino acid modification selected from Group A and one or more amino acid modifications selected from Group B and/or Group C,
  • Group A is:
  • 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;
  • Group B is:
  • DPP- IV dipeptidyl peptidase IV
  • DPP- IV dipeptidyl peptidase IV
  • 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.
  • 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 (SEQ ID NO: 1) at the glucagon receptor.
  • a glucagon peptide comprising a glutamine analog at position 3 may exhibit about 20%, about 50%, about 75%, about 100%, about 200% or about 500% or greater the activity, of a corresponding glucagon peptide having the same amino acid sequence as the peptide comprising the glutamine analog, except for the modified amino acid at position 3 (e.g.
  • a glucagon peptide comprising a glutamine analog at position 3 exhibits enhanced activity at the glucagon receptor, but the enhanced activity is no more than 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon or of a corresponding glucagon peptide having the same amino acid sequence as the peptide comprising the glutamine analog, except for the modified amino acid at position 3.
  • 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 C 1-3 alkyl
  • R 3 is C 1-3 alkyl
  • R 4 is H or C 1-3 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 Q heteroalkyl.
  • R 2 is NHR 4 or C alkyl.
  • R 4 is H or C 1 alkyl.
  • 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 R2 is CH 3 (acetyldiaminobutanoic acid, Dab(Ac)); R 1 is C 0 alkyl, X is NH, R 2 is NHR 4 , and R 4 is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R 1 is CH 2 -CH 2 , X is NH, and R is CH 3 (acetylornithine, Orn(Ac)).
  • glucagon agonists can comprise the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74.
  • 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 glucagon peptide for glucagon receptor vs. 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).
  • the 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 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.
  • DMIA alpha
  • N-methyl histidine alpha-methyl imidiazole acetic acid
  • imidazole acetic acid desaminohistidine
  • hydroxyl-histidine hydroxyl-histidine
  • acetyl- histidine acetyl- histidine and homo-histidine.
  • 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, and aminoisobutyric acid.
  • an intramolecular bridge in the C-terminal portion (amino acids 12-29) of the peptide 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
  • i is an integer from 12 to 25
  • Hydrophilic moieties such as PEG groups can be attached to the glucagon peptides under any suitable conditions used to react a protein with an activated polymer molecule. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, a- haloacetyl, maleimido or hydrazino group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydra
  • Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, 5-pyridyl. and alpha-halogenated acyl group (e.g., alpha-iodo acetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid).
  • the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).
  • an amino acid residue on the glucagon peptide having a thiol is modified with a hydrophilic moiety such as PEG.
  • the thiol is modified with maleimide- activated PEG in a Michael addition reaction to result in a PEGylated peptide comprising the thioether linkage shown below:
  • the thiol is modified with a haloacetyl-activated PEG in a nucleophilic substitution reaction to result in a PEGylated peptide comprising the thioether linkage shown below:
  • Suitable hydrophilic moieties include polyethylene glycol (PEG),
  • polypropylene glycol polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG),
  • POG polyoxyethylated polyols
  • POG polyoxyethylated glycerol
  • polyoxyalkylenes polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol, carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, poly ( ⁇ -amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene
  • the polyethylene glycol chain in accordance with some embodiments has a molecular weight selected from the range of about 500 to about 40,000 Daltons. In one embodiment the polyethylene glycol chain has a molecular weight selected from the range of about 500 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons. In another embodiment the polyethylene glycol chain has a molecular weight of about 10,000 to about 20,000 Daltons. In yet other exemplary embodiments the
  • polyethylene glycol chain has a molecular weight of about 20,000 to about 40,000 Daltons.
  • Dextrans are polysaccharide polymers of glucose subunits, predominantly linked by ccl-6 linkages. Dextran is available in many molecular weight ranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD.
  • Linear or branched polymers are contemplated.
  • Resulting preparations of conjugates may be essentially monodisperse or polydisperse, and may have about 0.5, 0.7, 1, 1.2, 1.5 or 2 polymer moieties per peptide.
  • the glucagon peptide is modified to comprise an acyl group, e.g., an acyl group which is not naturally-occurring on an amino acid (e.g., an acyl group which is non-native to a naturally-occurring amino acid).
  • an acyl group causes the peptide to have one or more of a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved resistance to proteases, such as DPP- IV, and increased potency at the GLP-1 and glucagon receptors.
  • acylation of the glucagon peptide does not lead to decreased activity at the glucagon and GLP-1 receptors.
  • acylation actually increases the activity at the GLP-1 and glucagon receptors. Accordingly, the potency of the acylated analogs is comparable to the unacylated versions of the glucagon co-agonist analogs, if not enhanced.
  • the glucagon peptide is modified to comprise an acyl group which is attached to the glucagon peptide via an ester, thioester, or amide linkage for purposes of prolonging half-life in circulation and/or delaying the onset of and/or extending the duration of action and/or improving resistance to proteases such as DPP-IV.
  • Acylation can be carried out at any position within the glucagon peptide, including any of positions 1-29, a position within a C-terminal extension, or the C- terminal amino acid, provided that glucagon and/or GLP-1 activity is retained, if not enhanced.
  • Nonlimiting examples include positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, or 29.
  • acylation occurs at position 10 of the glucagon peptide and the glucagon peptide lacks an intramolecular bridge, e.g., a covalent intramolecular bridge (e.g., a lactam bridge).
  • acylated peptides lacking an intramolecular bridge demonstrate enhanced activity at the GLP-1 and glucagon receptors as compared to the corresponding non-acylated peptides lacking a covalent intramolecular bridge and in comparison to the corresponding peptides lacking an intramolecular bridge acylated at a position other than position 10.
  • acylation at position 10 can even transform a glucagon analog having little activity at the glucagon receptor to a glucagon analog having activity at both the glucagon and GLP-1 receptors. Accordingly, the position at which acylation occurs can alter the overall activity profile of the glucagon analog.
  • Glucagon peptides may be acylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.
  • Nonlimiting examples include acylation at position 10 and pegylation at one or more positions in the C-terminal portion of the glucagon peptide, e.g., position 24, 28 or 29, within a C- terminal extension, or at the C-terminus (e.g., through adding a C-terminal Cys).
  • the acyl group can be covalently linked directly to an amino acid of the glucagon peptide, or indirectly to an amino acid of the glucagon peptide via a spacer, wherein the spacer is positioned between the amino acid of the glucagon peptide and the acyl group.
  • the glucagon peptide is modified to comprise an acyl group by direct acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the glucagon peptide.
  • the glucagon peptide is directly acylated through the side chain amine, hydroxyl, or thiol of an amino acid.
  • acylation is at position 10, 20, 24, or 29.
  • the acylated glucagon peptide can comprise the amino acid sequence of SEQ ID NO : 1, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the direct acylation of the glucagon peptide occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position 10.
  • the amino acid comprising a side chain amine is an amino acid of Formula I:
  • the amino acid of Formula I is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).
  • the amino acid comprising a side chain hydroxyl is an amino acid of Formula II:
  • the amino acid of Formula II is the amino acid wherein n is 1 (Ser).
  • the amino acid comprising a side chain thiol is an amino acid of Formula III:
  • the amino acid of Formula III is the amino acid wherein n is 1 (Cys).
  • the amino acid comprising a side chain amine, hydroxyl, or thiol is a disubstituted amino acid comprising the same structure of Formula I, Formula II, or Formula III, except that the hydrogen bonded to the alpha carbon of the amino acid of Formula I, Formula II, or Formula III is replaced with a second side chain.
  • the acylated glucagon peptide comprises a spacer between the peptide and the acyl group.
  • the glucagon peptide is covalently bound to the spacer, which is covalently bound to the acyl group.
  • the amino acid to which the spacer is attached can be any amino acid (e.g., a singly a-substituted amino acid or an ⁇ , ⁇ -disubstituted amino acid) comprising a moiety which permits linkage to the spacer.
  • an amino acid comprising a side chain NH 2 , -OH, or -COOH e.g., Lys, Orn, Ser, Asp, or Glu
  • the acylated glucagon peptide can comprise the amino acid sequence of SEQ ID NO: 1, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or carboxylate.
  • the spacer is an amino acid comprising a side chain amine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the acylation can occur through the alpha amine of the amino acid or a side chain amine.
  • the amino acid of the spacer can be any amino acid.
  • the amino acid of the spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5- aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid.
  • the amino acid of the spacer can be an acidic residue, e.g., Asp and Glu.
  • the amino acid of the spacer is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • a side chain amine e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • both the alpha amine and the side chain amine of the amino acid of the spacer to be acylated, such that the glucagon peptide is diacylated.
  • Embodiments of the invention include such diacylated molecules.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula II.
  • the amino acid is Ser.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula III.
  • the amino acid is Cys.
  • the spacer is a hydrophilic bifunctional spacer.
  • the hydrophilic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof.
  • the hydrophilic bifunctional spacer comprises a hydroxyl group and a carboxylate.
  • the hydrophilic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydrophilic bifunctional spacer comprises a thiol group and a carboxylate.
  • the spacer comprises an amino
  • the spacer can comprise, for example, 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, such as, e.g., 8-amino-3,6-dioxaoctanoic acid, which is commercially available from Peptides International, Inc. (Louisville, KY).
  • the spacer is a hydrophobic bifunctional spacer.
  • Hydrophobic bifunctional spacers are known in the art. See, e.g., Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated by reference in its entirety.
  • the hydrophobic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof.
  • the hydrophobic bifunctional spacer comprises a hydroxyl group and a carboxylate.
  • the hydrophobic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydrophobic bifunctional spacer comprises a thiol group and a carboxylate.
  • Suitable hydrophobic bifunctional spacers comprising a carboxylate, and a hydroxyl group or a thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.
  • the bifunctional spacer is not a dicarboxylic acid comprising an unbranched, methylene of 1-7 carbon atoms between the carboxylate groups. In some embodiments, the bifunctional spacer is a dicarboxylic acid comprising an unbranched, methylene of 1-7 carbon atoms between the carboxylate groups.
  • the spacer e.g., amino acid, dipeptide, tripeptide, hydrophilic or hydrophobic bifunctional spacer
  • the spacer in specific embodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or 10 atoms) in length.
  • the spacer is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12 to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acyl group, such that the total length of the spacer and acyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms. In some embodiments, the length of the spacer and acyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.
  • the bifunctional spacer can be a synthetic or naturally occurring amino acid (including, but not limited to, any of those described herein) comprising an amino acid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid, 5 -amino valeric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid).
  • the spacer can be a dipeptide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length.
  • Each amino acid of the dipeptide or tripeptide spacer can be the same as or different from the other amino acid(s) of the dipeptide or tripeptide and can be independently selected from the group consisting of: naturally-occurring and/or non-naturally occurring amino acids, including, for example, any of the D or L isomers of the naturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or L isomers of the non- naturally occurring amino acids selected from the group consisting of: ⁇ -alanine ( ⁇ - Ala), N-a-methyl- alanine (Me-Ala), aminobutyric acid (Abu), ⁇ -aminobutyric acid ( ⁇ - Abu), aminohexanoic acid ( ⁇ -Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid, amino
  • ACHPA 4-amino-3-hydroxy-5-phenylpentanoic acid
  • AHPPA 1,2,3,4,-tetrahydro- isoquinoline-3-carboxylic acid
  • Tic tetrahydropyranglycine
  • Thienylalanine Thi
  • O- benzyl-phosphotyrosine O-Phosphotyrosine
  • methoxytyrosine ethoxytyrosine
  • O- (bis-dimethylamino-phosphono)-tyrosine tyrosine sulfate tetrabutylamine, methyl- valine (MeVal), and alkylated 3-mercaptopropionic acid.
  • the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids.
  • the dipeptide is not any of the dipeptides of general structure A-B, wherein A is selected from the group consisting of Gly, Gin, Ala, Arg, Asp, Asn, lie, Leu, Val, Phe, and Pro, wherein B is selected from the group consisting of Lys, His, Trp.
  • the dipeptide spacer is selected from the group consisting of: Ala- Ala, ⁇ -Ala- ⁇ -Ala, Leu-Leu, Pro-Pro, ⁇ -aminobutyric acid- ⁇ -aminobutyric acid, and ⁇ - Glu- ⁇ -Glu.
  • the glucagon peptide is modified to comprise an acyl group by acylation of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position 10, 20, 24, or 29, or at the C-terminal amino acid of the glucagon peptide.
  • the acyl group is attached to the amino acid at position 10 of the glucagon peptide and the length of the spacer and acyl group is 14 to 28 atoms.
  • the amino acid at position 10 in some aspects, is an amino acid of Formula I, e.g., Lys, or a disubstituted amino acid related to Formula I.
  • the glucagon peptide lacks an intramolecular bridge, e.g., a covalent intramolecular bridge.
  • the glucagon peptide for example, can be a peptide comprising one or more alpha, alpha-disubstituted amino acids, e.g., AIB, for stabilizing the alpha helix of the peptide.
  • AIB alpha, alpha-disubstituted amino acids
  • such peptides comprising an acylated spacer covalently attached to the side chain of the amino acid at position 10 exhibit enhanced potency at both the GLP-1 and glucagon receptors.
  • Suitable methods of peptide acylation via amines, hydroxyls, and thiols are known in the art.
  • the acyl group of the acylated glucagon peptide can be of any size, e.g., any length carbon chain, and can be linear or branched.
  • the acyl group is a C4 to C30 fatty acid.
  • the acyl group can be any of a C4 fatty acid, C6 fatty acid, C8 fatty acid, CIO fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid.
  • the acyl group is a C8 to C20 fatty acid, e.g., a C14 fatty acid or a C16 fatty acid.
  • the acyl group is a bile acid.
  • the bile acid can be any suitable bile acid, including, but not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • the glucagon peptide is modified to comprise an acyl group by acylation of a long chain alkane by the glucagon peptide.
  • the long chain alkane comprises an amine, hydroxyl, or thiol group (e.g. octadecylamine, tetradecanol, and hexadecanethiol) which reacts with a carboxyl group, or activated form thereof, of the glucagon peptide.
  • the carboxyl group, or activated form thereof, of the glucagon peptide can be part of a side chain of an amino acid (e.g., glutamic acid, aspartic acid) of the glucagon peptide or can be part of the peptide backbone.
  • an amino acid e.g., glutamic acid, aspartic acid
  • the glucagon peptide is modified to comprise an acyl group by acylation of the long chain alkane by a spacer which is attached to the glucagon peptide.
  • the long chain alkane comprises an amine, hydroxyl, or thiol group which reacts with a carboxyl group, or activated form thereof, of the spacer.
  • Suitable spacers comprising a carboxyl group, or activated form thereof, are described herein and include, for example, amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers and hydrophobic bifunctional spacers.
  • activated forms of a carboxyl groups may include, but are not limited to, acyl chlorides, anhydrides, and esters.
  • the activated carboxyl group is an ester with a N- hydroxysuccinimide ester (NHS) leaving group.
  • the long chain alkane in which a long chain alkane is acylated by the glucagon peptide or the spacer, the long chain alkane may be of any size and can comprise any length of carbon chain.
  • the long chain alkane can be linear or branched.
  • the long chain alkane is a C4 to C30 alkane.
  • the long chain alkane can be any of a C4 alkane, C6 alkane, C8 alkane, CIO alkane, C12 alkane, C14 alkane, C16 alkane, CI 8 alkane, C20 alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane.
  • the long chain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane, C16 alkane, or a C18 alkane.
  • an amine, hydroxyl, or thiol group of the glucagon peptide is acylated with a cholesterol acid.
  • the glucagon peptide is linked to the cholesterol acid through an alkylated des-amino Cys spacer, i.e., an alkylated 3-mercaptopropionic acid spacer.
  • the acylated glucagon peptides described herein can be further modified to comprise a hydrophilic moiety.
  • the hydrophilic moiety can comprise a polyethylene glycol (PEG) chain.
  • PEG polyethylene glycol
  • the acylated glucagon peptide can comprise SEQ ID NO: 1, including any of the modifications described herein, in which at least one of the amino acids at position 10, 20, 24, and 29 comprise an acyl group and at least one of the amino acids at position 16, 17, 21, 24, or 29, a position within a C-terminal extension, or the C-terminal amino acid are modified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalently bonded to a hydrophilic moiety (e.g., PEG).
  • a hydrophilic moiety e.g., PEG
  • the acyl group is attached to position 10, optionally via a spacer comprising Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cys residue at position 24.
  • the acylated glucagon peptide can comprise a spacer, wherein the spacer is both acylated and modified to comprise the hydrophilic moiety.
  • suitable spacers include a spacer comprising one or more amino acids selected from the group consisting of Cys, Lys, Orn, homo-Cys, and Ac- Phe.
  • the glucagon peptide is modified to comprise an alkyl group, e.g., an alkyl group which is not naturally-occurring on an amino acid (e.g., an alkyl group which is non-native to a naturally-occurring amino acid).
  • an alkyl group e.g., an alkyl group which is not naturally-occurring on an amino acid (e.g., an alkyl group which is non-native to a naturally-occurring amino acid).
  • alkylation of glucagon peptides will achieve similar, if not the same, effects as acylation of the glucagon peptides, e.g., a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved resistance to proteases, such as DPP-IV, and increased potency at the GLP-1 and glucagon receptors.
  • Alkylation can be carried out at any position within the glucagon peptide, including any of positions 1-29, a position within a C-terminal extension, or the C- terminal amino acid, provided that the glucagon activity is retained.
  • Nonlimiting examples include positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, or 29.
  • the alkyl group can be covalently linked directly to an amino acid of the glucagon peptide, or indirectly to an amino acid of the glucagon peptide via a spacer, wherein the spacer is positioned between the amino acid of the glucagon peptide and the alkyl group.
  • Glucagon peptides may be alkylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.
  • Nonlimiting examples include alkylation at position 10 and pegylation at one or more positions in the C-terminal portion of the glucagon peptide, e.g., position 24, 28 or 29, within a C-terminal extension, or at the C-terminus (e.g., through adding a C-terminal Cys).
  • the glucagon peptide is modified to comprise an alkyl group by direct alkylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the glucagon peptide.
  • alkylation is at position 10, 20, 24, or 29.
  • the alkylated glucagon peptide can comprise the amino acid sequence of SEQ ID NO : 1, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the direct alkylation of the glucagon peptide occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position 10.
  • the amino acid comprising a side chain amine is an amino acid of Formula I.
  • the amino acid of Formula I is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).
  • the amino acid comprising a side chain hydroxyl is an amino acid of Formula II.
  • the amino acid of Formula II is the amino acid wherein n is 1 (Ser).
  • the amino acid comprising a side chain thiol is an amino acid of Formula III.
  • the amino acid of Formula III is the amino acid wherein n is 1 (Cys).
  • the amino acid comprising a side chain amine, hydroxyl, or thiol is a disubstituted amino acid comprising the same structure of Formula I, Formula II, or Formula III, except that the hydrogen bonded to the alpha carbon of the amino acid of Formula I, Formula II, or Formula III is replaced with a second side chain.
  • the alkylated glucagon peptide comprises a spacer between the peptide and the alkyl group.
  • the glucagon peptide is covalently bound to the spacer, which is covalently bound to the alkyl group.
  • the glucagon peptide is modified to comprise an alkyl group by alkylation of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position 10, 20, 24, or 29 of the glucagon peptide.
  • the amino acid to which the spacer is attached can be any amino acid (e.g., a singly a-substituted amino acid or an ⁇ , ⁇ -disubstituted amino acid) comprising a moiety which permits linkage to the spacer.
  • an amino acid comprising a side chain NH 2 , -OH, or -COOH e.g., Lys, Orn, Ser, Asp, or Glu
  • the alkylated glucagon peptide can comprise the amino acid sequence of SEQ ID NO: 1, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or carboxylate.
  • the spacer is an amino acid comprising a side chain amine, hydroxyl, or thiol or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the alkylation can occur through the alpha amine of an amino acid or a side chain amine.
  • the amino acid of the spacer can be any amino acid.
  • the amino acid of the spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5- aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid.
  • the amino acid of the spacer can be an acidic residue, e.g., Asp and Glu, provided that the alkylation occurs on the alpha amine of the acidic residue.
  • the amino acid of the spacer is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • Embodiments of the invention include such dialkylated molecules.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula II.
  • the amino acid is Ser.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula III.
  • the amino acid is Cys.
  • the spacer is a hydrophilic bifunctional spacer.
  • the hydrophilic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof.
  • the hydrophilic bifunctional spacer comprises a hydroxyl group and a carboxylate.
  • the hydrophilic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydrophilic bifunctional spacer comprises a thiol group and a carboxylate.
  • the spacer comprises an amino
  • the spacer can comprise, for example, 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, such as, e.g., 8-amino-3,6-dioxaoctanoic acid, which is commercially available from Peptides International, Inc. (Louisville, KY).
  • the spacer is a hydrophobic bifunctional spacer.
  • the hydrophobic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof.
  • the hydrophobic bifunctional spacer comprises a hydroxyl group and a carboxylate. In other embodiments, the
  • hydrophobic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydrophobic bifunctional spacer comprises a thiol group and a carboxylate.
  • Suitable hydrophobic bifunctional spacers comprising a carboxylate, and a hydroxyl group or a thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.
  • the spacer e.g., amino acid, dipeptide, tripeptide, hydrophilic or hydrophobic bifunctional spacer
  • the spacer in specific embodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or 10 atoms)) in length.
  • the spacer is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C12 to C18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, such that the total length of the spacer and alkyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms.
  • the length of the spacer and alkyl is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.
  • the bifunctional spacer can be a synthetic or non-naturally occurring amino acid comprising an amino acid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid).
  • the spacer can be a dipeptide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length.
  • the dipeptide or tripeptide spacer can be composed of naturally-occurring and/or non-naturally occurring amino acids, including, for example, any of the amino acids taught herein.
  • the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids.
  • the dipeptide spacer is selected from the group consisting of: Ala- Ala, ⁇ -Ala- ⁇ -Ala, Leu-Leu, Pro-Pro, ⁇ -aminobutyric acid- ⁇ - aminobutyric acid, and ⁇ -Glu- ⁇ -Glu.
  • Suitable methods of peptide alkylation via amines, hydroxyls, and thiols are known in the art.
  • a Williamson ether synthesis can be used to form an ether linkage between a hydroxyl group of the glucagon peptide and the alkyl group.
  • a nucleophilic substitution reaction of the peptide with an alkyl halide can result in any of an ether, thioether, or amino linkage.
  • the alkyl group of the alkylated glucagon peptide can be of any size, e.g., any length carbon chain, and can be linear or branched.
  • the alkyl group is a C4 to C30 alkyl.
  • the alkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl, CIO alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl.
  • the alkyl group is a C8 to C20 alkyl, e.g., a C14 alkyl or a C16 alkyl.
  • the alkyl group comprises a steroid moiety of a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • a bile acid e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • the glucagon peptide is modified to comprise an alkyl group by reacting a nucleophilic, long chain alkane with the glucagon peptide, wherein the glucagon peptide comprises a leaving group suitable for nucleophilic substitution.
  • the nucleophilic group of the long chain alkane comprises an amine, hydroxyl, or thiol group (e.g. octadecylamine, tetradecanol, and hexadecanethiol).
  • the leaving group of the glucagon peptide can be part of a side chain of an amino acid or can be part of the peptide backbone.
  • Suitable leaving groups include, for example, N-hydroxysuccinimide, halogens, and sulfonate esters.
  • the glucagon peptide is modified to comprise an alkyl group by reacting the nucleophilic, long chain alkane with a spacer which is attached to the glucagon peptide, wherein the spacer comprises the leaving group.
  • the long chain alkane comprises an amine, hydroxyl, or thiol group.
  • the spacer comprising the leaving group can be any spacer discussed herein, e.g., amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers and hydrophobic bifunctional spacers further comprising a suitable leaving group.
  • the long chain alkane in which a long chain alkane is alkylated by the glucagon peptide or the spacer, the long chain alkane may be of any size and can comprise any length of carbon chain.
  • the long chain alkane can be linear or branched.
  • the long chain alkane is a C4 to C30 alkane.
  • the long chain alkane can be any of a C4 alkane, C6 alkane, C8 alkane, CIO alkane, C12 alkane, C14 alkane, C16 alkane, CI 8 alkane, C20 alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane.
  • the long chain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane, C16 alkane, or a C18 alkane.
  • alkylation can occur between the glucagon peptide and a cholesterol moiety.
  • the hydroxyl group of cholesterol can displace a leaving group on the long chain alkane to form a cholesterol-glucagon peptide product.
  • the alkylated glucagon peptides described herein can be further modified to comprise a hydrophilic moiety.
  • the hydrophilic moiety can comprise a polyethylene glycol (PEG) chain.
  • PEG polyethylene glycol
  • the alkylated glucagon peptide can comprise SEQ ID NO: 1 or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, in which at least one of the amino acids at position 10, 20, 24, and 29 comprise an alkyl group and at least one of the amino acids at position 16, 17, 21, 24, and 29, a position within a C-terminal extension or the C-terminal amino acid are modified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalently bonded to a hydrophilic moiety (e.g., PEG).
  • a hydrophilic moiety e.g., PEG
  • the alkyl group is attached to position 10, optionally via a spacer comprising Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cys residue at position 24.
  • the alkylated glucagon peptide can comprise a spacer, wherein the spacer is both alkylated and modified to comprise the hydrophilic moiety.
  • suitable spacers include a spacer comprising one or more amino acids selected from the group consisting of Cys, Lys, Orn, homo-Cys, and Ac- Phe.
  • the glucagon peptide, or analog thereof comprises an amino acid modification which selectively reduces GLP-1 activity.
  • the acylated or alkylated glucagon peptide, or analog thereof comprises a C-terminal alpha carboxylate group; a substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He; deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28 (e.g., deletion of the amino acid at position 28, deletion of the amino acid at positions 28 and 29) to yield a peptide 27 or 28 amino acids in length, or a combination thereof.
  • the present disclosure also encompasses other conjugates in which glucagon peptides of the invention are linked, optionally via covalent bonding and optionally via a linker, to a conjugate moiety.
  • Linkage can be accomplished by covalent chemical bonds, physical forces such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.
  • a variety of non-covalent coupling systems may be used, including biotin-avidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.
  • the peptide can be linked to conjugate moieties via direct covalent linkage by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these targeted amino acids.
  • Reactive groups on the peptide or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group.
  • Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
  • the conjugate moieties can be linked to the peptide indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers.
  • polysaccharide carriers include aminodextran.
  • suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier.
  • Cysteinyl residues are most commonly are reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid, chloroacetamide to give
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo- -(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1 ,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O- methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon- amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4- azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or
  • conjugate moieties that can be linked to any of the glucagon peptides described herein include but are not limited to a heterologous peptide or polypeptide (including for example, a plasma protein), a targeting agent, an immunoglobulin or portion thereof (e.g. variable region, CDR, or Fc region), a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents.
  • a conjugate comprising a glucagon peptide of the present invention and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferin, fibrinogen and globulins.
  • the plasma protein moiety of the conjugate is albumin or transferin.
  • the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate.
  • the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell.
  • the length of the linker is long enough to reduce the potential for steric hindrance.
  • the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide
  • the entire conjugate can be a fusion protein.
  • peptidyl linkers may be any length. Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.
  • Such fusion proteins may alternatively be produced by recombinant genetic engineering methods known to one of ordinary skill in the art.
  • the glucagon peptides are conjugated, e.g., fused to an immunoglobulin or portion thereof (e.g. variable region, CDR, or Fc region).
  • immunoglobulins e.g. variable region, CDR, or Fc region.
  • immunoglobulins include IgG, IgA, IgE, IgD or IgM.
  • the Fc region is a C-terminal region of an Ig heavy chain, which is responsible for binding to Fc receptors that carry out activities such as recycling (which results in prolonged half-life), antibody dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC).
  • the human IgG heavy chain Fc region stretches from Cys226 to the C-terminus of the heavy chain.
  • the "hinge region” generally extends from Glu216 to Pro230 of human IgGl (hinge regions of other IgG isotypes may be aligned with the IgGl sequence by aligning the cysteines involved in cysteine bonding).
  • the Fc region of an IgG includes two constant domains, CH2 and CH3.
  • the CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341.
  • the CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447.
  • the Fc region may comprise one or more native or modified constant regions from an
  • immunoglobulin heavy chain other than CHI, for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE.
  • Suitable conjugate moieties include portions of immunoglobulin sequence that include the FcRn binding site.
  • FcRn a salvage receptor, is responsible for recycling immunoglobulins and returning them to circulation in blood.
  • the region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379).
  • the major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains.
  • Fc-FcRn contacts are all within a single Ig heavy chain.
  • the major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH 2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain.
  • FcyR are responsible for ADCC and CDC.
  • positions within the Fc region that make a direct contact with FcyR are amino acids 234-239 (lower hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino acids 327-332 (F/G) loop (Sondermann et al., Nature 406: 267-273, 2000).
  • the lower hinge region of IgE has also been implicated in the FcRI binding (Henry, et al., Biochemistry 36, 15568-15578, 1997).
  • Such variant Fc regions comprise at least one amino acid modification in the CH3 domain of the Fc region (residues 342-447) and/or at least one amino acid modification in the CH2 domain of the Fc region (residues 231-341).
  • Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
  • Other mutations may reduce binding of the Fc region to FcyRI, FcyRIIA, FcyRIIB, and/or FcyRIIIA without significantly reducing affinity for FcRn.
  • substitution of the Asn at position 297 of the Fc region with Ala or another amino acid removes a highly conserved N-glycosylation site and may result in reduced immunogenicity with concomitant prolonged half-life of the Fc region, as well as reduced binding to FcyRs (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68: 1632; Shields et al. 1995, J. Biol. Chem. 276:6591).
  • Amino acid modifications at positions 233-236 of IgGl have been made that reduce binding to FcyRs (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613).
  • Some exemplary amino acid substitutions are described in US Patents 7,355,008 and 7,381,408, each incorporated by reference herein in its entirety.
  • the present disclosure also encompasses glucagon fusion peptides or proteins wherein a second peptide or polypeptide has been fused to a terminus, e.g., the carboxy terminus of the glucagon peptide.
  • exemplary candidates for C-terminal fusion include SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to amino acid 29 of the glucagon peptide.
  • 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, 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 intramolecular bridge is formed between two amino acid side chains to stabilize the three dimensional structure of the carboxy terminal portion (e.g., amino acids 12-29) of the glucagon peptide.
  • 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 peptide may be considered herein as comprising a covlent intramolecular bridge.
  • the peptide may be considered herein as comprising a non-covalent intramolecular bridge.
  • the size of the linker is about 8 atoms, or about 7-9 atoms.
  • the intramolecular bridge is formed between two amino acids that are two amino acids apart, e.g., amino acids at positions j and j+3, wherein j is any integer between 12 and 26 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In some specific embodiments, j is 17.
  • the size of the linker is about 6 atoms, or about 5 to 7 atoms.
  • the intramolecular bridge is formed between two amino acids that are 6 amino acids apart, e.g., amino acids at positions k and k+7, wherein k is any integer between 12 and 22 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). In some specific embodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.
  • amino acid pairings that are capable of covalently bonding to form a seven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp (lactam); or Homo ser-Homo glu (lactone).
  • amino acid pairings that may form an eight- atom linker include Lys-Glu (lactam); Homolys-Asp (lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp (lactone).
  • amino acid pairings that may form a nine-atom linker include Homolys-Glu (lactam); Lys-
  • the size of a lactam ring can vary depending on the length of the amino acid side chains, and in one embodiment the lactam is formed by linking the side chains of a lysine amino acid to a glutamic acid side chain.
  • Further exemplary embodiments include the following pairings, optionally with a lactam bridge: Glu at position 12 with Lys at position 16; native Lys at position 12 with Glu at position 16; Glu at position 16 with Lys at position 20; Lys at position 16 with Glu at position 20; Glu at position 20 with Lys at position 24; Lys at position 20 with Glu at position 24; Glu at position 24 with Lys at position 28; Lys at position 24 with Glu at position 28.
  • a lactam ring can be formed between the side chains of a Lysl2 and a Glul6 or alternatively between a Glu 12 and a Lysl6).
  • Intramolecular bridges other than a lactam bridge can be used to stabilize the alpha helix of the glucagon analog peptides.
  • the intramolecular bridge is a hydrophobic bridge.
  • the intramolecular bridge optionally is between the side chains of two amino acids that are part of the hydrophobic face of the alpha helix of the glucagon analog peptide.
  • one of the amino acids joined by the hydrophobic bridge can be the amino acid at position 10, 14, and 18.
  • olefin metathesis is used to cross-link one or two turns of the alpha helix of the glucagon peptide using an all-hydrocarbon cross-linking system.
  • the glucagon peptide in this instance can comprise a-methylated amino acids bearing olefinic side chains of varying length and configured with either R or S stereochemistry at the i and i+4 or i+7 positions.
  • the olefinic side can can comprise (CH 2 )n, wherein n is any integer between 1 to 6. In one embodiment, n is 3 for a cross-link length of 8 atoms. Suitable methods of forming such
  • the glucagon peptide can comprise O-allyl Ser residues located on adjacent helical turns, which are bridged together via ruthenium-catalyzed ring closing metathesis.
  • Such procedures of cross-linking are described in, for example, Blackwell et al, Angew, Chem., Int. Ed. 37: 3281-3284 (1998).
  • lanthionine which has been widely adopted as a peptidomimetic of cystine, is used to cross-link one turn of the alpha helix.
  • Suitable methods of lanthionine-based cyclization are known in the art. See, for instance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004); Mayer et al., J. Peptide Res. 51: 432-436 (1998);
  • a, ⁇ -diaminoalkane tethers e.g., 1,4-diaminopropane and 1,5-diaminopentane
  • tethers lead to the formation of a bridge 9-atoms or more in length, depending on the length of the diaminoalkane tether. Suitable methods of producing peptides cross-linked with such tethers are described in the art. See, for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460 (1997).
  • a disulfide bridge is used to crosslink one or two turns of the alpha helix of the glucagon peptide.
  • a modified disulfide bridge in which one or both sulfur atoms are replaced by a methylene group resulting in an isosteric macrocyclization is used to stabilize the alpha helix of the glucagon peptide.
  • Suitable methods of modifying peptides with disulfide bridges or sulfur-based cyclization are described in, for example, Jackson et al., /. Am. Chem. Soc. 113: 9391-9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).
  • the alpha helix of the glucagon peptide is stabilized via the binding of metal atom by two His residues or a His and Cys pair positioned at i and i+4.
  • the metal atom can be, for example, Ru(III), Cu(II), Zn(II), or Cd(II).
  • Such methods of metal binding-based alpha helix stabilization are known in the art. See, for example, Andrews and Tabor, Tetrahedron 55: 11711-11743
  • the alpha helix of the glucagon peptide can alternatively be stabilized through other means of peptide cyclizing, which means are reviewed in Davies, /. Peptide. Sci. 9: 471-501 (2003).
  • the alpha helix can be stabilized via the formation of an amide bridge, thioether bridge, thioester bridge, urea bridge, carbamate bridge, sulfonamide bridge, and the like.
  • a thioester bridge can be formed between the C-terminus and the side chain of a Cys residue.
  • a thioester can be formed via side chains of amino acids having a thiol (Cys) and a carboxylic acid (e.g., Asp, Glu).
  • a cross-linking agent such as a dicarboxylic acid, e.g. suberic acid (octanedioic acid), etc. can introduce a link between two functional groups of an amino acid side chain, such as a free amino, hydroxyl, thiol group, and combinations thereof.
  • the alpha helix of the glucagon peptide is stabilized through the incorporation of hydrophobic amino acids at positions i and i+4.
  • i can be Tyr and i+4 can be either Val or Leu;
  • i can be Phe and i+4 can be Cys or Met;
  • I can be Cys and i+4 can be Met; or
  • i can be Phe and i+4 can be He.
  • the above amino acid pairings can be reversed, such that the indicated amino acid at position i could alternatively be located at i+4, while the i+4 amino acid can be located at the i position.
  • the alpha helix is stabilized through incorporation (either by amino acid substitution or insertion) of one or more alpha helix- stabilizing amino acids at the C-terminal portion of the glucagon peptide (around amino acids 12-29).
  • the alpha helix- stabilizing amino acid is an a, a-disubstitued amino acid, including, but not limited to any of 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).
  • AIB amino iso-butyric acid
  • 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).
  • one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of the glucagon peptide is substituted with an a, a-disubstituted amino acid.
  • one, two, three or all of positions 16, 20, 21, and 24 are substituted with AIB.
  • the glucagon peptide can comprise a substitution of position 16 with AIB in the absence of an intramolecular bridge, e.g., a non-covalent
  • intramolecular bridge e.g., a salt bridge
  • covalent intramolecular bridge e.g., a lactam
  • the glucagon peptide lacking an intramolecular bridge comprises one or more substitutions within amino acid positions 12-29 with an ⁇ , ⁇ -disubstituted amino acid and an acyl or alkyl group covalently attached to the side chain of an amino acid of the glucagon peptide, e.g., the amino acid at position 10 of the glucagon peptide.
  • the acyl or alkyl group is non-native to a naturally occurring amino acid.
  • the acyl or alkyl group is non-native to the amino acid at position 10.
  • Such acylated or alkylated glucagon peptides lacking an intramolecular bridge exhibit enhanced activity at the GLP-1 and glucagon receptors as compared to the non- acylated counterpart peptides. Further enhancement in activity at the GLP-1 and glucagon receptors can be achieved by the acylated glucagon peptides lacking an intramolecular bridge by incorporating a spacer between the acyl or alkyl group and the side chain of the amino acid at position 10 of the peptide. Acylation and alkylation, with or without incorporating spacers, are further described herein.
  • the acylated or alkylated glucagon peptide, or analog thereof further comprises a modification which selectively reduces activity at the GLP-1 receptor.
  • the acylated or alkylated glucagon peptide, or analog thereof comprises one or a combination of: a C-terminal alpha carboxylate, a deletion of the amino acids C-terminal to the amino acid at position 27 or 28 (e.g., deletion of the amino acid at position 29, deletion of the amino acids at positions 28 and 29), a substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or He.
  • glucagon analogs each comprising a modified amino acid sequence of native human glucagon (which native sequence is set forth herein as SEQ ID NO: 1).
  • the glucagon analog is one which exhibits increased solubility in an aqueous solution at a pH of about 6 to about 7.5 relative to other glucagon analogs, and relative to native glucagon.
  • the glucagon analog exhibits increased solubility in an aqueous solution at pH of about 6.5 to about 7.5, or about 6, about 7, or about 7.5.
  • the glucagon analog exhibits a maximum solubility in an aqueous solution at a pH of about 6 to about 8.5 which is at least or about 1.5-fold (e.g., at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 50-fold, at least or about 100-fold better) than the maximum solubility of native glucagon in an aqueous solution at a physiological pH.
  • Methods of testing maximum solubility are known in the art and described herein at Example 30.
  • the glucagon analog is one which exhibits increased stability (i.e. reduced degradation) in an aqueous solution at a pH of about 6 to about 7.5 compared to native glucagon.
  • the stability of the glucagon analog in an aqueous solution at physiological pH is at least or about 1.5-fold (e.g., at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 10-fold, at least or about 20-fold, at least or about 50-fold, at least or about 100-fold better) than the stability of native glucagon in an aqueous solution at a physiological pH.
  • Methods of testing stability of peptides are known in the art and include the method described herein at Example 31.
  • the use of the stabilized glucagon analogs disclosed herein allow for the preparation and storage of glucagon agonist solutions at physiological pH for long periods of time.
  • the glucagon analog may retain at least 95% of the original peptide after 24 hours at 25 °C.
  • the glucagon analogs of the invention exhibit improved stability, such that at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, more than 95%, up to 100%) of a concentration of the analog or less than about 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%, 4%, 3%, 2%, 1%, down to 0%) of degraded peptide is detectable at 280 nm by an ultraviolet (UV) detector after 1 or more weeks (e.g., 2 weeks, 4 weeks, 1 month, two months, four months, six months, eight months, ten months, twelve months) in solution at ambient room temperature (e.g., about 20 to about 25 °C) or refrigerated temperature (e.g., about 5 °C), or higher temperatures intended to accelerate degradation (e.g. about UV) detector after 1
  • the glucagon analogs exhibit superior solubility and/or stability in aqueous solutions at physiological pH, yet demonstrate activity at the glucagon receptor which is comparable if not better than the activity of wildtype glucagon at the glucagon receptor.
  • the activity of the glucagon analog at the glucagon receptor relative to the activity of native glucagon at the glucagon receptor is not substantially reduced.
  • the activity of the glucagon analog at the glucagon receptor is greater than or about 50%, greater than or about 55%, greater than or about 60%, greater than or about 65%, greater than or about 70%, greater than or about 75%, greater than or about 80%, greater than or about 85%, greater than or about 90%, greater than or about 91%, greater than or about 92%, greater than or about 93%, greater than or about 94%, greater than or about 95%, greater than or about 96%, greater than or about 97%, greater than or about 98%, or greater than or about 99% of the activity of native glucagon at the glucagon receptor.
  • the activity of the glucagon analog at the glucagon receptor is substantially the same as or greater than the activity of native glucagon at the glucagon receptor.
  • the glucagon analog exhibits activity at the glucagon receptor which is about 100% or more, about 110% or more, about 120% or more about 130% or more, about 140% or more, abour 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, about 200% or more, about 250% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, about 1000% or more of the activity of native glucagon at the glucagon receptor.
  • the glucagon analog of any of the examples of embodiments described herein exhibits at least about 20% of the activity of native glucagon at the glucagon receptor when the glucagon analog lacks a hydrophilic moiety.
  • the glucagon analog does not activate the GLP-1 receptor to any appreciable degree.
  • the glucagon agonist is a peptide which exhibits about 10% or less (e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less) of the activity of native GLP-1 at the GLP-1 receptor.
  • the glucagon analog exhibits less than 10% of the activity of native GLP-1 at the GLP-1 receptor when the glucagon analog thereof lacks a hydrophilic moiety.
  • the glucagon analog exhibits no more than about 0.5% of the activity of native GLP-1 at the GLP- 1 receptor, when the glucagon analog thereof lacks a hydrophilic moiety.
  • the glucagon agonist when the glucagon agonist is conjugated to a heterologous moiety (e.g., a hydrophilic moiety), as further described herein, the glucagon agonist exhibits a decreased activity (e.g., a lower potency or higher EC50) than when the glucagon agonist is in a free or unconjugated form. In some aspects, when the glucagon agonist is free or unconjugated, the glucagon agonist exhibits a potency at the glucagon receptor that is about 10-fold or greater than the potency of the glucagon agonist when the glucagon agonist is conjugated to a heterologous moiety (e.g., a hydrophilic moiety).
  • a heterologous moiety e.g., a hydrophilic moiety
  • the glucagon agonist when unconjugated, exhibits a potency at the glucagon receptor that is about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40- fold, about 45-fold, about 50-fold, about 100-fold or more higher than the potency of the glucagon agonist when conjugated to a heterologous moiety.
  • the glucagon analog comprises a modified amino acid sequence of native human glucagon (SEQ ID NO: 1) with a stabilized peptide bond between amino acids 15 and 16, an amino acid substitution of the Gin at position 3 with another amino acid which does not substantially reduce the peptide's ability to activate the glucagon receptor, an amino acid substitution of the Arg at position 17 with a negative charged amino acid, optionally, at least one charged amino acid C- terminal to the amino acid at position 27, and optionally, up to 10 (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1) additional amino acid modifications (relative to SEQ ID NO: 1).
  • SEQ ID NO: 1 modified amino acid sequence of native human glucagon
  • the glucagon analog comprises a modified amino acid sequence of native human glucagon (SEQ ID NO: 1) with (a) a substitution of the amino acid at position 16 with an ⁇ , ⁇ -disubstituted amino acid; (b) a substitution of the Gin at position 3 of native human glucagon (SEQ ID NO: 1) with a glutamine analog; (c) an amino acid substitution of Arg at position 17 with a negative-charged amino acid; (d) optionally, at least one charged amino acid C-terminal to the amino acid at position 27 of the glucagon peptide; and (e) optionally, up to 10 additional amino acid modifications.
  • the negative-charged amino acid may be any amino acid that comprises a side chain that is negative-charged (i.e., de-protonated) in aqueous solution at physiological pH.
  • the negative-charged amino acid in some aspects is a naturally-occurring amino acid, including any of the negative-charged amino acids among the 20 amino acids commonly found in human proteins (Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr).
  • the negative-charged amino acid may be one which is considered a coded amino acid.
  • Coded refers to an amino acid that is an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp,
  • the negative-charged amino acid is atypical, synthetic, non-coded, or a non-naturally occurring amino acid.
  • 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.
  • Non-coded refers to an amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr.
  • the negative-charged amino acid which replaces the Gin at position 17 (according to the numbering of SEQ ID NO: 1) of the glucagon analog is selected from the group consisting of: a glutamic acid, a sulfonic acid derivative of Cys, homoglutamic acid, ⁇ -homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of:
  • OOH wherein X5 is Ci-C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl.
  • the glucagon analog comprises a modified amino acid sequence of native human glucagon (SEQ ID NO: 1) with a stabilized peptide bond between amino acids 15 and 16. Accordingly, in some embodiments, the glucagon analog comprises an ⁇ , ⁇ -disubstituted amino acid at position 16.
  • the ⁇ , ⁇ -disubstituted amino acid comprises R 1 and R 2 , each of which is bonded to the alpha carbon, wherein each of R 1 and R 2 is independently selected from the group consisting of C1-C4 alkyl, optionally substituted with a hydroxyl, amide, thiol, halo, or R 1 and R 2 together with the alpha carbon to which they are attached form a ring.
  • the ⁇ , ⁇ -disubstituted amino acid is alpha, amino isobutyric acid (AIB).
  • the ⁇ , ⁇ -disubstituted amino acid is alpha-methyl serine.
  • the glucagon analogs described herein comprise an amino acid substitution of the Gin at position 3 with another amino acid which does not substantially reduce the activity of the peptide at the glucagon receptor.
  • the glucagon analog comprises at position 3 a glutamine analog, including, but not limited to a glutamine analog which comprises 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 C 1-3 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.
  • the glutamine analog comprises a side chain of: (i) Structure I and R is CH 2 - S, X is NH, and R 2 is CH 3 (C(Acm); (ii) Structure I and R 1 is CH 2 , X is NH, R 2 is CH 3 (Dab(Ac)), (iii) Structure I and Rl is CO alkyl, X is NH, R2 is NHR4, and R4 is H (Dap(urea)); (iv) Structure II and Rl is CH2, Y is NHR4, and R4 is CH3 9 (Q(Me); (v) Structure III and Rl is CH2 and R4 of Structure III is H (M(O)); or (vi) Structure I and R 1 is CH 2 -CH 2 , X is NH, and R 2 is CH 3 (Orn(Ac)).
  • the glucagon analog (e.g., any of the glucagon analogs described herein as Examples of Embodiments) comprises at least one charged amino acid C-terminal to the amino acid at position 27 of the glucagon peptide.
  • the analog comprises a modified amino acid sequence based on SEQ ID NO: 1 in which the sequence comprises 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.
  • 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 glucagon analog comprises (a) a substitution of Asn at position 28 with a charged amino acid; (b) a substitution of Thr at position 29 with a charged amino acid; (c) an addition of 1-3 charged amino acids after position 29; and (d) a combination thereof.
  • the glucagon analog comprises a charged amino acid C-terminal to position 27, wherein the negative- charged amino acid is selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid.
  • the glucagon analog comprises Asn, Asp, or Glu at position 28 and/or Asp, Glu, or Lys at position 29.
  • the glucagon analog comprises an addition of one, two or three amino acids after position 29, e.g. a charged amino acid such as a negatively charged amino acid.
  • the two or three amino acids comprise Gly amino acids and/or Lys amino acids.
  • Gly-Lys or Lys-Lys is added to the glucagon analog after position 29.
  • the glucagon analog comprises at least one and up to 10 additional amino acid modification(s) as compared to SEQ ID NO: 1 and in addition to those described above under “Examples of Embodiments.” In some aspsects, the glucagon analog comprises at least one amino acid modification selected from the group consisting of:
  • the glucagon analog further comprises (i) a 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), (ii) 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), or (iii) both (i) and (ii).
  • DPP-IV dipeptidyl peptidase IV
  • the His at position 1 is substituted with 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 Ser at position 2 is substituted with D-Ser, D-Ala, Gly, n-methyl-Ser, Val, or alpha, amino isobutyric acid (AIB).
  • the glucagon analog further comprises an amino acid modification selected from the group consisting of: substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, optionally, He or aminobutyric acid (Abu); deletion of 1 or 2 amino acids C-terminal to the amino acid at position 27; a C-terminal alpha carboxylate; and a combination thereof.
  • an amino acid modification selected from the group consisting of: substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, optionally, He or aminobutyric acid (Abu); deletion of 1 or 2 amino acids C-terminal to the amino acid at position 27; a C-terminal alpha carboxylate; and a combination thereof.
  • the glucagon analog further comprises an amino acid modification selected from the group consisting of: substitution of Tyr at position 10 with Phe or Val;
  • amino acid sequence of SEQ ID NO: 20 to the C-terminus, wherein the amino acid at position 29 is Thr or Gly;
  • the glucagon analog comprises an amino acid comprising a side chain covalently attached to an acyl group or an alkyl group, which acyl group or alkyl group is non-native to a naturally-occurring amino acid.
  • Acylation and alkylation of glucagon peptides are described further herein under the sections entitled "Acylation” and "Alkylation” respectively.
  • the glucagon analog comprises an acylated or alkylated amino acid, wherein the amino acid to which the acyl group or alkyl group is attached is the amino acid at position 10 of the analog or the C-terminal amino acid.
  • the acyl group or alkyl group is attached to the side chain of the amino acid through a spacer.
  • the acyl group is a C 4 to C30 fatty acyl group or the alkyl group is a C 4 to C 30 alkyl.
  • the glucagon analog further comprises a hydrophilic moiety.
  • Hydrophilic moieties and their attachment to a glucagon analog or peptide is further described herein under the section entitled "Addition of Hydwphillic
  • the hydrophilic moiety is covalently linked to any of amino acid positions 16, 17, 20, 21, 24, or 29, or at the C-terminal amino acid of the analog. In some aspects, the hydrophilic moiety is covalently linked to Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine. In some embodiments, the hydrophilic moiety is a polyethylene glycol (PEG), although the hydrophilic moiety is limited to this one type of moiety. In some aspects, the PEG has a molecular weight of about 1,000 Daltons to about 40,000 Daltons. In further aspects, the PEG has a molecular weight of about 20,000 Daltons to about 40,000 Daltons.
  • PEG polyethylene glycol
  • the glucagon analog in some aspects, comprises the amino acid sequence of any of SEQ ID NOS: 117-120, 122-125, and 127-130, optionally modified to include an amino acid linked to a hydrophilic moiety or an amino acid comprising a non-native acyl or alkyl group, and/or optionally modified at positions 1 and/or 2 for reduced susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV).
  • DPP-IV dipeptidyl peptidase IV
  • the glucagon analog in some aspects, comprises the amino acid sequence of any of SEQ ID NOS: 117-120, 122-125, and 127-130, optionally, with one, two, three, four, five, six, seven, eight, nine, or ten conservative amino acid substitutions.
  • the present invention also provides a dimer comprising two peptides bound via a linker, wherein at least one of the two peptides is a glucagon analog as described herein. More specifically, in some embodiments, the dimer comprises two peptides bound via a linker, wherein at least one of the two peptides is any of the above-described glucagon analogs.
  • the glucagon analog present as part of the dimer may be further modified as described herein.
  • the dimer is a homodimer.
  • the dimer comprises a linker and the linker is selected from the group consisting of a bifunctional thiol crosslinker and a bio-functional amine crosslinker.
  • the present invention moreover provides a conjugate comprising a conjugate moiety and a glucagon analog as described herein, a dimer as described herein, or a combination thereof. Further provided is a fusion peptide comprising a glucagon analog as described herein, a dimer as described herein, or a combination thereof, wherein the glucagon analog is fused to a heterologous peptide.
  • the invention provides pharmaceutical compositions comprising any of the foregoing glucagon analogs, dimmers, conjugates or fusion proteins, with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • present disclosures provides further descriptions relating to pharmaceutical compositions, e.g., formulations, routes of administration, etc.
  • the invention furthermore provides a kit for administering a glucagon agonist to a patient in need thereof, said kit comprising a pharmaceutical composition as described herein and a device for administering said pharmaceutical composition to the patient.
  • the device comprises a syringe and a needle, wherein the pharmaceutical composition is pre-packaged within the syringe. Kits are further described herein.
  • the pharmaceutical compositions are believed to be useful in methods of treating a patient. Accordingly, the invention provides a method of causing temporary paralysis of the intestinal tract in a patient in need thereof. The method comprises administering to the patient a pharmaceutical composition as described herein in an amount effective to cause temporary paralysis of the intestinal tract in the patient.
  • the invention further provides a method of treating or preventing hypoglycemia in a patient in need thereof.
  • the method comprises administering to the patient a pharmaceutical composition as described herein in an amount effective to treat or prevent hypoglycemia in the patient.
  • the invention additionally provides a method of stabilizing a blood glucose level in a patient in need thereof, wherein the patient is on a treatment regimen comprising administration of insulin.
  • the method comprises administering to the patient a pharmaceutical composition as described herein in an amount effective to stabilize the blood glucose level of the patient.
  • the pharmaceutical composition further comprises the insulin.
  • the glucagon agonists of the present invention have enhanced biophysical stability and aqueous solubility in solutions of phisiological pH, while retaining or demonstrating enhanced bioactivity relative to the native peptide. Accordingly, the glucagon agonists of the present invention are believed to be suitable for any use that has previously been described for the native glucagon peptide. Therefore, the modified glucagon peptides described herein can be used to treat hypoglycemia, to increase blood glucose level, to induce temporary paralysis of the gut for radiological uses, to reduce and maintain body weight, as adjunctive therapy with insulin, or to treat other metabolic diseases that result from low blood levels of glucagon.
  • glucagon peptides described herein also are expected to be used to reduce or maintain body weight, or to treat hyperglycemia, or to reduce blood glucose level, or to normalize blood glucose level, and/or to stabilize blood glucose level.
  • Normalizing blood level means that the blood glucose level is returned to normal (e.g. lowering blood glucose level if it is higher than normal, or raising blood glucose level if it is lower than normal).
  • Stabilizing blood glucose level means reducing the maximal variation in blood glucose level over a period of time, e.g., 8 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week. For example, administration of glucagon peptide causes the blood glucose level over time to be maintained closer to the normal range of glucose values than it would be in the absence of administration of glucagon peptide.
  • glucagon peptides of the invention may be administered alone or in combination with other anti-diabetic or anti-obesity agents.
  • Anti-diabetic agents known in the art or under investigation include insulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),
  • glipizide Glucotrol
  • glyburide Diabeta, Micronase, Glynase
  • glimepiride Amaryl
  • gliclazide Diamicron
  • meglitinides such as repaglinide (Prandin) or nateglinide (Starlix)
  • biguanides such as metformin
  • Glucophage or phenformin
  • thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors
  • alpha glucosidase inhibitors that inhibit carbohydrate digestion such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide
  • Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin
  • SGLT sodium- dependent glucose transporter 1 inhibitors
  • GKA glucokinase activators
  • GAA glucagon receptor antagonists
  • FBPase fructtose 1,6-bisphosphatase
  • Anti-obesity agents known in the art or under investigation include appetite suppressants, including phenethylamine type stimulants, phenteraiine (optionally with fenfluramine or dexfenfluramine), diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide) or Contrave (bupropion and naltrexone); or lipase inhibitors, similar to xenical (Orlistat) or Cetilistat (also known as ATL-962), or GT 389-255.
  • appetite suppressants including phenethylamine type
  • One aspect of the present disclosure is directed to a pre-formulated aqueous solution of the presently disclosed glucagon agonist for use in treating hypoglycemia.
  • the improved stability and/or solubility of the agonist compositions described herein allow for the preparation of pre-formulated aqueous solutions of glucagon for rapid administration and treatment of hypoglycemia. Accordingly, in one embodiment a solution comprising a glucagon agonist of the present invention is provided for administration to a patient suffering from hypoglycemia.
  • a solution comprising a pegylated glucagon agonist as disclosed herein for administration to a patient suffering from hypoglycemia, wherein the total molecular weight of the PEG chains linked to the pegylated glucagon agonist is between about 500 to about 5,000 Daltons.
  • the pegylated glucagon agonist comprises a peptide selected from the group consisting of 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, and glucagon agonist analogs of thereof, wherein the side chain of an amino acid residue at position 16, 17, 21, 24 or 29, within a C-terminal extension, or at the C-terminal amino acid of said glucagon peptide is covalently bound to the polyethylene glycol chain.
  • the pegylated glucagon agonist comprises the peptide of SEQ ID NO: 16, wherein the amino acid residue at position 21 of the peptide is covalently linked to polyethylene glycol.
  • the pegylated glucagon agonist comprises the peptide of SEQ ID NO: 17, wherein the amino acid residue at position 24 of the peptide is covalently linked to polyethylene glycol.
  • the treatment methods in accordance with the present invention may comprise the steps of administering the presently disclosed glucagon agonists to a patient using any standard route of administration, including parenterally, such as intravenously, intraperitoneally, subcutaneously or intramuscularly, intrathecally, transdermally, rectally, orally, nasally or by inhalation.
  • parenterally such as intravenously, intraperitoneally, subcutaneously or intramuscularly, intrathecally, transdermally, rectally, orally, nasally or by inhalation.
  • the composition is administered subcutaneously or intramuscularly.
  • the composition is administered parenterally and the glucagon composition is prepackaged in a syringe.
  • the composition is prepackaged in an inhaler or other aerosolized drug deliver device.
  • the aqueous stable glucagon analogs disclosed herein exhibit superior stability and solubility in aqueous buffers in the broadest pH range used for pharmacological purposes, relative to native glucagon.
  • the use of the stabilized glucagon analogs disclosed herein allow for the preparation and storage of glucagon agonist solutions at physiological pH for long periods of time.
  • pegylated glucagon peptides can be prepared that retain the parent peptide's bioactivity and specificity. However, increasing the length of the PEG chain, or attaching multiple PEG chains to the peptide, such that the total molecular weight of the linked PEG is greater than 5,000 Daltons, begins to delay the time action of the modified glucagon.
  • a glucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 13, or a glucagon agonist analog thereof wherein the peptide comprises one or more polyethylene glycol chains, wherein the total molecular weight of the linked PEG is greater than 5,000 Daltons, and in one embodiment is greater than 10,000 Daltons, but less than 40,000 Daltons.
  • modified glucagon peptides have a delayed time of activity but without loss of the bioactivity. Accordingly, such compounds can be administered prophylactically to extend the effect of the administered glucagon peptide.
  • Glucagon peptides that have been modified to be covalently bound to a PEG chain having a molecular weight of greater than 10,000 Daltons can be administered in conjunction with insulin to buffer the actions of insulin and help to maintain stable blood glucose levels in diabetics.
  • the modified glucagon peptides of the present disclosure can be co-administered with insulin as a single composition,
  • the insulin and the modified glucagon peptide can be administered at different time relative to one another.
  • the composition comprising insulin and the composition comprising the modified glucagon peptide are administered within 12 hours of one another.
  • the exact ratio of the modified glucagon peptide relative to the administered insulin will be dependent in part on determining the glucagon levels of the patient, and can be determined through routine experimentation.
  • a composition comprising insulin and a modified glucagon peptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and glucagon agonist analogs thereof, wherein the modified glucagon peptide further comprises a polyethylene glycol chain covalently bound to an amino acid side chain at position 16, 17, 21, 24, 29, within a C-terminal extension, or at the C-terminal amino acid.
  • the composition is an aqueous solution comprising insulin and the glucagon analog.
  • the glucagon peptide comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 13
  • the peptide may further comprise a polyethylene glycol chain covalently bound to an amino acid side chain at position 16, 17, 21, 24, 29, within a C-terminal extension, or at the C-terminal amino acid.
  • the molecular weight of the PEG chain of the modified glucagon peptide is greater than 10,000 Daltons.
  • the pegylated glucagon peptide comprises a peptide selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 13 wherein the side chain of an amino acid residue at position 21 or 24 of said glucagon peptide is covalently bound to the polyethylene glycol chain.
  • the polyethylene glycol chain has a molecular weight of about 10,000 to about 40,000.
  • the modified glucagon peptides disclosed herein are used to induce temporary paralysis of the intestinal tract. This method has utility for radiological purposes and comprises the step of administering an effective amount of a pharmaceutical composition comprising a pegylated glucagon peptide, a glucagon peptide comprising a c-terminal extension or a dimer of such peptides.
  • the glucagon peptide comprises a sequence selected from the group consisting 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, 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.
  • the glucagon peptide further comprises a PEG chain, of about 1,000 to 40,000 Daltons is covalently bound to an amino acid residue at position 21, 24 or 29, within a C-terminal extension, or at the C-terminal amino acid.
  • the glucagon peptide is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.
  • the PEG chain has a molecular weight of about 500 to about 5,000 Daltons.
  • the composition used to induce temporary paralysis of the intestinal tract comprises a first modified glucagon peptide and a second modified glucagon peptide, wherein the first modified peptide comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13, optionally linked to a PEG chain of about 500 to about 5,000 Daltons, and the second peptide comprises a covalently linked PEG chain of about 10,000 to about 40,000 Daltons.
  • the PEG chain of each peptide is covalently bound to an amino acid residue at either position 21, 24 or 29, within a C-terminal extension, or at the C-terminal amino acid, of the respective peptide, and independent of one another.
  • Oxyntomodulin a naturally occurring digestive hormone found in the small intestine, has been reported to cause weight loss when administered to rats or humans (see Diabetes 2005;54:2390-2395).
  • 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: 23 (KRNRNNIA).
  • a truncated oxyntomodulin molecule comprising a glucagon peptide of the invention, having the terminal four amino acids of oxyntomodulin removed, will also be effective in suppressing appetite and inducing weight loss/weight
  • the present invention also encompasses the modified glucagon peptides of the present invention that have a carboxy terminal extension of SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22.
  • a glucagon agonist analog of SEQ ID NO: 33 further comprising the amino acid sequence of SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 linked to amino acid 29 of the glucagon peptide, is administered to individuals to induce weight loss or prevent weight gain.
  • a glucagon agonist analog of SEQ ID NO: 11 or SEQ ID NO: 13, further comprising the amino acid sequence of SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 linked to amino acid 29 of the glucagon peptide, is administered to individuals to induce weight loss or prevent weight gain.
  • a method of reducing weight gain or inducing weight loss in an individual comprises administering an effective amount of a composition comprising a glucagon agonist comprising a glucagon peptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, wherein amino acid 29 of the glucagon peptide is bound to a second peptide through a peptide bond, and said second peptide comprises the sequence of SEQ ID NO: 24 (KRNRNNIA) or SEQ ID NO: 25, and wherein a PEG chain of about 1,000 to 40,000 Daltons is covalently bound to an amino acid residue at position 21 and/or 24.
  • a composition comprising a glucagon agonist comprising a glucagon peptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, wherein amino acid 29 of the glucagon peptide is bound to a second peptid
  • the glucagon peptide segment of the glucagon agonist is selected from the group consisting of 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 of about 1,000 to 40,000 Daltons is covalently bound to an amino acid residue at position 16, 17, 21, 24, or 29, within a C-terminal extension, or at the C-terminal amino acid.
  • Exendin-4 is a peptide made up of 39 amino acids. It is a powerful stimulator of a receptor known as GLP-1. This peptide has also been reported to suppress appetite and induce weight loss.
  • the terminal sequence of Exendin-4 when added at the carboxy terminus of glucagon improves the solubility and stability of glucagon without compromising the bioactivy of glucagon.
  • the terminal ten amino acids of Exendin-4 i.e. the sequence of SEQ ID NO: 20 (GPSSGAPPPS)
  • GPSSGAPPPS sequence of SEQ ID NO: 20
  • the sequence of SEQ ID NO: 20 is linked to the C-terminus of the glucagon peptide and the amino acid at position 29 is either Thr or Gly.
  • the terminal amino acid of the SEQ ID NO: 20 extension comprises an amide group in place of the carboxy group (i.e., SEQ ID NO: 23) and this sequence is linked to the carboxy terminus of a glucagon peptide of the present disclosure.
  • a method of reducing weight gain or inducing weight loss in an individual comprises administering an effective amount of a composition comprising a glucagon agonist comprising a glucagon peptide of SEQ ID NO: 33 wherein amino acid 29 of the glucagon peptide is bound to a second peptide through a peptide bond, and said second peptide comprises the sequence of SEQ ID NO: 20 (GPSSGAPPPS) or SEQ ID NO: 23.
  • the glucagon peptide is selected from the group consisting 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, 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 wherein amino acid 29 of the glucagon peptide is bound to a second peptide through a peptide bond, and said second peptide comprises the sequence of SEQ ID NO: 20 (GPSSGAPPPS) or SEQ ID NO: 23.
  • the glucagon peptide of the glucagon agonist is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 13.
  • the glucagon peptide segment of the fusion peptide is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein the molecular weight of the PEG chain is selected from the range of 500 to 40,000 Daltons. More particularly, in one embodiment the glucagon peptide of the fusion peptide is selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 17 wherein the molecular weight of the PEG chain is selected from the range of 1,000 to 5,000.
  • a composition is administered to a patient to suppress appetite, prevent weight gain and/or induce weight loss by the administration of a pharmaceutical composition comprising a first pegylated glucagon peptide and a second pegylated glucagon peptide, wherein the first and second peptide are fusion peptides comprising a c-terminal peptide extension comprising SEQ ID NO: 20 (GPSSGAPPPS) or SEQ ID NO: 23.
  • the present disclosure also encompasses multimers of the modified glucagon peptides disclosed herein.
  • Two or more of the modified glucagon peptides can be linked together using standard linking agents and procedures known to those skilled in the art.
  • dimers can be formed between two modified glucagon peptides through the use of bifunctional thiol crosslinkers and bi-functional amine crosslinkers, particularly for the glucagon peptides that have been substituted with cysteine, lysine ornithine, homocysteine or acetyl phenylalanine residues (e.g. SEQ ID NO: 4 and SEQ ID NO: 5).
  • the dimer can be a homodimer or alternatively can be a
  • the dimer comprises a homodimer of a glucagon fusion peptide wherein the glucagon peptide portion comprises an agonist analog of SEQ ID NO: 11 and an amino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to amino acid 29 of the glucagon peptide.
  • the dimer comprises a homodimer of a glucagon agonist analog of SEQ ID NO: 11, wherein the glucagon peptide further comprises a polyethylene glycol chain covalently bound to position 21, 24, 29, within a C-terminal extension, or at the C-terminal amino acid of the glucagon peptide.
  • a dimer comprising a first glucagon peptide bound to a second glucagon peptide via a linker, wherein the first glucagon peptide comprises a peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and the second glucagon peptide comprises SEQ ID NO: 33.
  • the second glucagon peptide when the amino acid at position 28 is asparagine and the amino acid at position 29 is threonine, the second glucagon peptide further comprises one to two amino acids (independently selected from the group consisting of Lys, Arg, His, Asp or Glu), added to the carboxy terminus of the second glucagon glucagon peptide, and pharmaceutically acceptable salts of said glucagon polypeptides.
  • a dimer comprising a first glucagon peptide bound to a second glucagon peptide via a linker, wherein said first glucagon peptide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, 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 and the second glucagon peptide is independently selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 13, and pharmaceutically acceptable salts of said glucagon polypeptides.
  • the first glucagon peptide is selected from the group consisting of SEQ ID NO: 7 and the second glucagon peptide is independently selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • the dimer is formed between two peptides wherein each peptide comprising the amino acid sequence of SEQ ID NO: 11.
  • composition comprising a glucagon agonist analog of the present disclosure, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise any
  • pharmaceutically acceptable ingredient including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing
  • the pharmaceutical composition comprises any one or a combination of the following components: acacia, acesulfame potassium,
  • acetyltributyl citrate acetyltriethyl citrate, agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate, amylopectin, a-amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, butylparaben sodium, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, dibasic anhydrous calcium phosphate, dibasic dehydrate calcium phosphate, tribasic calcium
  • hydrochloride chlorodifluoroethane (HCFC), chlorodifluoromethane
  • chlorofluorocarbons CFCchlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl- ⁇ -cyclodextrin, cyclodextrin-type compounds such as Captisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate, disodium edentate, disodium hydrogen phosphate, docusate calcium, docus
  • ethylcellulose ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium, ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructose milled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, glyceride mixtures of saturated vegetable fatty acids, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitostearate, glycine, glycols, glycofurol, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromid
  • methylparaben sodium, microcrystalline cellulose and carboxymethylcellulose sodium mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceutical glaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol, phenylethyl alcohol,
  • polymethacrylates polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates, polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, postassium citrate, potassium citrate anhydrous, potassium hydrogen phosphate, potassium metabisulfite, monobasic potassium phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl gallate, propylparaben, propylparaben potassium, propylparaben sodium, protamine sulfate, rapeseed oil, Ringer's solution, saccharin, saccharin ammonium, saccharin calcium, saccharin sodium, safflower oil, saponite, serum proteins
  • the pharmaceutical formulations disclosed herein may be designed to be short-acting, fast-releasing, long-acting, or sustained-releasing as described below.
  • the pharmaceutical formulations may also be formulated for immediate release, controlled release or for slow release.
  • the instant compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect.
  • the disclosed pharmaceutical formulations may be
  • any regime including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, every three weeks, monthly, or bi-monthly.
  • the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v.
  • A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v.
  • the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at most B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In other embodiments, the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at most B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001%
  • compositions at any concentration range, such as, for example from about A to about B.
  • A is 0.0001% and B is 90%.
  • the pharmaceutical compositions may be formulated to achieve a
  • the pH of the pharmaceutical composition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 11, depending on the formulation and route of administration.
  • the pharmaceutical compositions may comprise one or more buffering agents to achieve a physiological compatible pH.
  • the buffering agents may include any compounds capable of buffering at the desired pH such as, for example, phosphate buffers (e.g. PBS), triethanolamine, Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES, and others.
  • the strength of the buffer is at least 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 120 mM, at least 150 mM, or at least 200 mM. In some embodiments, the strength of the buffer is no more than 300 mM (e.g.
  • At most 200 mM at most 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM, at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10 mM, at most 5 mM, at most 1 mM).
  • the pharmaceutical composition comprises a 1 mg/ml concentration of the glucagon agonist analog and 10-50 mM Triethanolamine at pH 7.0-8.5, or 6-9, or 7-9. In one embodiment the pharmaceutical composition comprises a 1 mg/ml concentration of the glucagon agonist analog and 20 mM Triethanolamine at pH 8.5.
  • the modified glucagon peptides of the present invention can be provided in accordance with one embodiment as part of a kit.
  • a kit for administering a glucagon agonist to a patient in need thereof is provided wherein the kit comprises any of the glucagon peptides of the invention in aqueous solution.
  • Exemplary glucagon peptides for inclusion in such kits include a glucagon peptide selected from the group consisting of 1) a glucagon peptide comprising the sequence of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: l 1 or SEQ ID NO: 13 or SEQ ID NO: 33; 2) a glucagon fusion peptide comprising a glucagon agonist analog of SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 33, and an amino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to amino acid 29 of the glucagon peptide; and 3) a pegylated glucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 33, further comprising an amino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21 (KRN
  • the kit is provided with a device for administering the glucagon composition to a patient, e.g. syringe needle, pen device, jet injector or other needle-free injector.
  • a device for administering the glucagon composition to a patient e.g. syringe needle, pen device, jet injector or other needle-free injector.
  • the kit may alternatively or in addition include one or more of a variety of containers, e.g. , vials, tubes, bottles, single or multi-chambered pre-filled syringes, cartridges, infusion pumps (external or implantable), jet injectors, pre-filled pen devices and the like, optionally containing the glucagon peptide in a lyophilized form or in aqueous solution.
  • the kits will also include instructions for use.
  • the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device.
  • the kit comprises a syringe and a needle, and in one embodiment the sterile glucagon composition is prepackaged within the syringe.
  • the compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Although certain non-natural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.
  • Glucagon analogs were synthesized using HBTU-activated "Fast Boc" single coupling starting from 0.2 mmole of Boc Thr(OBzl)Pam resin on a modified Applied Biosystem 430 A peptide synthesizer. Boc amino acids and HBTU were obtained from Midwest Biotech (Fishers, IN). Side chain protecting groups used were: Arg(Tos), Asn(Xan), Asp(OcHex), Cys(pMeBzl), His(Bom), Lys(2Cl-Z), Ser(OBzl), Thr(OBzl), Tyr(2Br-Z), and Trp(CHO). The side-chain protecting group on the N- terminal His was Boc.
  • Each completed peptidyl resin was treated with a solution of 20% piperdine in dimethylformamide to remove the formyl group from the tryptophan.
  • Liquid hydrogen fluoride cleavages were performed in the presence of p-cresol and dimethyl sulfide. The cleavage was run for 1 hour in an ice bath using an HF apparatus (Penninsula Labs). After evaporation of the HF, the residue was suspended in diethyl ether and the solid materials were filtered.
  • the glucagon Cys analog is dissolved in phosphate buffered saline
  • the completed peptidyl resin was treated with 20% piperidine/dimethylformamide to remove the Trp formyl protection then transferred to an HF reaction vessel and dried in vacuo.
  • 1.0ml p-cresol and 0.5 ml dimehyl sulfide were added along with a magnetic stir bar.
  • the vessel was attached to the HF apparatus (Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, and aprox. 10ml liquid hydrogen fluoride was condensed in.
  • the reaction was stirred in an ice bath for lhr then the HF was removed in vacuo.
  • the vessel was attached to the HF apparatus (Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, and aprox. 10ml liquid hydrogen fluoride was condensed in.
  • the reaction was stirred in an ice bath for lhr then the HF was removed in vacuo. The residue was suspended in ethyl ether; the solids were filtered, washed with ether, and the peptide extracted into 50 ml aqueous acetic acid.
  • Glucagon Cys 17 (1-29) and 27.3mg methoxy poly(ethyleneglycol) maleimide avg. M.W.5000 (mPEG-Mal-5000,Nektar Therapeutics) were dissolved in 3.5ml phosphate buffered saline (PBS) and 0.5ml 0.01M ethylenediamine tetraacetic acid (EDTA) was added.
  • PBS phosphate buffered saline
  • EDTA ethylenediamine tetraacetic acid
  • reaction mixture was loaded onto 2.2 x 25 cm Kromasil C18 preparastive reverse phase column.
  • An acetonitrile gradient was run on a Pharmacia FPLC while monitoring the UV wavelength at 214nm and collecting 5 min fractions.
  • the fractions corresponding to the product were combined, frozen and lyophilized to give 25.9 mg.
  • MALDI matrix assisted laser desorption ionization
  • a solution (lmg/ml or 3mg/ml) of glucagon (or an analog) is prepared in 0.01N HCl.
  • lOOul of stock solution is diluted to 1ml with 0.01N HCl and the UV absorbance (276nm) is determined.
  • the pH of the remaining stock solution is adjusted to pH7 using
  • UV absorbance is determined (in duplicate).
  • the initial absorbance reading is compensated for the increase in volume and the following calculation is used to establish percent solubility:
  • Glucagon-Cex represents wild type glucagon (SEQ ID NO: 1) plus a carboxy terminal addition of SEQ ID NO: 20 and Glucagon-
  • Cex R 12 represents SEQ ID NO: 1 wherein the Lys at position 12 is substituted with Arg and a peptide of SEQ ID NO: 20 is added to the carboxy terminus.
  • the data (shown in Figs. 3 & 4) demonstrates the superior solubility of the D28, E29, E30, E15D28, D28E30, D28E29 analogs relative to native glucagon at pH values of 5.5 and 7.0.
  • the data presented in Fig. 3 represents the solubility measured after 60 hours at 25 °C
  • the data presented in Fig. 4 represents the solubility measured after 24 hours at 25 °C and then 24 hours at 4 °C.
  • Fig. 5 represents data regarding the maximum solubility of the glucagon analogs D28, D28E30 and E15D28.
  • the affinity of peptides to the glucagon receptor was measured in a competition binding assay utilizing scintillation proximity assay technology.
  • Serial 3- fold dilutions of the peptides made in scintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clear bottom plate (Corning Inc., Acton, MA) with 0.05 nM (3-[ 125 I]- iodotyrosyl) TyrlO glucagon (Amersham Biosciences, Piscataway, NJ), 1-6 micrograms per well, plasma membrane fragments prepared from cells over- expressing human glucagon receptor, and 1 mg/well polyethyleneimine-treated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, NJ).
  • glucagon analogs to induce cAMP was measured in a firefly luciferase-based reporter assay.
  • HEK293 cells co-transfected with either glucagon- or GLP-1 receptor and luciferase gene linked to cAMP responsive element were serum deprived by culturing 16h in DMEM (Invitrogen, Carlsbad, CA) supplemented with 0.25% Bovine Growth Serum (HyClone, Logan, UT) and then incubated with serial dilutions of either glucagon, GLP-1 or novel glucagon analogs for 5 h at 37°C, 5% C0 2 in 96 well poly-D-Lysine-coated "Biocoat” plates (BD Biosciences, San Jose, CA).
  • LucLite luminescence substrate reagent Perkin-Elmer, Wellesley, MA
  • the plate was shaken briefly, incubated 10 min in the dark and light output was measured on MicroBeta-1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA).
  • the luminescent light output indicates activation of the luciferase reporter gene, which in turn is a measure of the activation of the receptor.
  • Effective 50% concentrations (“EC50") were calculated by using Origin software (OriginLab, Northampton, MA. Results are shown in Tables 2 and 3.
  • EC50 is the concentration of the peptide that produces 50% of the peptide's maximum activation response at the indicated receptor. A relatively lower EC50 indicates that a peptide is relatively more potent at that receptor, while a higher EC50 indicates that a peptide is less potent.
  • Figure 11 displays data on cAMP induction at the glucagon and GLP-1 receptors for a glucagon analog with the following modifications: T16,A20,E21,A24,Nle27,D28, and E29 (SEQ ID NO: 56).
  • the data shows that a glucagon analog containing multiple modifications (seven substitutions) retains substantial glucagon activity.
  • glucagon analog was dissolved in water or PBS and an initial HPLC analysis was conducted. After adjusting the pH ( 4, 5, 6, 7), the samples were incubated over a specified time period at 37°C and re-analyzed by HPLC to determine the integrity of the peptide. The concentration of the specific peptide of interest was determined and the percent remaining intact was calculated relative to the initial analysis. Results for Glucagon Cys 21 -maleimidoPEGs K are shown in Figs. 1 and 2.
  • Lys(Cl-Z) was used to protect the native Lys at position 12 if lactams were constructed from 16-20, 20-24, or 24-28.
  • the completed peptidyl resin was treated with 20% piperidine/dimethylformamide for one hour with rotation to remove the Trp formyl group as well as the Fmoc and OFm protection from Lys 12 and Glul6.
  • dimethylformamide and diisopropylethylamine DIEA
  • DIEA diisopropylethylamine
  • the reaction proceeded for 8-10 hours and the cyclization was confirmed by a negative ninhydrin reaction.
  • the resin was washed with dimethylformamide, followed by dichloromethane and subsequently treated with trifluoroacetic acid for 10 minutes. The removal of the Boc group was confirmed by a positive ninhydrin reaction.
  • the resin was washed with dimethylformamide and dichloromethane and dried before being transferred to a hydrofluoric acid (HF) reaction vessel. 500 p-cresol was added along with a magnetic stir bar.
  • HF hydrofluoric acid
  • the vessel was attached to the HF apparatus (Peninsula Labs), cooled in a dry ice/methanol bath, evacuated, and approximately 10 mL of liquid hydrofluoric acid was condensed into the vessel.
  • the reaction was stirred for 1 hour in an ice bath and the HF was subsequently removed in vacuo.
  • the residue was suspended in ethyl ether; the solids were filtered, washed with ether, and the peptide was solubilized with 150 mL 20% acetonitrile/1% acetic acid.
  • HPLC analysis of the purified peptide demonstrated greater than 95% purity and electrospray ionization mass spectral analysis confirmed a mass of 3506 Da for the 12-16 lactam. Lactams from 16-20, 20-24, and 24-28 were prepared similarly.
  • Glucagon peptides with the following sequences were constructed generally as described herein:
  • Glucagon peptides with the following modifications from SEQ ID NO: 1 was constructed generally as described herein:
  • any of these peptides may comprise AIB20 and/or AIB24 instead of the A20 and/or A24 substitutions.
  • Any of these peptides may further comprise a T16 or AIB16 amino acid substitution.
  • T16,A20,E21,A24,Nle27,D28,E29 (SEQ ID NO: 56) was constructed.
  • a glucagon peptide with the following modifications from SEQ ID NO: 1 was constructed generally as described herein: DMIA1, El 6, K20-glucagon-COOH (C24-PEG, E16 to K20 lactam). Its sequence is set forth below:
  • Acylated and/or PEGylated peptides are prepared as follows. Peptides are synthesized on a solid support resin using either a CS Bio 4886 Peptide Synthesizer or Applied Biosystems 430A Peptide Synthesizer. In situ neutralization chemistry is used as described by Schnolzer et al., Int. J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides, the target amino acid residue to be acylated (e.g., position ten) is substituted with an N ⁇ -FMOC lysine residue. Treatment of the completed N- terminally BOC protected peptide with 20% piperidine in DMF for 30 minutes removes FMOC/formyl groups.
  • CS Bio 4886 Peptide Synthesizer or Applied Biosystems 430A Peptide Synthesizer. In situ neutralization chemistry is used as described by Schnolzer et al., Int. J. Peptide Protein
  • Coupling to the free ⁇ -amino Lys residue is achieved by coupling a ten-fold molar excess of either an FMOC-protected spacer amino acid (ex. FMOC-(N-BOC)-Tryptophan-OH) or acyl chain (ex. C17-COOH) and PyBOP or DEPBT coupling reagent in DMF/DIEA. Subsequent removal of the spacer amino acid's FMOC group is followed by repetition of coupling with an acyl chain. Final treatment with 100% TFA results in removal of any side chain protecting groups and the N-terminal BOC group.
  • Peptide resins are neutralized with 5% DIEA/DMF, are dried, and then are cleaved from the support using HF/p-cresol, 95:5, at 0°C for one hour. Following ether extraction, a 5% HOAc solution is used to solvate the crude peptide. A sample of the solution is then verified to contain the correct molecular weight peptide by ESI-MS. Correct peptides are purified by RP-HPLC using a linear gradient of 10% CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm x 250 mm protein column is used for the purification.
  • peptide pegylation 40 kDa methoxy poly(ethylene glycol) maleimido- propionamide (Chirotech Technology Ltd.) is reacted with a molar equivalent of peptide in 7M Urea, 50mM Tris-HCl buffer using the minimal amount of solvent needed to dissolve both peptide and PEG into a clear solution (generally less than 2 mL for a reaction using 2-3 mg peptide). Vigorous stirring at room temperature commences for 4-6 hours and the reaction is analyzed by analytical RP-HPLC.
  • PEGylated products appear distinctly from the starting material with decreased retention times. Purification is performed on a Vydac C4 column with conditions similar to those used for the initial peptide purification. Elution typically occurs around buffer ratios of 50:50. Fractions of pure PEGylated peptide are collected and lyophilized.
  • Peptides are assayed for biological activity as described above in Example 13.
  • Canine/Beagle dogs of 8-12 kg, being of 8-16 months of age and good health were used to determine the pharmacokinetics and pharmacodynamics of glucagon action. Every animal was fasted overnight and bled at the following time points after each dose: 0 hr. (pre-dose), 5, 10, 20, 30, 45, 60, 90, 120, 240 minutes post dose. Six animals were used for each dose group and approximately l-2ml of whole blood was withdrawn at each time point. About 1.0 ml whole blood was added to K 2 EDTA tubes containing a sufficient volume of Trasylol (aprotinin) to yield at least 500 KIU/mL of whole blood.
  • trastinin Trasylol
  • Glucagon and the analogs were dissolved in 0.01N HC1 at a concentration of 0.1667 mg/ml and the animals were dosed at 0.03 ml/kg.
  • the animals were administered a 0.005 mg/kg dose intramuscularly of either glucagon, a glucagon analog comprising glucagon with the sequence of SEQ ID NO: 31 linked to the carboxy terminus of glucagon (glucagon-CEX) or a glucagon analog comprising an aspartic acid substitution at amino acid 28 (glucagon-Asp28) SEQ ID NO: 11.
  • glucagon-CEX the sequence of SEQ ID NO: 31 linked to the carboxy terminus of glucagon
  • glucagon-Asp28 glucagon-Asp28
  • A A peptide comprising the amino acid sequence of SEQ ID NO: 1 with an amino acid substitution at position 16 with AIB ("AIB 16
  • AIB 16 Glucagon 0.37 0.05 2 32.43 8.63 4
  • AIB 19 Glucagon 13.52 0.64 3 64.75 11.38 4
  • D28/E29 Glucagon comprising the amino acid sequence of SEQ ID NO: 1 modified to comprise Asp at position 28 and Glu at position 29:
  • Peptides of Set A each comprising the amino acid sequence of SEQ ID NO: 1 with the modifications listed in Table 6, are made as essentially described herein.
  • Dab(Ac) AIB Ala Ala Leu Asn Glu n/a Absent
  • Dab(Ac) AIB Ser Ser Leu Asp Glu Glu Absent C-terminal
  • Peptides of a second set are made with the same structures of the peptides of Set A, except that the peptides of Set B comprise a Cys at position 24 wherein the Cys residue is covalently attached to a 40 kDa PEG.
  • the peptides of Sets A and B are tested for in vitro activity at the glucagon receptor as essentially described in Example 13.
  • Q(Me) methylglutamine
  • M(O) methionine- sulfoxide
  • Orn(Ac) acetylornithine.
  • Glucagon analog peptides comprising Dab(Ac) at position 3 on various glucagon analog backbones were made as essentially described herein and the in vitro activity at the glucagon receptor was tested. The structures and activities of each peptide are shown in Table 8.
  • Dab(Ac) lie AIB 10 C14 Asp Thr Absent
  • Dab(Ac) lie AIB 10 C14 Asn Glu Absent
  • Dab(Ac) lie AIB 10 C14 Asp Glu Absent
  • Dab(Ac) lie AIB 10 C14 Glu Thr Absent
  • Dab(Ac) lie AIB 10 C14 Glu Glu Absent
  • Dab(Ac) lie AIB 30 C14 Asp Thr Absent
  • Dab(Ac) lie AIB 30 C14 Asn Glu Absent C-

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Abstract

L'invention concerne des analogues du glucagon comprenant une séquence d'acides aminés du glucagon humain natif modifiée par (a) substitution de Ser en position 16 du glucagon natif (SEQ ID N° 1) par un acide aminé α,α-disubstitué, facultativement l'acide alpha-aminoisobutyrique ; substitution de Gln en position 3 du glucagon humain natif (SEQ ID N° 1) par un analogue de la glutamine ; substitution d'Arg en position 17 du glucagon humain natif (SEQ ID N° 1) par un acide aminé chargé négativement ; facultativement au moins un acide aminé chargé en position C-terminale par rapport à l'acide aminé en position 27 du peptide glucagon ; et facultativement jusqu'à 10 autres modifications d'acides aminés. L'invention concerne également des dimères apparentés, des conjugués apparentés, des peptides de fusion apparentés, des compositions pharmaceutiques apparentées, des kits apparentés, ainsi que des procédés apparentés d'utilisation des compositions pharmaceutiques.
PCT/US2011/041623 2010-06-25 2011-06-23 Analogues du glucagon présentant une solubilité et une stabilité améliorées dans des tampons à ph physiologique WO2011163473A1 (fr)

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US10220075B2 (en) 2015-06-04 2019-03-05 Rezolute, Inc. Amine pegylation methods for the preparation of site-specific protein conjugates
US10413593B2 (en) 2014-10-24 2019-09-17 Merck Sharp & Dohme Corp. Co-agonists of the glucagon and GLP-1 receptors
US10570184B2 (en) 2014-06-04 2020-02-25 Novo Nordisk A/S GLP-1/glucagon receptor co-agonists for medical use
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US10806797B2 (en) 2015-06-05 2020-10-20 Sanofi Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate
JP2022519620A (ja) * 2019-02-05 2022-03-24 イーライ リリー アンド カンパニー グルカゴン類似体アゴニストおよびその使用方法

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