WO2011094337A1 - Glucagon antagonist - gip agonist conjugates and compositions for the treatment of metabolic disorders and obesity - Google Patents

Glucagon antagonist - gip agonist conjugates and compositions for the treatment of metabolic disorders and obesity Download PDF

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Publication number
WO2011094337A1
WO2011094337A1 PCT/US2011/022608 US2011022608W WO2011094337A1 WO 2011094337 A1 WO2011094337 A1 WO 2011094337A1 US 2011022608 W US2011022608 W US 2011022608W WO 2011094337 A1 WO2011094337 A1 WO 2011094337A1
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Prior art keywords
amino acid
peptide
seq
peptide combination
acid
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PCT/US2011/022608
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English (en)
French (fr)
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WO2011094337A8 (en
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Richard D. Dimarchi
Tao Mat
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Indiana University Research And Technology Corporation
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Priority to CA2788304A priority Critical patent/CA2788304A1/en
Application filed by Indiana University Research And Technology Corporation filed Critical Indiana University Research And Technology Corporation
Priority to KR1020127021672A priority patent/KR20120123443A/ko
Priority to BR112012018585A priority patent/BR112012018585A2/pt
Priority to IN6437DEN2012 priority patent/IN2012DN06437A/en
Priority to JP2012551270A priority patent/JP2013518115A/ja
Priority to EP11737598.0A priority patent/EP2528618A4/en
Priority to MX2012008603A priority patent/MX2012008603A/es
Priority to RU2012136450/10A priority patent/RU2012136450A/ru
Priority to US13/575,363 priority patent/US8551946B2/en
Priority to CN2011800170897A priority patent/CN102834108A/zh
Publication of WO2011094337A1 publication Critical patent/WO2011094337A1/en
Publication of WO2011094337A8 publication Critical patent/WO2011094337A8/en
Priority to US14/026,671 priority patent/US9487571B2/en

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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • GIP Glucose-dependent insulinotropic peptide
  • GLP-1 Glucagon-like Peptide- 1
  • GIP exerts glucose-dependent stimulatory effects on insulin secretion, thereby ensuring prompt insulin-mediated uptake of glucose into tisssues
  • GLP-1 stimulates insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake.
  • GLP-1 agonists While agonist peptide analogs of both incretins have been made and tested, GLP-1 agonists have been and remain the central focus of research and development for treating of metabolic diseases, such as Type 2 diabetes. This is not surprising, since in Type 2 diabetes, GIP no longer modulates glucose-dependent insulin secretion, whereas GLP- 1 retains insulinotropic activities even in Type 2 diabetetic patients. Also, research by some groups McLean et al., Am J Physiol Endocrinol Metab 296(6): E1746-1755 (epub 2007) have suggested the use of GIP antagonists and not GIP agonists for the treatment of diabetes.
  • glucagon When blood glucose levels begin to fall, glucagon is produced by the pancreas and the binding of this hormone to its receptor signals the liver to break down glycogen and release glucose. The actions of glucagon cause blood glucose levels to rise toward a normal level. Because glucagon exerts actions which oppose incretins, many glucagon antagonists have been made and tested for the treatment of metabolic diseases, including Type 2 diabetes.
  • the peptide combinations comprise a GIP agonist peptide which exhibits agonist activity at the GIP receptor and a glucagon antagonist peptide which exhibits antagonist or inhibitory activity at the glucagon receptor.
  • the GIP agonist peptide exhibits at least 0.1% activity (e.g., at least 0.5%, at least 0.75%, at least 1%, at least 5%, at least 10%) of native GIP at the GIP receptor and the glucagon antagonist peptide exhibits at least 60% inhibition of the maximum response achieved by glucagon at the glucagon receptor.
  • the IC50 at the glucagon receptor of the glucagon antagonist peptide is within about 10-fold (higher or lower) of the EC50 at the GIP receptor of the GIP agonist peptide.
  • either or both of the GIP agonist peptide and glucagon antagonist peptide additionally exhibit agonist activity at the GLP-1 receptor.
  • the peptide combinations are provided as a composition, such as, for example, a pharmaceutical composition.
  • the composition comprises the GIP agonist peptide in admixture with the glucagon antagonist peptide and the two peptides are not attached to one another.
  • the peptide combinations are provided as a conjugate in which the GIP agonist peptide is attached via covalent or non-covalent bonds (or a mixture of both types of bonds) to the glucagon antagonist peptide.
  • the GIP agonist peptide is covalently attached to the glucagon antagonist peptide via peptide bonds.
  • the conjugate is a single polypeptide chain (e.g., a fusion peptide) comprising the GIP agonist peptide and glucagon antagonist peptide.
  • the fusion peptide can be produced recombinantly.
  • the GIP agonist peptide is attached to the glucagon antagonist peptide via one or more side chain functional groups of one or more amino acids of the GIP agonist peptide and/or glucagon antagonist peptide.
  • the conjugate is a heterodimer (or multimer) comprising the GIP agonist peptide and glucagon antagonist peptide attached to one another.
  • the GIP agonist peptide is attached to the glucagon antagonist peptide via a linker, e.g., a bifunctional linker.
  • the bifunctional linker is a hydrophilic polymer, e.g., polyethylene glycol.
  • the bifunctional linker connects a Cys residue of one of the GIP agonist peptide and glucagon antagonist peptide to a Lys of the other peptide.
  • each of the Cys and Lys is located at the C-terminus of the peptide or within the C-terminal region of the peptide.
  • the peptide combination is provided as a kit.
  • the GIP agonist peptide is packaged together with the glucagon antagonist peptide.
  • the GIP agonist peptide is packaged separately from the glucagon antagonist peptide.
  • the kit in some aspects comprises instructions for administering the GIP agonist peptide and glucagon antagonist peptide.
  • the kit comprises instructions for co-administering the GIP agonist peptide and glucagon antagonist peptide.
  • compositions e.g., pharmaceutical compositions
  • conjugates e.g., fusion peptides, heterodimers
  • kits each of which comprise a GIP agonist peptide and a glucagon antagonist peptide.
  • Methods of using such compositions, conjugates, and kits are further provided herein.
  • the present disclosures provide a method of treating a metabolic disease (e.g., diabetes, obesity) in a patient, comprising administering to the patient any of the compositions or conjugates described herein in an amount effective to treat the metabolic disease in the patient.
  • a metabolic disease e.g., diabetes, obesity
  • the treatment of other diseases is further contemplated herein.
  • Figure 1 is a graph of the % change in body weight in mice as a function of time (days) after administration of vehicle alone (closed upright triangles), GLP-1 E 16 agonist at 10 nmol/kg (closed inverted triangles) or 35 nmol/kg (open squares), triagonist peptide MT- 170 at 10 nmol/kg (open inverted triangles) or 35 nmol/kg (closed diamonds), or GLP-l/GIP co-agonist peptide MT-178 at 10 nmol/kg (grey inverted triangles) or at 35 nmol/kg (grey squares).
  • Figure 2 is a graph of the change in blood glucose levels (mg/dL) in mice at Day 7 after administration of vehicle alone (black bar), GLP-1 E 16 agonist at 10 nmol/kg (white bar) or 35 nmol/kg (grey bar), triagonist peptide MT-170 at 10 nmol/kg (horizontal lined bar) or 35 nmol/kg (vertical lined bar), or GLP-l/GIP co-agonist peptide MT-178 at 10 nmol/kg (right- left diagonal lined bar) or at 35 nmol/kg (left-right diagonal lined bar).
  • Figure 3 represents a graph of the blood glucose levels (mg/dL) as a function of time before and after a glucose injection (administered at timepoint 0) of mice injected (at timepoint -60) with a vehicle control, a GLP-1 agonist peptide control, a lactam-containing (cyclic), pegylated, GIP-active glucagon analog ("mt-178"), or a lactam-lacking (linear), pegylated, GIP-active glucagon analog (“mt-274”) at 1, 3, or 10 nmol/kg/week.
  • the data of this figure excludes the data of four mice, as these mice exhibited aggressive behavior and substantial weight loss.
  • Figure 4 represents a graph of the blood glucose levels (mg/dL) as a function of time before and after a glucose injection (administered at timepoint 0) of mice injected (24 hours before the glucose injection) with a vehicle control, a GLP-1 agonist peptide control, mt-178, or mt-274 at 1, 3, or 10 nmol/kg/week.
  • the data of this figure excludes the data of four mice, as these mice exhibited aggressive behavior and substantial weight loss.
  • Figure 5 represents a graph of the blood glucose levels (mg/dL) of mice 0 or 7 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, or mt-274 at 1, 3, or 10 nmol/kg/week.
  • the data of this figure excludes the data of four mice, as these mice exhibited aggressive behavior and substantial weight loss.
  • Figure 6 represents a graph of the percent change in body weight of mice 0, 1, 3, 5, and 7 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, or mt-274 at 1, 3, or 10 nmol/kg/week.
  • the data of this figure excludes the data of four mice, as these mice exhibited aggressive behavior and substantial weight loss.
  • Figure 7 represents a graph of the blood glucose levels (mg/dL) of mice 0 or 7 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35 nmol/kg/week.
  • TE indicates a PEG group attached to the Cys at position 40.
  • Figure 8 represents a graph of the change in blood glucose (mg/dL) of mice 7 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35 nmol/kg/week.
  • TE indicates a PEG group attached to the Cys at position 40.
  • Figure 9 represents a graph of the percent change in body weight of mice 0, 1, 3, 5, 7, and 10 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35 nmol/kg/week.
  • TE indicates a PEG group attached to the Cys at position 40.
  • Figure 10 represents a graph of the percent change in body weight of mice 7 days after injection with a vehicle control, a GLP-1 agonist peptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35 nmol/kg/week.
  • TE indicates a PEG group attached to the Cys at position 40.
  • Figure 11 represents a graph of the change in blood glucose levels (mg/dL) of mice 0 and 7 days after QD injections for 7 days with a vehicle control, liraglutide (an acylated GLP-1 analog), a C14 fatty acylated, unpegylated linear peptide ("mt-260"), a C16 fatty acylated, unpegylated linear peptide ("mt-261”), or a C18 fatty acylated, unpegylated linear peptide ("mt-262”) at 25 or 125 nmol/kg.
  • liraglutide an acylated GLP-1 analog
  • mt-260 C14 fatty acylated, unpegylated linear peptide
  • mt-261 C16 fatty acylated, unpegylated linear peptide
  • mt-262 C18 fatty acylated, unpegylated linear peptide
  • Figure 12 represents a graph of the percent change in body weight of mice 0, 1, 3, 5, and 7 days after injection with a vehicle control, liraglutide, mt-260, mt-261, or mt-262 at 25 or 125 nmol/kg.
  • Figure 13 represents a graph of the percent change in body weight of mice 7 days after injection with a vehicle control, liraglutide, mt-260, mt-261, or mt-262 at 25 or 125 nmol/kg.
  • Figure 14 represents a graph of the change in body weight (g) of mice 0, 1, 3, 5, and 7 days after the first injection with a vehicle control, liraglutide (30 nmol/kg/day), or mt-261 (0.3, 1, 3, 10, or 30 nmol/kg/day).
  • Figure 15 represents a graph of the fat mass of mice 7 days after the first injection with a vehicle control, liraglutide (30 nmol/kg/day), or mt-261 (0.3, 1, 3, 10, or 30 nmol/kg/day).
  • Figure 16 represents a graph of the blood glucose levels (mg/dL) of mice 0 and 7 days after the first injection with a vehicle control, liraglutide (30 nmol/kg/day), or mt-261 (0.3, 1, 3, 10, or 30 nmol/kg/day).
  • Figure 17 represents a line graph of the change in body weight ( change) as a function of time of mice injected with mt-263, Exendin-4, or a vehicle control at the doses (nmol/kg/day) indicated in ().
  • Figure 18 represents a bar graph of the total change in body weight ( ) (as measured on Day 7 in comparison to Day 0) of mice injected with mt-263, Exendin-4, or a vehicle control at the doses (nmol/kg/day) indicated in ().
  • Figure 19 represents a bar graph of the change in blood glucose levels (mg/dL) (as measured on Day 7 in comparison to Day 0) of mice injected with mt-263, Exendin-4, or a vehicle control at the doses (nmol/kg/day) indicated in ().
  • Figure 20 represents a graph of the % change in body weight of mice 0, 1, 3, 5, and 7 days after the first injection with a vehicle control, liraglutide, mt-277, mt-278, or mt-279.
  • Figure 21 represents a graph of the blood glucose levels (mg/dL) of mice 0 and 7 days after the first injection with a vehicle control, liraglutide, mt-277, mt-278, or mt-279.
  • Figure 22 represents a graph of the total change in body weight ( ) of mice as measured 7 days after administration of mt-331, mt-311, or a vehicle control.
  • Figure 23 represents a graph of the total food intake (g) by mice as measured 7 days after administration of mt-331, mt-311, or a vehicle control. Doses (nmol/kg) are indicated in () ⁇ [0032]
  • Figure 24 represents a graph of the total change in blood glucose levels of mice as measured 7 days after administration of mt-331, mt-311, or a vehicle control. Doses
  • Figure 25 represents a graph of the total change in body weight of mice as measured 7 days after administration of mt-331, mt-353, or a vehicle control at the indicated dose (nmol/kg) shown in ().
  • Figure 26 represents a graph of the total food intake (g) by mice as measured 7 days after administration of mt-331, mt-353, or a vehicle control at the indicated dose (nmol/kg) shown in ().
  • Figure 27 represents a graph of the change in blood glucose levels (mg/dL) of mice as measured 7 days after administration of mt-331, mt-353, or a vehicle control at the indicated dose (nmol/kg) shown in ().
  • Figure 28 represents a graph of the total change in body weight (%) of mice as measured 7 days after the first administration of mt-277, mt-278, mt-279, or a vehicle control.
  • Figure 29 represents a graph of the total change in body weight (%) of mice as measured 6 days after the first administration of mt-261, mt-309, or a vehicle control.
  • Figure 30 represents a graph of the blood glucose levels (mg/dL) of mice as measured 6 days after the first administration of mt-261, mt-309, or a vehicle control.
  • the first bar of each pair of bars of the same pattern is the blood glucose levels as measured on Day 0 and the second bar of each pair is the levels on Day 6.
  • Figure 31 represents a bar graph of the total change in body weight (%)as measured 6 days after the first administration of mt-261 (in comparison to the body weight as measured on the first day of administration) of mice injected with a vehicle control or mt-261 as further described herein.
  • Figure 32 represents a graph of the total change in body weight (%) of mice injected with different acylated peptides (MT-261, MT-367, MT-270, and MT-369) as calculated by subtracting the body weight on Day 0 from the body weight on Day 7.
  • Figure 33 represents a graph of the total change in blood glucose levels of mice injected with different acylated peptides (MT-261, MT-367, MT-270, and MT-369) as calculated by subtracting the blood glucose levels on Day 0 from that on Day 7.
  • Figure 34 represents a graph of the total change in body weight (%) of mice injected with MT-367, MT-369, MT-368, MT-384, MT-385, or MT-364 as calculated by subtracting the body weight on Day 0 from the body weight on Day 7.
  • Figure 35 represents a graph of the total change in blood glucose levels of mice injected with MT-367, MT-369, MT-368, MT-384, MT-385, or MT-364 as calculated by subtracting the blood glucose levels on Day 0 from that on Day 7.
  • Figure 36 represents a graph of the change in body weight (%) of mice injected with an Exendin-4-like peptide or with MT-263, MT-280, MT-356, or MT-357, or with a vehicle control, as a function of time (days).
  • Figure 37 represents a graph of a graph of the blood glucose levels (ml/dL) of mice injected with an Exendin-4-like peptide or with MT-263, MT-280, MT-356, or MT-357, or with a vehicle control, as measured on Day 0 and Day 7 of the study.
  • Figure 38 represents a graph of the % change in body weight of mice injected with vehicle only (daily or once every 3 days) or MT-263 (daily, once every 2 days, or once every 3 days).
  • Figure 39 represents a graph of the blood glucose levels as measured on Days 1 and 6 of the study of mice injected with vehicle only (daily or once every 3 days) or MT-263 (daily, once every 2 days, or once every 3 days).
  • Figure 40 represents a graph of the total change in body weight observed in mice upon administration with acylated or pegylated compounds as further described herein.
  • Figure 41 represents a graph of the change in blood glucose observed in mice upon administration with acylated or pegylated compounds as further described herein.
  • Figure 42 represents a graph of the total change in body weight observed in mice upon administration of MT-261 or MT-278 as further described herein.
  • Figure 43 represents a graph of the change in blood glucose observed in mice upon administration of MT-261 or MT-278 as further described herein.
  • 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.
  • the term "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.
  • An alternative desired effect for the glucagon peptides of the present disclosure would include treating hyperglycemia, e.g., as measured by a change in blood glucose level closer to normal, or inducing weight loss/preventing weight gain, e.g., as measured by reduction in body weight, or preventing or reducing an increase in body weight, or normalizing body fat distribution.
  • 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, e.g., subcutaneous, intramuscular, intraspinal, or intravenous.
  • isolated means having been removed from its natural environment.
  • the analog is made through recombinant methods and the analog is isolated from the host cell.
  • purified relates 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 and means having been increased in purity as a result of being separated from other components of the original composition.
  • 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.
  • peptide encompasses a sequence of 2 or more amino acids and typically less than 50 amino acids, wherein the amino acids are naturally occurring or coded or non-naturally occurring or non-coded 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.
  • Non-coded refer 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.
  • polypeptide and “protein” are terms that are used interchangeably to refer to a polymer of amino acids, without regard to the length of the polymer. Typically, polypeptides and proteins have a polymer length that is greater than that of "peptides.” In some instances, a protein comprises more than one polypeptide chain covalently or noncovalently attached to each other.
  • a "peptide combination” encompasses a composition, conjugate, or kit comprising a GIP agonist peptide and a glucagon antagonist peptide.
  • the GIP agonist peptide and a glucagon antagonist peptide may be separated or mixed together, or may be linked covalently or non-covalently.
  • the peptide combination is referred to as a "conjugate.”
  • positions 28 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 an analog of glucagon 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 analog of glucagon in which one amino acid has been added before the N-terminus of SEQ ID NO: 1.
  • amino acid modification refers to (i) a substitution or replacement of an amino acid of SEQ ID NO: 1 with a different amino acid (naturally- occurring or coded or non-coded or non-naturally-occurring amino acid), (ii) an addition of an amino acid (naturally- occurring or coded or non-coded or non-naturally-occurring amino acid), to SEQ ID NO: 1 or (iii) a deletion of one or more amino acids of SEQ ID NO: 1.
  • the amino acid substitution or replacement is a conservative amino acid substitution, e.g., a conservative substitution of the amino acid 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.
  • conservative amino acid substitution is the replacement of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity,
  • hydrophilicity, and/or aromaticity and includes exchanges within one of the following five groups:
  • the amino acid substitution is not a conservative amino acid substitution, e.g., is a non-conservative amino acid substitution.
  • charged amino acid refers to an amino acid that comprises a side chain that is negative-charged (i.e., de-protonated) or positive-charged (i.e., protonated) in aqueous solution at physiological pH.
  • negative-charged amino acids include aspartic acid, glutamic acid, cysteic acid, homocysteic acid, and homoglutamic acid
  • positive-charged amino acids include arginine, lysine and histidine.
  • Charged amino acids include the charged amino acids among the 20 coded amino acids, as well as atypical or non-naturally occurring or non-coded amino acids.
  • acidic amino acid refers to an amino acid that comprises a second acidic moiety (other than the alpha carboxylic acid of the amino acid), including for example, a side chain carboxylic acid or sulfonic acid group.
  • sulfonic acid derivative of cysteine refers to compounds of the general structure:
  • X 6 is C C 4 alkyl, C 2 -C 4 alkenyl or C 2 -C 4 alkynyl.
  • C C n alkyl wherein n can be from 1 through 6, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms.
  • Typical C -C alkyl groups include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
  • C 2 -C n alkenyl wherein n can be from 2 through 6, as used herein, represents an olefinically unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one double bond.
  • C 2 -C n alkynyl wherein n can be from 2 to 6, refers to an unsaturated branched or linear group having from 2 to n carbon atoms and at least one triple bond.
  • pH stabilized glucagon antagonist refers to a glucagon antagonist that exhibits superior stability and solubility, relative to native glucagon, in aqueous buffers in the broadest pH range used for pharmacological purposes.
  • the term "selectivity" of a molecule for a first receptor relative to a second receptor refers to the following ratio: EC50 of the molecule at the second receptor divided by the EC50 of the molecule at the first receptor. For example, a molecule that has an EC50 of 1 nM at a first receptor and an EC50 of 100 nM at a second receptor has 100-fold selectivity for the first receptor relative to the second receptor.
  • the term “native glucagon” refers to a peptide consisting of the sequence of SEQ ID NO: 1 and the term “native GLP-1” is a generic term that designates GLP- 1(7-36) amide, GLP-1 (7-37) acid or a mixture of those two compounds.
  • the term “native GIP” refers to a peptide consisting of SEQ ID NO: 2.
  • GIP potency or “potency compared to native GIP” of a molecule refers to the ratio of the EC50 of the molecule at the GIP receptor divided by the EC50 of native GIP at the GIP receptor.
  • glucagon potency or “potency compared to native glucagon” of a molecule refers to the ratio of the EC50 of the molecule at the glucagon receptor divided by the EC50 of native glucagon at glucagon receptor.
  • GLP-1 potency or “potency compared to native GLP-1" of a molecule refers to the ratio of the EC50 of the molecule at GLP-1 receptor divided by the EC50 of native GLP-1 at GLP-1 receptor.
  • GIP agonist peptide refers to a compound that binds to and activates downstream signaling of the GIP receptor. However, this term should not be construed as limiting the compound to having activity at only the GIP receptor. Rather, the GIP agonist peptides of the present disclosures may exhibit additional activities at other receptors, as further discussed herein. GIP agonist peptides, for example, may exhibit activity (e.g., agonist activity) at the GLP-1 receptor. Also, the term “GIP agonist peptide” should not be construed as limiting the compound to only peptides. Rather, compounds other than peptides are encompassed by this term.
  • the GIP agonist peptide in some aspects is a peptide in conjugate form (a heterodimer, a multimer, a fusion peptide), a chemically- derivatized peptide, a pharmaceutical salt of a peptide, a peptidomimetic, and the like.
  • glucagon antagonist peptide refers to a compound that counteracts glucagon activity or prevents glucagon function.
  • a glucagon antagonist exhibits at least 60% inhibition (e.g., at least 70% inhibition) and preferably, at least 80% inhibition, of the maximum response achieved by glucagon at the glucagon receptor.
  • at least 60% inhibition e.g., at least 70% inhibition
  • preferably, at least 80% inhibition of the maximum response achieved by glucagon at the glucagon receptor.
  • the glucagon antagonist exhibits at least 90% inhibition of the maximum response achieved by glucagon at the glucagon receptor. In a specific embodiment, the glucagon antagonist exhibits 100% inhibition of the maximum response achieved by glucagon at the glucagon receptor. Additionally, a glucagon antagonist at a concentration of about 1 ⁇ exhibits less than about 20% of the maximum agonist activity achieved by glucagon at the glucagon receptor. In one embodiment, the glucagon antagonist exhibits less than about 10% of the maximum agonist activity achieved by glucagon at the glucagon receptor. In a specific embodiment, the glucagon antagonist exhibits less than about 5% of the maximum agonist activity achieved by glucagon at the glucagon receptor. In yet another specific embodiment, the glucagon antagonist exhibits 0% of the maximum agonist activity achieved by glucagon at the glucagon receptor.
  • glucagon antagonist peptide should not be construed as limiting the compound to having activity at only the glucagon receptor. Rather, the glucagon antagonist peptides of the present disclosures may exhibit additional activities at the glucagon receptor (e.g., partial agonism) or other receptor. Glucagon antagonist peptides, for example, may exhibit activity (e.g., agonist activity) at the GLP-1 receptor. Also, the term “glucagon antagonist peptide” should not be construed as limiting the compound to only peptides.
  • the GIP agonist peptide is a peptide in conjugate form, a chemi]cally- derivatized peptide, a pharmaceutical salt of a peptide, a peptidomimetic, and the like.
  • a "pure glucagon antagonist” is a glucagon antagonist that does not produce any detected stimulation of glucagon or GLP- 1 receptor activity, as measured by cAMP production using a validated in vitro model assay, such as that described in Example 2.
  • a pure glucagon antagonist exhibits less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0%) of the maximum agonist activity achieved by glucagon at the glucagon receptor and exhibits less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, and about 0%) of the maximum agonist activity achieved by GLP-1 at the GLP-1 receptor.
  • EMBODIMENTS EMBODIMENTS
  • the present disclosures provide peptide combinations comprising a GIP agonist peptide and a glucagon antagonist peptide.
  • the activity of the GIP agonist peptide at the GIP receptor can be in accordance with any of the teachings set forth herein.
  • the activity of the glucagon antagonist peptide at the glucagon receptor can be in accordance with any of the teachings set forth herein.
  • the GIP agonist peptide exhibits at least 0.1% activity of native GIP at the GIP receptor and the glucagon antagonist peptide exhibits at least 60% inhibition of the maximum response achieved by glucagon at the glucagon receptor.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is within about 50-fold (e.g., within about 40-fold, within about 30-fold, within about 20-fold, within about 10-fold, within about 5-fold, within about 2-fold) of the EC50 at the GIP receptor of the GIP agonist peptide. In some embodiments, the EC50 at the GIP receptor of the GIP agonist peptide is greater than the IC50 of the glucagon antagonist peptide at the glucagon peptide. In alternative aspects, the EC50 at the GIP receptor of the GIP agonist peptide is less than the IC50 of the glucagon antagonist peptide at the glucagon peptide.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor divided by the EC50 of the GIP agonist peptide at the GIP receptor is less than or about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In certain aspects, the EC50 of the GIP agonist peptide at the GIP receptor divided by the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than or about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • either or both of the GIP agonist peptide and glucagon antagonist peptide additionally exhibit agonist activity at the GLP-1 receptor.
  • the activity at the GLP- 1 receptor of either or both of the GIP agonist peptide and glucagon antagonist peptide may be in accordance with any of the teachings described herein.
  • the peptide combinations are provided as a composition, such as, for example, a pharmaceutical composition.
  • the GIP agonist peptide is in admixture with the glucagon antagonist peptide.
  • the pharmaceutical composition in some aspects comprises a pharmaceutical acceptable carrier.
  • the peptide combinations are provided as a conjugate in which the GIP agonist peptide is attached via covalent or non-covalent bonds (or a mixture of both types of bonds) to the glucagon antagonist peptide.
  • the GIP agonist peptide is covalently attached to the glucagon antagonist peptide via peptide bonds.
  • the conjugate is a single polypeptide chain (e.g., a fusion peptide) comprising the GIP agonist peptide and glucagon antagonist peptide.
  • the fusion peptide can be produced recombinantly.
  • the GIP agonist peptide is attached to the glucagon antagonist peptide via one or more side chain functional groups of one or more amino acids of the GIP agonist peptide and/or glucagon antagonist peptide.
  • the conjugate is a heterodimer (or multimer) comprising the GIP agonist peptide and glucagon antagonist peptide attached to one another.
  • the GIP agonist peptide is attached to the glucagon antagonist peptide via a linker, e.g., a bifunctional linker.
  • the bifunctional linker is a hydrophilic polymer, e.g., polyethylene glycol.
  • the bifunctional linker connects a Cys residue of one of the GIP agonist peptide and glucagon antagonist peptide to a Lys of the other peptide.
  • each of the Cys and Lys is located at the C-terminus of the peptide or within the C-terminal region of the peptide.
  • the peptide combination is provided as a kit.
  • the GIP agonist peptide is packaged together with the glucagon antagonist peptide.
  • the GIP agonist peptide is packaged separately from the glucagon antagonist peptide.
  • the kit in some aspects comprises instructions of administering the GIP agonist peptide and glucagon antagonist peptide.
  • the GIP agonist peptide and glucagon antagonist peptide may be co-administered together or separately, simultaneously or sequentially (so long as both peptides exert the desired activity during an overlapping time period).
  • Methods of co-administering the GIP agonist peptide and glucagon antagonist peptide for therapeutic purpose(s) are provided herein. Also provided are the use of a GIP agonist peptide in the preparation of a
  • glucagon antagonist peptide for co-administration with a glucagon antagonist peptide and the use of a glucagon antagonist peptide in the preparation of a medicament for co-administration with a GIP agonist peptide.
  • the GIP agonist peptide exhibits at least or about 0.1% activity of native GIP at the GIP receptor.
  • the GIP agonist peptide exhibits at least or about 0.2%, at least or about 0.3%, at least or about 0.4%, at least or about 0.5%, at least or about 0.6%, at least or about 0.7%, at least or about 0.8%, at least or about 0.9%, at least or about 1%, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 100% of the activity of native GIP at the GIP receptor.
  • the GIP agonist peptide exhibits activity at the GIP receptor which is greater than that of native GIP.
  • the GIP agonist peptide exhibits at least or about 101%, at least or about 105%, at least or about 110%, at least or about 125%, at least or about 150%, at least or about 175% at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500% or higher % of the activity of native GIP at the GIP receptor.
  • the GIP agonist peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GIP receptor relative to native GIP.
  • a peptide's activity at the GIP receptor relative to native GIP is calculated as the inverse ratio of EC50s for the GIP agonist peptide vs. native GIP.
  • the GIP agonist peptide exhibits an EC50 for GIP receptor activation which is in the nanomolar range.
  • the EC50 of the GIP agonist peptide at the GIP receptor is less than 1000 nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM.
  • the EC50 of the peptide at the GIP receptor is about 100 nM or less, e.g., about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • the GIP agonist peptide exhibits an EC50 for GIP receptor activation which is in the picomolar range.
  • the EC50 of the GIP agonist peptide at the GIP receptor is less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM.
  • the EC50 of the peptide at the GIP receptor is about 100 pM or less, e.g., about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 8 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1 pM or less.
  • Receptor activation can be measured by in vitro assays measuring cAMP induction in HEK293 cells over- expressing the GIP receptor, e.g. assaying HEK293 cells co-transfected with DNA encoding the receptor and a luciferase gene linked to cAMP responsive element as described in Example 2.
  • the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the GIP agonist peptide does not activate the glucagon receptor to any appreciable degree. Accordingly, in some aspects of the present disclosures, the
  • the GIP agonist peptide is a GIP agonist 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 glucagon at the glucagon receptor.
  • the GIP agonist peptide is a co- agonist peptide insofar as it activates a second receptor different from the GIP receptor, in addition to the GIP receptor.
  • the GIP agonist peptide in some aspects exhibits activity at both the GIP receptor and the GLP-1 receptor ("GLP-1/GIP receptor co- agonists").
  • the EC50 of the GIP agonist peptide at the GIP receptor is within about 50- or less fold (higher or lower) than the EC50 of the GIP agonist peptide at the GLP-1 receptor.
  • the EC50 of the GIP agonist peptide at the GIP receptor is within about 40-fold, about 30-fold, about 20-fold (higher or lower) from its EC50 at the GLP-1 receptor.
  • the GIP potency of the GIP agonist peptide is less than or about 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its GLP-1 potency.
  • the ratio of the EC50 of the GIP agonist peptide at the GIP receptor divided by the EC50 of the GIP agonist peptide at the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the ratio of the GIP potency of the GIP agonist peptide compared to the GLP- 1 potency of the GIP agonist peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the EC50 of the GIP agonist peptide at the GLP-1 receptor divided by the EC50 of the GIP agonist peptide at the GIP receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the ratio of the GLP-1 potency of the GIP agonist peptide compared to the GIP potency of the GIP agonist peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the selectivity of the GIP agonist peptide does not have at least 100- fold selectivity for the human GLP-1 receptor versus the GIP receptor.
  • the selectivity of the GIP agonist peptide for the human GLP-1 receptor versus the GIP receptor is less than 100-fold (e.g., less than or about 90-fold, less than or about 80- fold, less than or about 70-fold, less than or about 60-fold, less than or about 50-fold, less than or about 40-fold, less than or about 30-fold, less than or about 20-fold, less than or about 10-fold, less than or about 5-fold).
  • the GIP agonist peptide exhibits at least or about 0.1% activity of native GLP-1 at the GLP-lreceptor.
  • the GIP agonist peptide exhibits at least or about 0.2%, at least or about 0.3%, at least or about 0.4%, at least or about 0.5%, at least or about 0.6%, at least or about 0.7%, at least or about 0.8%, at least or about 0.9%, at least or about 1%, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 100% of the activity of native GLP-1 at the GLP-1 receptor.
  • the GIP agonist peptide exhibits activity at only the GIP receptor, and not the GLP-1 receptor.
  • the GIP agonist peptide is a GIP agonist 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 glucagon GLP-1.
  • the GIP agonist peptide exhibits a decreased activity (e.g., a lower potency or higher EC50) than when the GIP agonist peptide is in a free or unconjugated form.
  • the GIP agonist peptide when the GIP agonist peptide is free or unconjugated, the GIP agonist peptide exhibits a potency at the GIP receptor that is about 10-fold or greater than the potency of the GIP agonist peptide when the GIP agonist peptide is conjugated to a heterologous moiety (e.g., a hydrophilic moiety).
  • the GIP agonist peptide when unconjugated, exhibits a potency at the GIP 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 GIP agonist peptide when conjugated to a heterologous moiety.
  • the GIP agonist peptide when unconjugated, exhibits a potency at the GIP 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 GIP agonist peptide when conjugated to a glucagon antagonist peptide.
  • the GIP receptor agonist is an analog of native human GIP, the amino acid sequence of which is provided herein as SEQ ID NO: 2. Accordingly, in some embodiments, the GIP agonist peptide comprises an amino acid sequence which is based on the amino acid sequence of SEQ ID NO: 2 but is modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.), amino acid modifications.
  • the GIP analog comprises 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 amino acid modifications relative to the native human GIP sequence (SEQ ID NO: 2).
  • the modifications are any of those described herein, e.g., acylation, alkylation, pegylation, truncation at C-terminus, substitution of the amino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.
  • Exemplary GIP receptor agonists are known in the art.
  • the GIP agonist peptide of the present disclosures comprises an amino acid sequence which has at least 25% sequence identity to the amino acid sequence of native human GIP (SEQ ID NO: 2).
  • the GIP agonist peptide comprises an amino acid sequence which is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to SEQ ID NO: 2.
  • the amino acid sequence of the GIP agonist peptide which has the above-referenced % sequence identity is the full-length amino acid sequence of the GIP agonist peptide.
  • the amino acid sequence of the GIP agonist peptide which has the above-referenced % sequence identity is only a portion of the amino acid sequence of the GIP agonist peptide.
  • the GIP agonist peptide comprises an amino acid sequence which has about A% or greater sequence identity to a reference amino acid sequence of at least 5 contiguous amino acids (e.g., at least 6, at least 7, at least 8, at least 9, at least 10 amino acids) of SEQ ID NO: 2, wherein the reference amino acid sequence begins with the amino acid at position C of SEQ ID NO: 2 and ends with the amino acid at position D of SEQ ID NO: 2, wherein A is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8,
  • the GIP agonist peptide is an analog of native GIP comprising an amino acid modification at position 1, position 2, or at both positions 1 and 2, wherein the amino acid modification confers the peptide with increased resistance to DPP-rV protease cleavage.
  • the amino acid modification which confers the peptide with increased resistance to DPP-IV protease cleavage are any of those described herein with regard to analogs of native human glucagon.
  • the amino acid modification may be a substitution of the Tyr at position 1 of SEQ ID NO: 2 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.
  • D-histidine alpha
  • alpha-dimethyl imidiazole acetic acid (DMIA) N- methyl histidine
  • alpha-methyl histidine alpha-methyl histidine
  • imidazole acetic acid desaminohistidine
  • hydroxyl- histidine acetyl-histidine
  • homo-histidine homo-histidine
  • the amino acid modification is a substitution of the Ala at position 2 of SEQ ID NO: 2 with an amino acid selected from the group consisting of D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB).
  • the GIP agonist peptide which is an analog of native GIP comprises or further comprises a C-terminal extension comprising 1-21 amino acids.
  • extensions are known in the art and include those described herein with regard to fusion peptides of glucagon analogs.
  • the extension comprises the amino acid sequence of any of SEQ ID NOs: 3 to 9.
  • the Xaa of SEQ ID NO: 4, 6, or 7 is a small, aliphatic residue, e.g., a Gly.
  • the C-terminal extension comprises 1-6 positive-charged amino acids, e.g., Arg, an analog of Arg, an amino acid of Formula IV, e.g., Lys, d-Lys, Orn, Dab, etc.
  • GIP agonist peptide is an analog of GIP and comprises an amino acid modification which reduces agonist activity at the GIP receptor to, e.g., a level such that the EC50 at the GIP receptor is within 10-fold of the IC50 of the glucagon antagonist peptide of the peptide combination.
  • Suitable amino acid modifications that reduce GIP agonist activity include, for example, substitution of the Tyr at position 1 with a small aliphatic amino acid residue, e.g., Ala, Gly, or with an imidazole containing amino acid, e.g., His, or an analog thereof.
  • the amino acid modification that reduce GIP agonist activity is a deletion of the amino acid at position 1 or a deletion of the amino acids at positions 1 and 2.
  • GIP amino acid sequence SEQ ID NO: 2
  • GIP agonist peptide which is an analog of native glucagon, e.g., any of those taught as affecting activity at the GIP receptor, GLP-1 receptor, and/or glucagon receptor, increasing stability, solubility, half-life, time of action, and the like, are contemplated.
  • the GIP agonist peptide is structurally similar to native human glucagon (SEQ ID NO: 1), e.g., is an analog of native human glucagon (or "glucagon analog").
  • native human glucagon or "glucagon analog”
  • Such analogs of glucagon exhibiting GIP receptor agonist activity are known in the art. See, for example, the teachings of International Patent Application No. PCT
  • the GIP agonist peptide is an analog of native human glucagon (SEQ ID NO: 1) which comprises an amino acid sequence based on the amino acid sequence of SEQ ID NO: 1 but is modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.), amino acid modifications.
  • the GIP agonist peptide comprises 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 amino acid
  • modifications relative to the native human glucagon sequence are any of those described herein, e.g., acylation, alkylation, pegylation, truncation at C-terminus, substitution of the amino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.
  • the GIP agonist peptide of the present disclosures comprises an amino acid sequence which has at least 25% sequence identity to the amino acid sequence of native human glucagon (SEQ ID NO: 1). In some embodiments, the GIP agonist peptide comprises an amino acid sequence which is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the GIP agonist peptide which has the above-referenced % sequence identity is the full-length amino acid sequence of the GIP agonist peptide.
  • the amino acid sequence of the GIP agonist peptide which has the above-referenced % sequence identity is only a portion of the amino acid sequence of the GIP agonist peptide.
  • the GIP agonist peptide comprises an amino acid sequence which has about A% or greater sequence identity to a reference amino acid sequence of at least 5 contiguous amino acids (e.g., at least 6, at least 7, at least 8, at least 9, at least 10 amino acids) of SEQ ID NO: 1, wherein the reference amino acid sequence begins with the amino acid at position C of SEQ ID NO: 1 and ends with the amino acid at position D of SEQ ID NO: 1, wherein A is 25, 30,
  • C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29. Any and all possible combinations of the foregoing parameters are envisioned, including but not limited to, e.g., wherein A is 90% and C and D are 1 and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and 27, or 16 and 27.
  • the GIP agonist peptides which are analogs of native human glucagon (SEQ ID NO: 1) described herein may comprise a peptide backbone of any number of amino acids, i.e., can be of any peptide length.
  • the GIP agonist peptides described herein are the same length as SEQ ID NO: 1, i.e., are 29 amino acids in length.
  • the GIP agonist peptide is longer than 29 amino acids in length, e.g., the GIP agonist peptide comprises a C-terminal extension of 1-21 amino acids, as further described herein. Accordingly, the GIP agonist peptide in some embodiments, is 30, 31, 32, 33, 34, 35,
  • the GIP agonist peptide is up to 50 amino acids in length. In some embodiments, the GIP agonist peptide is up to 50 amino acids in length. In some
  • the GIP agonist peptide is longer than 29 amino acids in length (e.g., greater than 50 amino acids, (e.g., at least or about 60, at least or about 70, at least or about 80, at least or about 90, at least or about 100, at least or about 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, at least or about 400, at least or about 450, at least or about 500 amino acids in length) due to fusion with another peptide.
  • the GIP agonist peptide is less than 29 amino acids in length, e.g., 28, 27, 26, 25, 24, 23, amino acids.
  • the GIP agonist peptide of the present disclosures is an analog of native human glucagon (SEQ ID NO: 1) comprising SEQ ID NO: 1 modified with one or more amino acid modifications which affect GIP activity, glucagon activity, and/or GLP-1 activity, enhance stability, e.g., by reducing degradation of the peptide (e.g., by improving resistance to DPP-IV proteases), enhance solubility, increase half-life, delay the onset of action, extend the duration of action at the GIP, glucagon, or GLP-1 receptor, or a combination of any of the foregoing.
  • amino acid modifications in addition to other modifications, are further described herein.
  • the GIP agonist peptide which is an analog of glucagon comprises SEQ ID NO: 1 with (a) an amino acid modification at position 1 that confers GIP agonist activity, (b) a modification which stabilizes the alpha helix structure of the C-terminal portion (amino acids 12-29) of the GIP agonist peptide, and (c) optionally, 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) further amino acid modifications.
  • the analog exhibits at least or about 0.1% (e.g., at least or about 0.25%, at least or about 0.5%, at least or about 0.75%, at least or about 1%) activity of native GIP at the GIP receptor or any other activity level at the GIP receptor described herein.
  • the modification which stabilizes the alpha helix structure is one which provides or introduces an intramolecular bridge, including, for example, a covalent intramolecular bridge, such as any of those described herein.
  • the covalent intramolecular bridge in some embodiments is a lactam bridge.
  • the lactam bridge of the GIP agonist peptide of these embodiments can be a lactam bridge as described herein.
  • the lactam bridge may be one which is between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17.
  • the lactam bridge can be between the amino acids at positions 16 and 20, wherein one of the amino acids at positions 16 and 20 is substituted with Glu and the other of the amino acids at positions 16 and 20 is substituted with Lys.
  • the modification which stabilizes the alpha helix structure is the introduction of one, two, three, or four ⁇ , ⁇ -disubstituted amino acids at position(s) 16, 20, 21, and 24 of the GIP agonist peptide.
  • the ⁇ , ⁇ - disubstituted amino acid is AIB.
  • the ⁇ , ⁇ -disubstituted amino acid e.g., AIB
  • the amino acid atposition 16 is substituted with a positive-charged amino acid, such as, for example, an amino acid of Formula IV, which is described herein.
  • the amino acid of Formula IV may be homo Lys, Lys, Orn, or 2,4-diaminobutyric acid (Dab).
  • the amino acid modification at position 1 that confers GIP agonist activity can be a substitution of His with an amino acid lacking an imidazole side chain.
  • the amino acid modification at position 1 can, for example, be a substitution of His with a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • the GIP agonist peptide comprises an amino acid modification at position 1 that confers GIP agonist activity and a modification which stabilizes the alpha helix structure of the C-terminal portion (amino acids 12-29) of the GIP agonist peptide
  • the GIP agonist peptide further comprises amino acid modifications at one, two or all of positions 27, 28 and 29.
  • the Met at position 27 is substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 is substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 is substituted with a small aliphatic amino acid, optionally Gly, or a combination of two or three of the foregoing.
  • the GIP agonist peptide which is a glucagon analog comprises Leu at position 27, Ala at position 28, and Gly or Thr at position 29.
  • the GIP agonist peptide comprises an amino acid modification at position 1 that confers GIP agonist activity and a modification which stabilizes the alpha helix structure of the C-terminal portion (amino acids 12-29) of the GIP agonist peptide
  • the GIP agonist peptide further comprises an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29.
  • the extension in some aspects comprises the amino acid sequence of SEQ ID NO: 3 or 4, for instance.
  • the GIP agonist peptide in some aspects comprises an extension of which 1-6 amino acids of the extension are positive-charged amino acids.
  • the positive- charged amino acids in some embodiments are amino acids of Formula IV, including, but not limited to Lys, d-Lys, homoLys, Orn, and Dab.
  • the positive-charged amino acid is Arg, or an analog thereof.
  • the extension comprises 1-6 aa that are negative-charged amino acids, e.g., Asp, Glu.
  • the GIP agonist peptide comprises an amino acid modification at position 1 that confers GIP agonist activity and a modification which stabilizes the alpha helix structure of the C-terminal portion (amino acids 12-29) of the GIP agonist peptide
  • the GIP agonist peptide is acylated or alkylated as described herein.
  • the acyl or alkyl group is attached to the GIP agonist peptide, with or without a spacer, at position 10 or 40 of the GIP agonist peptide, as further described herein.
  • the GIP agonist peptide in addition or alternative aspects is modified to comprise a hydrophilic moiety as further described herein.
  • the GIP agonist peptide comprises any one or a combination of the following modifications:
  • homocysteic acid Thr, Gly, or AIB
  • the GIP agonist peptide which is an analog of glucagon comprises the following modifications:
  • the EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • the lactam bridge of the GIP agonist peptide of these embodiments can be a lactam bridge as described herein. See, e.g., the teachings of lactam bridges under the section "Stabilization of the Alpha Helix Structure.”
  • the lactam bridge can be between the amino acids at positions 16 and 20, wherein one of the amino acids at positions 16 and 20 is substituted with Glu and the other of the amino acids at positions 16 and 20 is substituted with Lys.
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID NOs: 105-194.
  • the GIP agonist peptide comprises a modified amino acid sequence of SEQ ID NOs: 105-194.in which the amino acid at position 1 is substituted with Ala or is deleted.
  • the GIP agonist peptide which is an analog of glucagon (SEQ ID NO: 1) and which exhibits GIP agonist activity comprises the following modifications:
  • amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an ⁇ , ⁇ -disubstituted amino acid, amino acid modifications at one, two or all of positions 27, 28 and 29, e.g., amino acid modifications at position 27 and/or 28, and
  • the EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • the ⁇ , ⁇ -disubstituted amino acid of the GIP agonist peptide of these embodiments can be any ⁇ , ⁇ -disubstituted amino acid, including, but not limited to, amino iso-butyric acid (AIB), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g., 1-aminocyclooctane- 1-carboxylic acid).
  • the ⁇ , ⁇ -disubstituted amino acid is AIB.
  • the amino acid at position 20 is substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., AIB.
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID NOs: 199-241, 244-264, 266-269, and 273-278.
  • the GIP agonist peptide comprises a modified amino acid sequence of SEQ ID NOs: 199-241, 244-264, 266-269, and 273-278 in which the amino acid at position 1 is substituted with Ala or is deleted.
  • the GIP agonist peptide which is an analog of glucagon comprises the following modifications:
  • the EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • the amino acid of Formula IV of the analog of these embodiments may be any amino acid, such as, for example, the amino acid of Formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4, or 5, in which case, the amino acid is Dab, Orn, Lys, or homoLys respectively.
  • the alpha, alpha-disubstituted amino acid of the GIP agonist peptide of these embodiments may be any alpha, alpha-disubstituted amino acid, including, but not limited to, amino iso-butyric acid (AIB), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g., 1-aminocyclooctane-l-carboxylic acid).
  • 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).
  • the analog can comprise, for example, the amino acid sequence of any of SEQ ID NOs: 199-265.
  • the GIP agonist peptide comprises a modified amino acid sequence of SEQ ID NOs: 199-265 in which the amino acid at position 1 is substituted with Ala or is deleted.
  • the GIP agonist peptide which is an analog of glucagon comprises:
  • EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • the acylated or alkylated amino acid is an amino acid of Formula I, II, or III.
  • the amino acid of Formula I is Dab, Orn, Lys, or homoLys.
  • the extension of about 1 to about 21 amino acids comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) or
  • XGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 5) or XGPSSGAPPPK (SEQ ID NO: 6) or XGPSSGAPPPSK (SEQ ID NO: 7), wherein X is Gly or a small, aliphatic or non-polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to SEQ ID NOs: 3, 4, 5, 6, or 7. In some
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extended analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminally extended analog.
  • the GIP agonist peptide which is an analog of glucagon (SEQ ID NO: 1) further comprises amino acid modifications at one, two or all of positions 27, 28 and 29, e.g., amino acid modifications at position 27 and/or 28.
  • the amino acid modification at position 1 that confers GIP agonist activity can be a substitution of His with an amino acid lacking an imidazole side chain.
  • the amino acid modification at position 1 can, for example, be a substitution of His with a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • amino acid modifications at one, two, or all of positions 27, 28, and 29 can be any of the modifications at these positions described herein.
  • the Met at position 27 can be substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 can be substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 can be substituted with a small aliphatic amino acid, optionally Gly.
  • the analog can comprise such amino acid modifications at position 27 and/or 28.
  • the GIP agonist peptide of the above exemplary embodiments can further comprise 1-9 or 1-6 further, additional amino acid modifications, e.g.
  • the GIP agonist peptide which is an analog of glucagon in some aspects comprises one or more of the following modifications:
  • the GIP agonist peptide in some embodiments comprises a combination of the modifications (a) through (1).
  • the GIP agonist peptide can comprise an amino acid modification at position 3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gin with Glu), wherein the GIP agonist peptide has less than 1% of the activity of glucagon at the glucagon receptor.
  • the GIP agonist peptide can comprise an amino acid modification at position 7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), wherein the GIP agonist peptide has less than about 10% of the activity of GLP-1 at the GLP- 1 receptor.
  • an amino acid modification at position 7 of SEQ ID NO: 1 e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He
  • the GIP agonist peptide has less than about 10% of the activity of GLP-1 at the GLP- 1 receptor.
  • the GIP agonist peptide which is an analog of glucagon can be covalently linked to a hydrophilic moiety.
  • the GIP agonist peptide is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus.
  • the GIP agonist peptide comprises a C-terminal extension (e.g., an amino acid sequence of SEQ ID NO: 3) and an addition of an amino acid comprising the hydrophilic moiety, such that the hydrophilic moiety is covalently linked to the GIP agonist peptide at position 40.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the GIP agonist peptide.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to the glucagon sequence (SEQ ID NO: 1) or it may be an amino acid which is replacing a native amino acid of SEQ ID NO: 1.
  • the linkage to the hydrophilic moiety can comprise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the GIP agonist peptide which is an analog of glucagon (SEQ ID NO: 1) and which exhibits GIP agonist activity in some embodiments comprises a modified amino acid in which the side chain is covalently linked to an acyl or alkyl group (e.g., an acyl or alkyl group which is non-native to a naturally-occurring amino acid).
  • the acylated or alkylated analog can be in accordance with acylated or alkylated peptides described in the section "Acylation and alkylation.”
  • the acyl group is a C4 to a C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.
  • the acyl or alkyl group may be covalently attached to any amino acid of the analog, including, but not limited to the amino acid at position 10 or 40, or the C- terminal amino acid.
  • the analog comprises a C-terminal extension (e.g., an amino acid sequence of SEQ ID NO: 3) and an addition of an amino acid comprising the acyl or alkyl group, such that the acyl or alkyl group is covalently linked to the analog at position 40.
  • the acyl or alkyl group is covalently linked to the side chain of an amino acid of Formula I, II, or III, e.g., a Lys residue.
  • the acyl or alkyl group may be covalently linked to an amino acid which is native to the glucagon sequence (SEQ ID NO: 1) or may be linked to an amino acid which is added to the sequence of SEQ ID NO: 1 or to the sequence of SEQ ID NO: 1 followed by SEQ ID NO: 3 (at the N- or C-terminus) or may be linked to an amino acid which replaces a native amino acid, e.g., the Tyr at position 10 of SEQ ID NO: 1.
  • the GIP agonist peptide may be attached to the acyl or alkyl group via a spacer, as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-pAla, Leu-Leu, Pro-Pro, yGlu-yGlu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • the GIP agonist peptide which is an analog of glucagon comprises the amino acid sequence according to any one of SEQ ID NOs: 327, 328, 329, or 330 that further comprises the following modifications:
  • EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • the GIP agonist peptide comprises SEQ ID NOs: 327-330 in which the amino acid at position 1 is substituted with Ala or is deleted.
  • the acylated or alkylated amino acid is an amino acid of Formula I, II, or III.
  • the amino acid of Formula I is Dab, Orn, Lys, or homoLys.
  • the about 1 to about 21 amino acids comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 5) or XGPSSGAPPPK (SEQ ID NO: 6) or XGPSSGAPPPSK (SEQ ID NO: 7), wherein X is Gly or a small, aliphatic or non-polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to SEQ ID NO: 3, 4, 5, 6, or 7.
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally- extended analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminally extended analog.
  • the amino acid at position 1 that confers GIP agonist activity can be an amino acid lacking an imidazole side chain.
  • the amino acid at position 1 can, for example, be a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • the GIP agonist peptide of the above exemplary embodiments can further comprise 1-6 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half-life in circulation, delay the onset of action, or increase stability.
  • GIP agonist peptides described in the above exemplary embodiment comprise further amino acid modifications at one, two or all of positions 27, 28 and 29. Modifications at these positions can be any of the modifications described herein relative to these positions.
  • position 27 can be substituted with a large aliphatic amino acid (e.g., Leu, He or norleucine) or Met
  • position 28 can be substituted with another small aliphatic amino acid (e.g., Gly or Ala) or Asn
  • position 29 can be substituted with another small aliphatic amino acid (e.g., Ala or Gly) or Thr.
  • the analog can comprise such amino acid modifications at position 27 and/or 28.
  • the analog can further comprise one or more of the following additional modifications:
  • amino acid at position 2 is any one of D-Ser, Ala, D-Ala, Gly, N- methyl-Ser, AIB, Val, or a-amino-N-butyric acid;
  • amino acid at position 10 is Tyr, Trp, Lys, Orn, Glu, Phe, or Val;
  • amino acid at position 12 is He, Lys or Arg
  • amino acid at position 16 is any one of Ser, Glu, Gin, homo glutamic acid, homocysteic acid, Thr, Gly, or AIB;
  • amino acid at position 18 is any one of Ala, Arg, Ser, Thr, or Gly;
  • amino acid at position 20 is any one of Ala, Ser, Thr, Lys,
  • amino acid at position 21 is any one of Glu, Asp, homoglutamic acid, homocysteic acid;
  • amino acid at position 23 is Val or He;
  • amino acid at position 24 is any one of Gin, Asn, Ala, Ser, Thr, or
  • the GIP agonist peptide in some embodiments comprises a combination of the modifications (a) through (1).
  • the GIP agonist peptide can comprise an amino acid modification at position 3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gin with Glu), wherein the GIP agonist peptide has less than 1% of the activity of glucagon at the glucagon receptor.
  • the GIP agonist peptide can comprise an amino acid modification at position 7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), wherein the GIP agonist peptide has less than about 10% of the activity of GLP-1 at the GLP- 1 receptor.
  • an amino acid modification at position 7 of SEQ ID NO: 1 e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He
  • the GIP agonist peptide has less than about 10% of the activity of GLP-1 at the GLP- 1 receptor.
  • the analog can be covalently linked to a hydrophilic moiety.
  • the analog is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C- terminus.
  • the analog comprises a hydrophilic moiety covalently linked to the analog at position 24.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to SEQ ID NO: 1, 227, 228, 229 or 230 of Sequence Listing 2, or it may be a substituted amino acid.
  • the linkage may comprise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the GIP agonist peptide can comprise a modified amino acid within the C-terminal extension in which the side chain is covalently linked to an acyl or alkyl group.
  • the acylated or alkylated analog can be in accordance with acylated or alkylated peptides described in the section "Acylation and alkylation.”
  • the acyl group is a C4 to a C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.
  • the acyl or alkyl group may be covalently attached to any amino acid of the analog, including, but not limited to the amino acid at position 10 or 40, or the C- terminal amino acid.
  • the acyl or alkyl group is covalently linked to the side chain of an amino acid of Formula I, II, or III, e.g., a Lys residue.
  • the acyl or alkyl group is covalently linked to an amino acid which is native to SEQ ID NO: 1, 327, 328, 329, or 330 or it may be linked to a substituted amino acid.
  • the acyl or alkyl group is covalently linked to an amino acid which is native to SEQ ID NO: 3, 4, 6 or 7, or it may be linked to a substituted amino acid.
  • the GIP agonist peptide may be attached to the acyl or alkyl group via a spacer, as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-pAla, Leu-Leu, Pro-Pro, yGlu-yGlu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • an GIP agonist peptideof the present disclosures comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-241, 244-264, 266, 292-307, 309-321 and 323 or selected from the group consisting of SEQ ID NOs: 267-269, 273-278, and 325.
  • the GIP agonist peptide comprises a modified amino acid sequence of any of SEQ ID NOs: 199-241, 244- 264, 266, 292-307, 309-321, and 323 or any of SEQ ID NOs: 267-269, 273-278, and 325, in which the amino acid at position 1 is substituted with Ala or is deleted.
  • specific examples of GIP agonist peptides of the present disclosures include but are not limited to, any of SEQ ID NOs: 105-194, 199-246, 248-250, and 253-278.
  • the GIP agonist peptide which is an analog of glucagon comprises an acyl or alkyl group (e.g., an acyl or alkyl group which is non-native to a naturally occurring amino acid), wherein the acyl or alkyl group is attached to a spacer, wherein (i) the spacer is attached to the side chain of the amino acid at position 10 of the analog; or (ii) the analog comprises an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 and the spacer is attached to the side chain of an amino acid corresponding to one of positions 37-43 relative to SEQ ID NO: 1, wherein the EC50 of the analog for GIP receptor activation is about 10 nM or less.
  • SEQ ID NO: 1 comprises an acyl or alkyl group (e.g., an acyl or alkyl group which is non-native to a naturally occurring amino acid), wherein the acyl or alkyl group is attached to a spacer, wherein (i) the
  • the GIP agonist peptide may comprise an amino acid sequence of SEQ ID NO: 1 with (i) an amino acid modification at position 1 that confers GIP agonist activity, (ii) amino acid modifications at one, two, or all of positions 27, 28, and 29, (iii) at least one of:
  • the analog comprises a lactam bridge between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17;
  • the analog comprises (i) an amino acid substitution of Ser at position 16 with an amino acid of Formula IV:
  • n 1 to 7, wherein each of Rl and R2 is independently selected from the group consisting of H, CI -CI 8 alkyl, (CI -CI 8 alkyl)OH, (CI -CI 8 alkyl)NH2, (CI -CI 8 alkyl)SH, (C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl), wherein R7 is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group; and (ii) an amino acid substitution of the Gin at position 20 with an alpha, alpha-disubstituted amino acid, and (iii) up to 6 further amino acid modifications.
  • the alpha, alpha-disubstituted amino acid of the GIP agonist peptide of these embodiments may be any alpha, alpha-disubstituted amino acid, including, but not limited to, amino iso-butyric acid (AIB), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g., 1-aminocyclooctane-l-carboxylic acid).
  • 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).
  • the amino acid of Formula IV of the GIP agonist peptide of these embodiments may be any amino acid, such as, for example, the amino acid of Formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4, or 5, in which case, the amino acid is Dab, Orn, Lys, or homoLys respectively.
  • the amino acid modification at position 1 that confers GIP agonist activity can be a substitution of His with an amino acid lacking an imidazole side chain.
  • the amino acid modification at position 1 can, for example, be a substitution of His with a large, aromatic amino acid.
  • the large, aromatic amino acid is any of those described herein, including, for example, Tyr.
  • amino acid modifications at one, two, or all of positions 27, 28, and 29 can be any of the modifications at these positions described herein.
  • the Met at position 27 can be substituted with a large aliphatic amino acid, optionally Leu
  • the Asn at position 28 can be substituted with a small aliphatic amino acid, optionally Ala
  • the Thr at position 29 can be substituted with a small aliphatic amino acid, optionally Gly.
  • the GIP agonist peptide can comprise such amino acid modifications at position 27 and/or 28.
  • the GIP agonist peptide of the above exemplary embodiments can further comprise 1-9 or 1-6 further, additional amino acid modifications, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half-life in circulation, delay the onset of action, or increase stability.
  • additional amino acid modifications e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as, for example, any of the modifications described herein which increase or decrease the activity at any of the GIP, GLP-1, and glucagon receptors, improve solubility, improve duration of action or half-life in circulation, delay the onset of action, or increase stability.
  • the GIP agonist peptide can further comprise, for example, an amino acid modification at position 12, optionally, a substitution with lie, and/or amino acid modifications at positions 17 and 18, optionally substitution with Q at position 17 and A at position 18, and/or an addition of GPSSGAPPPS (SEQ ID NO: 3) or
  • the GIP agonist peptide which is an analog of glucagon comprises one or more of the following modifications:
  • homocysteic acid Thr, Gly, Lys, or AIB;
  • the GIP agonist peptide in some embodiments comprise a combination of the modifications (a) through (1).
  • the GIP agonist peptide can comprise an amino acid modification at position 3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gin with Glu), wherein the GIP agonist peptide has less than 1% of the activity of glucagon at the glucagon receptor.
  • the GIP agonist peptide can comprise an amino acid modification at position 7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He), a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28, yielding a 27- or 28-amino acid peptide, or a combination thereof, wherein the GIP agonist peptide has less than about 10% of the activity of GLP-1 at the GLP-1 receptor.
  • an amino acid modification at position 7 of SEQ ID NO: 1 e.g., an amino acid substitution of Thr with an amino acid lacking a hydroxyl group, e.g., Abu or He
  • a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28 yielding a 27- or 28-amino acid peptide, or a combination thereof, wherein the GIP agonist peptide has less than about 10% of the activity of GLP-1
  • the GIP agonist peptide can be covalently linked to a hydrophilic moiety.
  • the GIP agonist peptide is covalently linked to the hydrophilic moiety at any of amino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus.
  • the GIP agonist peptide comprises a C- terminal extension (e.g., an amino acid sequence of SEQ ID NO: 3) and an addition of an amino acid comprising the hydrophilic moiety, such that the hydrophilic moiety is covalently linked to the GIP agonist peptide at position 40.
  • the hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the GIP agonist peptide.
  • the Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an amino acid that is native to the glucagon sequence (SEQ ID NO: 1) or it may be an amino acid which is replacing a native amino acid of SEQ ID NO: 1.
  • the linkage to the hydrophilic moiety can comprise the structure
  • the hydrophilic moiety may be any of those described herein. See, e.g., the teachings under the section "Linkage of hydrophilic moieties.”
  • the hydrophilic moiety is a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG in certain embodiments has a molecular weight of about 1,000 Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000 Daltons.
  • the spacer can be any spacer as described herein.
  • the spacer may be 3 to 10 atoms in length and may be, for instance, an amino acid (e.g., 6-amino hexanoic acid, any amino acid described herein), a dipeptide (e.g., Ala-Ala, pAla-pAla, Leu-Leu, Pro-Pro, yGlu-yGlu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer.
  • the total length of the spacer and the acyl or alkyl group is about 14 to about 28 atoms.
  • the acyl or alkyl group is any acyl or alkyl group as described herein, such as an acyl or alkyl group which is non-native to a naturally occurring amino acid.
  • the acyl or alkyl group in some embodiments is a C4 to C30 fatty acyl group, such as, for example, a CIO fatty acyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group, or a C4 to C30 alkyl group.
  • the acyl group is a C12 to C18 fatty acyl group (e.g., a C14 or C16 fatty acyl group).
  • the extension of about 1 to about 21 amino acids C- terminal to the amino acid at position 29 of the GIP agonist peptide comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 5) or XGPSSGAPPPK (SEQ ID NO: 6) or XGPSSGAPPPSK (SEQ ID NO: 7), wherein X is Gly or a small, aliphatic or non-polar or slightly polar amino acid.
  • the about 1 to about 21 amino acids may comprise sequences containing one or more conservative substitutions relative to any of SEQ ID NO: 3, 4, 5, 6, or 7.
  • the acylated or alkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extended GIP agonist peptide.
  • the acylated or alkylated amino acid is located at position 40 of the C- terminally extended GIP agonist peptide.
  • the GIP agonist peptide which is an analog of glucagon is a peptide comprising the amino acid sequence of any of the amino acid sequences, e.g., SEQ ID NOs: 105-194, optionally with up to 1, 2, 3, 4, or 5 further modifications that retain GIP agonist activity.
  • the GIP agonist peptide which is an analog of glucagon (SEQ ID NO: 1) and which exhibits GIP agonist activity comprises the amino acids of any of SEQ ID NOs: 199-362.
  • the GIP agonist peptide comprises a modified amino acid sequence of SEQ ID NO: 199-362 in which the amino acid at position 1 is substituted with Ala or is deleted.
  • the GIP agonist peptide comprises the amino acid sequence of SEQ ID NO: 1 with at least one amino acid modification (optionally, up to 15 amino acid modifications), and an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 of the GIP agonist peptide.
  • the GIP agonist peptide comprises at least one amino acid modification and up to 15 amino acid modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid modifications, up to 10 amino acid modifications).
  • the GIP agonist peptides comprise at least one amino acid modification at up to 10 amino acid modifications and additional conservative amino acid modifications. Conservative amino acid
  • the GIP agonist peptide comprises an intramolecular bridge (e.g., a covalent intramolecular bridge, a non-covalent intramolecular bridge) between the side chains of two amino acids of the GIP agonist peptide.
  • an intramolecular bridge links the side chains of the amino acids at positions i and i+4, wherein i is 12, 13, 16, 17, 20, or 24. In other aspects, an intramolecular bridge connects the side chains of the amino acids at positions j and j+3, wherein j is 17, or at positions k and k+7" wherein k is any integer between 12 and 22.
  • the intramolecular bridge is a covalent intramolecular bridge, e.g., a lactam bridge. In specific aspects, the lactam bridge connects the side chains of the amino acids at positions 16 and 20. In particular aspects, one of the amino acids at positions 16 and 20 is a positive-charged amino acid and the other is a negative-charged amino acid.
  • the GIP agonist peptide can comprise a lactam bridge connecting the side chains of a Glu at position 16 and a Lys at position 20.
  • the negative-charged amino acid and the positive-charged amino acid form a salt bridge.
  • intramolecular bridge is a non-covalent intramolecular bridge.
  • the amino acid modification which confers a stabilized alpha helix is an insertion or substitution of an amino acid of SEQ ID NO: 1 with an ⁇ , ⁇ - disubstituted amino acid.
  • Suitable ⁇ , ⁇ -disubstituted amino acids for purposes of stabilizing the alpha helix are described herein and include, for example, AIB.
  • one, two, three, or more of the amino acids at positions 16, 20, 21, and 24 of SEQ ID NO: 1 are substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., AIB.
  • the amino acid at position 16 is AIB.
  • the GIP agonist peptide in some aspects comprises additional modifications, such as any of those described herein.
  • the amino acid modifications may increase or decrease activity at the GLP- 1 receptor or decrease activity at the glucagon receptor.
  • the amino acid modifications may increase stability of the peptide, e.g., increase resistance to DPP-IV protease degradation, stabilize the bond between amino acids 15 and 16.
  • the amino acid modifications may increase the solubility of the peptide and/or alter the time of action of the GIP agonist peptide at any of the GIP, glucagon, and GLP-1 receptors. A combination of any of these types of modifications may be present in the GIP agonist peptides which exhibit agonist activity at the GIP receptor.
  • the GIP agonist peptide comprises the amino acid sequence of SEQ ID NO: 1 with one or more of: Gin at position 17, Ala at position 18, Glu at position 21, He at position 23, and Ala, Asn, or Cys at position 24, or conservative amino acid substitutions thereof.
  • the GIP agonist peptide comprises a C-terminal amide in place of the C-terminal alpha carboxylate.
  • the GIP agonist peptide comprises an amino acid substitution at position 1, position 2, or positions 1 and 2, which substitution(s) achieve DPP-IV protease resistance. Suitable amino acid substitutions are described herein. For example, DMIA at position 1 and/or d-Ser or AIB at position 2.
  • the GIP agonist peptide may comprise one or a combination of: (a) Ser at position 2 substituted with Ala; (b) Gin at position 3 substituted with Glu or a glutamine GIP agonist peptide; (c) Thr at position 7 substituted with a He; (d) Tyr at position 10 substituted with Trp or an amino acid comprising an acyl or alkyl group which is non-native to a naturally-occurring amino acid; (e) Lys at position 12 substituted with He; (f) Asp at position 15 substituted with Glu; (g) Ser at position 16 substituted with Glu; (h) Gin at position 20 substituted with Ser, Thr, Ala, AIB; (i) Gin at position 24 substituted with Ser, Thr, Ala, AIB; j) Met at position 27 substituted with Leu or Nle; (k) Asn at position 29 substituted with a charged amino acid, optionally, Asp or Glu; and (1) Thr at position 29 substituted with a charged amino acid,
  • the GIP agonist peptide comprises the amino acid sequence of SEQ ID NO : 1 with at least one amino acid modification (optionally, up to 15 amino acid modifications), an extension of 1 to 21 amino acids C-terminal to the amino acid at position 29 of the GIP agonist peptide, and an amino acid modification at position 1 which
  • the GIP agonist peptide does not comprise an amino acid modification at position 1 which modification confers GIP agonist activity.
  • the amino acid at position 1 is not a large, aromatic amino acid, e.g., Tyr.
  • the amino acid at position 1 is an amino acid comprising an imidazole ring, e.g., His, analogs of His.
  • the GIP agonist peptide is not any of the compounds disclosed in U.S. Patent Application No. 61/151,349.
  • the GIP agonist peptide comprises the amino acid sequence of any of SEQ ID NOs: 1057-1069.
  • the GIP agonist peptide comprises a modified amino acid sequence of any of the glucagon-based sequences of any of SEQ ID NOs: 402-1056 in which the referenced amino acid sequence is modified to have (if it does not already have) (i) a stabilized alpha helix in the C-terminal portion of the peptide (e.g., amino acids 12-29), e.g., stabilized via an intramolecular bridge (e.g., a lactam bridge, a salt bridge) as described herein or stabilized via incorporation of one or more alpha, alpha disubstituted amino acids, e.g., AIB, at, for example, positions 16, 20, 21, 24 of the peptide; (ii) Leu at position 27, Ala at position 28, and Gly at position 29, or conservative amino acid substitutions thereof; (iii) He at position 12, or a conservative amino acid substitution thereof, and optionally, (iv) an amino acid modification at position 1 which confers GIP activity as described here
  • the extension of the GIP agonist peptide may comprise any amino acid sequence, provided that the extension is 1 to 21 amino acids. In some aspects, the extension is 7 to 15 amino acids and in other aspects, the extension is 9 to 12 amino acids.
  • the extension comprises (i) the amino acid sequence of SEQ ID NO: 426 or 1074, (ii) an amino acid sequence which has high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 98%, 99%) with the amino acid sequence of SEQ ID NO: 426 or 1074, or (iii) the amino acid sequence of (i) or (ii) with one or more conservative amino acid modifications.
  • At least one of the amino acids of the extension is acylated or alkylated.
  • the amino acid comprising the acyl or alkyl group may be located at any position of extension of the GIP agonist peptide.
  • the acylated or alkylated amino acid of the extension is located at one of positions 37, 38, 39, 40, 41, or 42 (according to the numbering of SEQ ID NO: 1) of the GIP agonist peptide.
  • the acylated or alkylated amino acid is located at position 40 of the GIP agonist peptide.
  • the acyl or alkyl group is an acyl or alkyl group which is non-native to a naturally- occurring amino acid.
  • the acyl or alkyl group may be a C4 to C30 (e.g., C12 to C18) fatty acyl group or C4 to C30 (e.g., C12 to C18) alkyl.
  • the acyl or alkyl group may be any of those discussed herein.
  • the acyl or alkyl group is attached directly to the amino acid, e.g., via the side chain of the amino acid.
  • the acyl or alkyl group is attached to the amino acid via a spacer (e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilic bifunctional spacer, a hydrophobic bifunctional spacer).
  • the spacer is 3 to 10 atoms in length.
  • the spacer is an amino acid or dipeptide comprising one or two of 6-aminohexanoic acid, Ala, Pro, Leu, beta- Ala, gamma- Glu (e.g., gamma-Glu-gamma-Glu).
  • the total length of the spacer is 14 to 28 atoms.
  • the amino acid to which the acyl or alkyl group is attached may be any of those described herein, including, for example, an amino acid of Fomula I, II, or III.
  • the amino acid which is acylated or alkylated may be a Lys, for example.
  • Suitable amino acids comprising an acyl or alkyl group, as well as suitable acyl groups, alkyl groups, and spacers are described herein. See, for example, the teachings under the sections entitled Acylation and Alkylation.
  • 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 amino acids) of the extension are positive-charged amino acids, e.g., Arg, amino acids of Formula ⁇ , such as, for example, Lys, D-Lys.
  • positive-charged amino acid refers to any amino acid, naturally-occurring or non-naturally occurring, comprising a positive charge on an atom of its side chain at a physiological pH.
  • the positive-charged amino acids are located at any of positions 37, 38, 39, 40, 41, 42, and 43.
  • a positive-charged amino acid is located at position 40.
  • 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 amino acids) of the extension are negative-charged amino acids, e.g., Asp, Glu.
  • Asp Asp
  • Glu Glu
  • negative-charged amino acid refers to any amino acid, naturally-occurring or non-naturally occurring, comprising a negative charge on an atom of its side chain at a physiological pH.
  • the negative-charged amino acids are located at any of positions 37, 38, 39, 40, 41, 42, and 43.
  • a negative-charged amino acid is located at position 40.
  • the extension is acylated or alkylated as described herein and comprises 1-6 positive charged amino acids as described herein.
  • the GIP agonist peptides which exhibit agonist activity at the GIP receptor comprises (i) SEQ ID NO: 1 with at least one amino acid modification, (ii) an extension of 1 to 21 amino acids (e.g., 5 to 18, 7 to 15, 9 to 12 amino acids) C-terminal to the amino acid at position 29 of the GIP agonist peptide, and (iii) an amino acid
  • the GIP agonist peptide comprises an acylated or alkylated amino acid at position 10.
  • the acyl or alkyl group is a C4 to C30 fatty acyl or C4 to C30 alkyl group.
  • the acyl or alkyl group is attached via a spacer, e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic
  • the GIP agonist peptide comprises an amino acid modification which stabilizes the alpha helix, such as a salt bridge between a Glu at position 16 and a Lys at position 20, or an alpha, alpha-disubstituted amino acid at any one, two, three, or more of positions 16, 20, 21, and 24.
  • the GIP agonist peptide additionally comprises amino acid modifications which confer DPP-IV protease resistance, e.g., DMIA at position 1, AIB at position 2. GIP agonist peptides comprising further amino acid modifications are contemplated herein.
  • the GIP agonist peptides having GIP receptor activity exhibit at least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%) activity of native GIP at the GIP receptor when the GIP agonist peptide lacks a hydrophilic moiety, e.g., PEG.
  • the GIP agonist peptides exhibit more than 10%, (e.g., more than 20%, more than 50%, more than 75%, more than 100%, more than 200%, more than 300%, more than 500%) activity of native GIP at the GIP receptor.
  • the GIP agonist peptide exhibits appreciable agonist activity at one or both of the GLP-1 and glucagon receptors.
  • the potency and/or selectivity for these receptors are within 1000-fold, 750-fold, 500- fold, 250-fold, or 100-fold (higher or lower).
  • the selectivity for the GLP-1 receptor of the GIP agonist peptides having GIP receptor activity can be less than 1000-fold, 500-fold, 100-fold, within 50-fold, within 25 fold, within 15 fold, within 10 fold) (higher or lower) the selectivity for the GIP receptor and/or the glucagon receptor.
  • the GIP agonist peptide is an analog of native glucagon (SEQ ID NO: 1) and the amino acid sequence of the peptide is SEQ ID NO: 1 with a stabilized alpha helix (e.g., a lactam bridge or alpha, alpha disubstituted amino acids), Leu-Ala-Gly as positions 27-29, lie at position 12, an amino acid at position 1 that confers the peptide with GIP activity or an amino acid which reduces GIP activity as described herein.
  • a stabilized alpha helix e.g., a lactam bridge or alpha, alpha disubstituted amino acids
  • the GIP agonist peptide is an analog of native glucagon (SEQ ID NO: 1) and the amino acid sequence of the peptide is SEQ ID NO: 1 with an amino acid of Formula IV at position 16, and alpha, alpha disubstituted amino acid (e.g., AIB) at position 20, Leu-Ala-Gly as positions 27-29, lie at position 12, an amino acid at position 1 that confers the peptide with GIP activity or an amino acid which reduces GIP activity as described herein.
  • the GIP agonist peptide comprises a modification that reduces activity at the GIP receptorat position 1 .
  • the GIP agonist peptide may comprise such an amino acid modification so that the activity levels at the GIP receptor (e.g., EC50 or potency at the GIP receptor) are within about 50-fold, about 40-fold, about 30-fold, about 20 fold, about 10 fold, or about 5-fold of the IC50 of the glucagon antagonist of the peptide combination.
  • the amino acid modification which reduces GIP agonist activity is a substitution of His at position 1 with a small aliphatic residue, e.g., Ala, Gly.
  • the amino acid modification which reduces GIP agonist activity is a deletion of the amino acid at position 1 or a deletion of the amino acids at positions 1 and 2.
  • the GIP agonist peptide is an analog of any of the amino acid sequences listed in Sequence Listing 2 in which Tyr is at position 1, wherein the analog comprises a small aliphatic residue at position 1, in lieu of the Tyr, or the analog lacks the amino acid at position 1 or at positions 1 and 2 of these amino acid sequences.
  • the GIP agonist peptide of the present disclosures which exhibits enhanced activity at the GIP receptor comprises an amino acid modification at position 1.
  • the His at position 1 of native glucagon is substituted with a large, aromatic amino acid, optionally Tyr, Phe, Trp, amino-Phe, nitro- Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3-amino Tyr in the GIP agonist peptide.
  • the GIP agonist peptide comprises a modification that reduces activity at the GIP receptor at position 1.
  • the GIP agonist peptide may comprise such an amino acid modification so that the activity levels at the GIP receptor (e.g., EC50 or potency at the GIP receptor) are within about 50-fold, about 40-fold, about 30-fold, about 20 fold, about 10 fold, or about 5-fold of the IC50 of the glucagon antagonist of the peptide combination.
  • the amino acid modification which reduces GIP agonist activity is a substitution of His at position 1 with a small aliphatic residue, e.g., Ala, Gly.
  • the amino acid modification which reduces GIP agonist activity is a deletion of the amino acid at position 1 or a deletion of the amino acids at positions 1 and 2.
  • the GIP agonist peptide which exhibits enhanced activity at the GIP receptor comprises an amino acid modification at one or all of positions 27, 28, and 29.
  • the GIP agonist peptide comprises the amino acid sequence of native glucagon in which (i) the Met at position 27 of the native glucagon amino acid sequence is substituted with a large aliphatic amino acid, optionally Leu, (ii) the Asn at position 28 of the native glucagon amino acid sequence is substituted with a small aliphatic amino acid, optionally Ala, (iii) the Thr at position 29 of the native glucagon amino acid sequence is substituted with a small aliphatic amino acid, optionally Gly, or any combination of (i), (ii), and (iii). Substitution with LAG at positions 27-29 provides increased GIP activity relative to the MNT sequence of native human glucagon at those positions.
  • the GIP agonist peptide which exhibits enhanced activity at the GIP receptor comprises an amino acid modification at position 12.
  • the amino acid at position 12 of the native glucagon amino acid sequence is substituted with a large, aliphatic, nonpolar amino acid, optionally He.
  • the GIP agonist peptide which exhibits enhanced activity at the GIP receptor comprises an amino acid modification at positions 17 and/or 18.
  • position 17 is substituted with a polar residue, optionally Gin
  • position 18 is substituted with a small aliphatic amino acid, optionally Ala.
  • a substitution with QA at positions 17 and 18 provides increased GIP activity relative to the native RR sequence at those positions.
  • the GIP agonist peptide which exhibits increased activity at the GIP receptor comprises an intramolecular bridge between the side chains of two amino acids located at any of positions from 12 to 29.
  • the intramolecular bridge is formed between two amino acids that are not present in the native amino acid sequence of human glucagon.
  • the GIP agonist peptide in some aspects comprises amino acid modifications, e.g., amino acid substitutions of the native human glucagon sequence, that permit bridge formation.
  • an intramolecular bridge is formed by a covalent bond between the side chains of two amino acids at positions i and i+4 or between positions j and j+3, or between positions k and k+7.
  • the bridge is between positions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20.
  • non-covalent interactions such as salt bridges can be formed between positively and negatively charged amino acids at these positions.
  • Intramolecular bridges within the C-terminal region of a glucagon-based peptide are further described herein. See “Stabilization of the Alpha Helix Structure"
  • 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 or analog thereof is substituted with an a, a-disubstituted amino acid.
  • substitution of position 16 of a glucagon peptide or analog thereof 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. Further discussion of this type of amino acid modification is provided herein. See “Stabilization of the Alpha Helix Structure"
  • the GIP agonist peptide comprises an extension of 1-21 amino acids C-terminal to the amino acid at position 29.
  • the extension in some aspects comprises the amino acid sequence of SEQ ID NO: 3 or 4, for instance.
  • the Xaa is a small aliphatic residue, e.g., Gly.
  • the extension comprises 1-6 charged amino acids.
  • the 1-6 amino acids are negative-charged amino acids, e.g., Asp, Glu.
  • the 1-6 amino acids are positive-charged amino acids, e.g., Arg, an amino acid of Formula IV (e.g., Dab, Orn, Lys, d-Lys, homoLys).
  • the charged amino acid may be located at any of positions 37, 38, 39, 40, 41, 42, and 43. In some aspects, the charged amino acid is located at position 40. In further aspects, the charged amino acid is modified with an acyl or alkyl group as described herein in "Acylation and alkylation.” In some aspects, the extension does not comprise a Lys at position 40.
  • the GIP agonist peptide which is an analog of native human glucagon comprises an amino acid modification that selectively reduces glucagon receptor activity.
  • the modification that reduces glucagon receptor activity is a modification of the amino acid at position 3, e.g. substitution of the naturally occurring glutamine at position 3, with an acidic, basic, or a hydrophobic amino acid.
  • the GIP agonist peptide comprises the native human glucagon amino acid sequence in which the amino acid at position 3 is substituted with glutamic acid, ornithine, or norleucine. Such modifications have been found to substantially reduce or destroy glucagon receptor activity. Without being bound to any particular theory, such amino acid substitutions at position 3 dominate any other amino acid modification which enhances glucagon receptor activity, such that the net result is reduced or destroyed activity at the glucagon receptor.
  • native human glucagon does not activate the GLP-1 receptor in the human body. Described herein are modifications of the native human glucagon amino acid sequence which alter this hormone, such that it exhibits appreciable activity at the GLP-1 receptor.
  • the GIP agonist peptide of the present disclosures exhibits enhanced activity at both the GIP receptor and GLP-1 receptor (as compared to the activity of native glucagon at these receptors).
  • the GIP agonist peptide may be considered as a GIP/GLP-1 co-agonist peptide.
  • the GIP agonist peptide which exhibits enhanced activity at the GLP-1 receptor in some embodiments comprises a charge-neutral group, such as an amide or ester, in place of the alpha carboxylic acid of the C-terminal amino acid.
  • the GIP agonist peptide comprises C-terminal amidation or comprises a C-terminal amide in place of the alpha carboxylate of the C-terminal residue.
  • the GIP agonist peptide which exhibits enhanced activity at the GLP-1 receptor comprises a stabilized alpha-helix structure in the C-terminal portion of glucagon (around amino acids 12-29), e.g., through formation of an intramolecular bridge between the side chains of two amino acids, or substitution and/or insertion of amino acids around positions 12-29 with an alpha helix- stabilizing amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid), as further described herein.
  • an alpha helix- stabilizing amino acid e.g., an ⁇ , ⁇ -disubstituted amino acid
  • the side chains of the amino acid pairs 12 and 16, 13 and 17, 16 and 20 , 17 and 21, 20 and 24 or 24 and 28 are linked to one another and thus stabilize the glucagon alpha helix.
  • the bridge or linker is about 8 (or about 7-9) atoms in length, particularly when the bridge is between positions i and i+4. In some embodiments, the bridge or linker is about 6 (or about 5-7) atoms in length, particularly when the bridge is between positions j and j+3.
  • intramolecular bridges are formed by (a) substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid, or a charged amino acid having a side chain containing at least one heteroatom, (e.g.
  • the side chains of such amino acids at positions 16 and 20 can form a salt bridge or can be covalently linked.
  • the two amino acids are bound to one another to form a lactam ring.
  • stabilization of the alpha helix structure in the C-terminal portion of the GIP agonist peptide is achieved through the formation of an intramolecular bridge other than a lactam bridge.
  • suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, the use of a, ⁇ -diaminoalkane tethers, the formation of metal-atom bridges, and other means of peptide cyclization are used to stabilize the alpha helix.
  • one or more a, a-disubstituted amino acids are inserted or substituted into this C-terminal portion (amino acids 12-29) at positions that retain the desired activity.
  • amino acids 12-29 amino acids 12-29
  • one, two, three or all of positions 16, 20, 21 or 24 are substituted with an a, a-disubstituted amino acid, e.g., AIB.
  • the GIP agonist peptide which exhibits increased activity at the GLP-1 receptor comprises an amino acid modification at position 20 as described herein.
  • the GIP agonist peptide which exhibits increased activity at the GLP-1 receptor comprises GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4) at the C-terminus.
  • GLP-1 activity in such analogs can be further increased by modifying the amino acid at position 18, 28 or 29, or at position 18 and 29, as described herein.
  • the GIP agonist peptide which exhibits increased activity at the GLP-1 receptor comprises a large, aromatic amino acid residue, optionally Trp, at position 10.
  • the GIP agonist peptide exhibits enhanced activity at the GLP-1 receptor
  • the GIP agonist peptide comprises an alanine at position 18 instead of an arginine which is native to the human glucagon amino acid sequence.
  • Any of the modifications described above in reference to a GIP agonist peptide which exhibit increased GLP-1 receptor activity can be applied individually or in
  • the present disclosures provides GIP agonist peptides that comprise modifications at position 16, at position 20, and at the C-terminal carboxylic acid group, optionally with a covalent bond between the amino acids at positions 16 and 20; GIP agonist peptides that comprise modifications at position 16 and at the C-terminal carboxylic acid group; GIP agonist peptides that comprise modifications at positions 16 and 20, optionally with a covalent bond between the amino acids at positions 16 and 20; and GIP agonist peptides that comprise modifications at position 20 and at the C-terminal carboxylic acid group.
  • GLP-1 activity may be reduced by specific amino acid modifications.
  • the GIP agonist peptide comprises (i) a C-terminal alpha carboxylate group, (ii) a substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group, e.g., Abu or lie, (iii) a 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 (iv) a combination thereof.
  • an amino acid substitution at position 7 which reduces, if not destroys, GLP-1 receptor activity dominates any other amino acid modification which is described herein as one which enhances GLP-1 receptor activity, such that the net effect of the modifications would be reduced or destroyed activity at the GLP- 1 receptor.
  • the GIP agonist peptide comprises (i) an amino acid substitution of Ser at position 16 with an amino acid of Formula IV:
  • each of Ri and R 2 is independently selected from the group consisting of H, C -Cn alkyl, (C C ⁇ alkyl)OH, (Ci-Cie alkyl)NH 2 , (Ci-Cie alkyl)SH, (C 0 -C 4 alkyl)(C 3 -C 6 )cycloalkyl, (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -C 10 aryl)R 7 , and (Ci-C 4 alkyl)(C 3 -C 9 heteroaryl), wherein R 7 is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group, and (ii) an amino acid substitution of the Gin at position 20 with an alpha, alpha-disub
  • the activity at each of the glucagon receptor, GLP- 1 receptor, and glucagon receptor of the GIP agonist peptide comprising an amino acid of Formula IV at position 16 and an alpha, alpha di-substituted amino acid at position 20 can be further enhanced by extending the length of the peptide, e.g. by fusion to a C-terminal extension peptide, e.g. of about 1-21, about 9 to 21, about 6-18, about 9-12, or about 10 or 11 amino acids in length.
  • the C-terminus of the GIP agonist peptide is extended by fusion to GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), wherein X is Gly or a small, aliphatic or non-polar or slightly polar amino acid.
  • the C- terminus of the GIP agonist peptide is extended by fusion to GPSSGAPPPS (SEQ ID NO: 3) and 1-11 amino acids are fused to the C-terminus of GPSSGAPPPS (SEQ ID NO: 3).
  • the C-terminal extension of the analog can comprise GPSSGAPPPS (SEQ ID NO: 3) followed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional amino acids at the C-terminus of SEQ ID NO: 3.
  • the 1-11 additional amino acids can be, for example, a small aliphatic amino acid, such as Ala.
  • the GIP agonist peptide in some embodiments comprises a C-terminal extension comprising, for example, the amino acid sequence of
  • GPSSGAPPPSA m wherein m is 1 to 11.
  • Enhancement of activity at each of the glucagon, GLP-1, and GIP receptors of the GIP agonist peptide can furthermore be achieved upon acylation or alkylation of an amino acid located within a C-terminal extension or at the C- terminal amino acid (e.g., an amino acid which is added to the C-terminus of the C-terminal extension).
  • the acylation or alkylation can be of an amino acid located, for example, at any of positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 of the C-terminally extended GIP agonist peptide.
  • the amino acid which is acylated or alkylated is located at position 37, 38, 39, 40, 41, 42, or 43 of the C- terminally extended GIP agonist peptide.
  • the acylated or alkylated amino acid is an amino acid of Formula I, II, or III, e.g., Lys, which is attached to an acyl or alkyl group, e.g.
  • the Lys is located C-terminal to a C- terminal extension consisting of SEQ ID NO: 3, such that the Lys, Dab, Orn, or homoLys is located at position 40 of the analog.
  • C-terminally extended peptides are also pegylated, e.g. at any of the positions described herein as suitable for pegylation (e.g., position 24).
  • Enhancement of the activity at each of the glucagon, GLP-1, and GIP receptors of a GIP-active, GIP agonist peptide can moreover be achieved by acylation or alkylation of an amino acid via a spacer (e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic bifunctional spacer).
  • a spacer e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic bifunctional spacer.
  • the GIP-active, GIP agonist peptide comprises an acyl or alkyl group via a spacer, which spacer is attached to the side chain of the amino acid at position 10 of the analog.
  • the GIP agonist peptide comprises a C-terminal extension of 1 to 21 amino acids (e.g., an extension comprising the amino acid sequence of SEQ ID NO: 3 or 4) C-terminal to the amino acid at position 29 and the spacer, which is covalently attached to an acyl or alkyl group, is attached to an amino acid of the extension at a position corresponding to one of positions 37-43 relative to SEQ ID NO: 1.
  • the spacer is attached to the amino acid at position 40 relative to SEQ ID NO: 1.
  • the spacer is 3 to 10 atoms in length.
  • the total length of the spacer and acyl or alkyl group is about 14 to about 28 atoms in length.
  • the spacer can be an amino acid, including, but not limited to, any of those described herein.
  • the spacer may be a dipeptide or tripeptide comprising amino acids described herein, e.g., a dipeptide or tripeptide spacer comprising acidic amino acids.
  • the spacer in specific aspects is one of the following dipeptides: Ala-Ala, pAla-pAla, or yGlu-yGlu. Additional suitable spacers for purposes of increasing activity at one or more of the glucagon, GLP-1, and GIP receptors are further described herein.
  • the GIP agonist peptide comprises any of the modifications above which achieve enhanced activity at each of the GIP, GLP-1, and glucagon receptors, in addition to an amino acid modification which reduces glucagon activity, e.g., Glu at position 3.
  • an amino acid substitution at position 3 which reduces, if not destroys, glucagon receptor activity dominates any other amino acid modification which enhances glucagon receptor activity, such that the net results would be reduced or destroyed activity at the glucagon receptor.
  • the GIP agonist peptide comprises an amino acid of Formula IV at position 16, an alpha, alpha-disubstituted amino acid at position 20, a C-terminal extension in accordance with the above teachings comprising an acylated Lys residue at position 40, and a Glu at position 3.
  • Such peptides exhibits little to no glucagon receptor activity.
  • Stabilization of the alpha-helix structure in the C-terminal portion of the GIP agonist peptide provides enhanced GLP-1 and/or GIP activity and restores glucagon activity which has been reduced by amino acid modifications at positions 1 and/or 2.
  • the alpha helix structure can be stabilized by, e.g., formation of a covalent or non-covalent intramolecular bridge, or substitution and/or insertion of amino acids around positions 12-29 with an alpha helix- stabilizing amino acid (e.g., an ⁇ , ⁇ - disubstituted amino acid).
  • 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 GIP agonist 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 bonds, e.g., hydrogen bonds, ionic interactions, the peptide may be considered herein as comprising a non-covalent
  • 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, ] 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 Homoser- Homoglu (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).
  • One of ordinary skill in the art can envision alternative pairings or alternative amino acid analogs, including chemically modified derivatives, that would create a stabilizing structure of similar size and desired effect.
  • a homocysteine - homocysteine disulfide bridge is 6 atoms in length and may be further modified to provide the desired effect.
  • the amino acid pairings described above or similar pairings that one of ordinary skill in the art can envision may also provide added stability to the alpha-helix through non-covalent bonds, for example, through formation of salt bridges or hydrogen-bonding interactions.
  • 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 Lys 12 and a Glu 16 or alternatively between a Glu 12 and a Lys 16).
  • Intramolecular bridges other than a lactam bridge can be used to stabilize the alpha helix of the GIP agonist 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 GIP agonist 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 GIP agonist peptide using an all-hydrocarbon cross-linking system.
  • the GIP agonist 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 intramolecular bridges are described in the art.
  • the GIP agonist 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
  • 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); Polinsky et al., J. Med. Chem. 35: 4185- 4194 (1992); Osapay et al., J. Med. Chem.
  • 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 cross-link one or two turns of the alpha helix of the GIP agonist 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 GIP agonist 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 GIP agonist 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).
  • metal binding-based alpha helix stabilization are known in the art. See, for example, Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); Ghadiri et al., /. Am. Chem. Soc. 112: 1630-1632 (1990); and Ghadiri et al., /. Am. Chem. Soc. 119: 9063-9064 (1997).
  • the alpha helix of the GIP agonist 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 GIP agonist 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 GIP agonist peptide (around amino acids 12-29).
  • the alpha helix-stabilizing amino acid is an a, a-disubstituted 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- 1-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- 1-carboxylic acid).
  • one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of the GIP agonist 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 an ⁇ , ⁇ -disubstituted amino acid, e.g., AIB, and the GIP agonist peptide optionally lacks any purposefully-introduced intramolecular bridges.
  • the GIP agonist peptide can comprise a substitution of position 16 or 20 with AIB in the absence of an intramolecular bridge, e.g., a non-covalent intramolecular bridge (e.g., a salt bridge) or a covalent intramolecular bridge (e.g., a lactam).
  • an intramolecular bridge e.g., a non-covalent intramolecular bridge (e.g., a salt bridge) or a covalent intramolecular bridge (e.g., a lactam).
  • a non-covalent intramolecular bridge e.g., a salt bridge
  • a covalent intramolecular bridge e.g., a lactam
  • the GIP agonist peptide lacking an intramolecular bridge comprises one or more substitutions within amino acid positions 12-29 with an a, a-disubstituted amino acid and an acyl or alkyl group covalently attached to the side chain of an amino acid of the GIP agonist peptide, e.g., the amino acid at positions 10 or 40 of the GIP agonist 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 GIP agonist 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 GIP agonist peptides lacking an intramolecular bridge by incorporating a spacer between the acyl or alkyl group and the side chain of the amino acid at positions 10 or 40 of the peptide. Acylation and alkylation, with or without incorporating spacers, are further described herein.
  • the acylated or alkylated GIP agonist peptide, or analog thereof further comprises a modification which selectively reduces activity at the GLP- 1 receptor.
  • the acylated or alkylated GIP agonist 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 a large, aliphatic, non-polar amino acid, e.g., He.
  • the GIP agonist peptide of the present disclosures comprises an amino acid modification which selectively reduces glucagon receptor activity. Such modifications are described further herein.
  • position 16 or position 20 is substituted with an ⁇ , ⁇ - disubstituted amino acid, e.g., AIB. In some embodiments, position 20 is substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., AIB. In certain embodiments, position 20 is substituted with an ⁇ , ⁇ -disubstituted amino acid, e.g., AIB, and position 16 is substituted with an amino acid of Formula IV
  • Ri and R 2 is independently selected from the group consisting of H, C -Cn alkyl, (C C ⁇ alkyl)OH, (Ci-Ci 8 alkyl)NH 2> (Ci-Ci 8 alkyl)SH, (C 0 -C 4 alkyl)(C 3 -C 6 )cycloalkyl, (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -C 10 aryl)R 7 , and (C C 4 alkyl)(C 3 -C 9 heteroaryl), wherein R 7 is H or OH, and the side chain of the amino acid of Formula IV comprises a free amino group.
  • the amino acid of Formula IV is 2,3 diamino propionic acid (DAP), 2,4-diaminobutyric acid (DAB), Orn, Lys or homoLys.
  • DAP 2,3 diamino propionic acid
  • DAB 2,4-diaminobutyric acid
  • Orn Lys or homoLys.
  • the combination of an amino acid of Formula IV at position 16 and an alpha, alpha disubstituted amino acid advantageously provides improved activity at each of the glucagon, GLP-1, and GIP receptors.
  • this peptide further comprises an amino acid modification which selectively reduces activity at the glucagon receptor, e.g., a substitution of the Gin at position 3 with Glu.
  • the GIP agonist peptide of the present disclosures are modified to comprise an acyl group or an 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 GIP agonist peptides in circulation.
  • Acylation or alkylation can advantageously delay the onset of action and/or extend the duration of action at the glucagon and/or GLP- 1 receptors and/or improve resistance to proteases such as DPP-IV and/or improve solubility.
  • Activity at the glucagon and/or GLP-1 and/or GIP receptors of the GIP agonist peptide may be maintained after acylation.
  • the potency of the acylated GIP agonist peptides is comparable to the unacylated versions of the GIP agonist peptides.
  • the potency of the acylated GIP agonist peptides is increased as compared to that of the unacylated version of the GIP agonist peptides.
  • the GIP agonist peptide is modified to comprise an acyl group or alkyl group covalently linked to the amino acid at position 10 of the GIP agonist peptide.
  • the GIP agonist peptide may further comprise a spacer between the amino acid at position 10 of the GIP agonist peptide and the acyl group or alkyl group.
  • the acyl group is a fatty acid or bile acid, or salt thereof, e.g. a C4 to C30 fatty acid, a C8 to C24 fatty acid, cholic acid, a C4 to C30 alkyl, a C8 to C24 alkyl, or an alkyl comprising a steroid moiety of a bile acid.
  • the spacer is any moiety with suitable reactive groups for attaching acyl or alkyl groups.
  • the spacer comprises an amino acid, a dipeptide, a tripeptide, a hydrophilic bifunctional, or a hydrophobic bifunctional spacer.
  • the spacer is selected from the group consisting of: Trp, Glu, Asp, Cys and a spacer comprising NH 2 (CH 2 CH 2 0)n(CH 2 )mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12.
  • acylated or alkylated GIP agonist peptides may also further comprise a hydrophilic moiety, optionally a polyethylene glycol. Any of the foregoing GIP agonist peptides may comprise two acyl groups or two alkyl groups, or a combination thereof.
  • Acylation can be carried out at any position within the GIP agonist peptide, including any of positions 1-29, a position within a C-terminal extension, or the N- or C- terminal amino acid, provided that GIP activity (and optionally GLP-1 and/or glucagon activity) is retained, if not enhanced. Acylation may occur, for example, at any amino acid which is added to the amino acid sequence (SEQ ID NO: 1), e.g., at the N- or C-terminus.
  • Nonlimiting examples include positions 1, 5, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the GIP agonist peptide.
  • the acyl group can be covalently linked directly to an amino acid of the GIP agonist peptide, or indirectly to an amino acid of the GIP agonist peptide via a spacer, wherein the spacer is positioned between the amino acid of the GIP agonist peptide and the acyl group.
  • GIP agonist 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 or position 40 and pegylation at one or more positions in the C-terminal portion of the GIP agonist 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 GIP agonist peptide is modified to comprise an extension of about 1 to about 21 amino acids C-terminal to the GIP agonist peptide of SEQ ID NO: 1 or an analog thereof and at least one of the amino acids of the extension is acylated or alkylated.
  • the modified GIP agonist peptide may comprise an extension of about 1 to about 21 amino acids C-terminal to the amino acid at position 29 of the GIP agonist peptide of SEQ ID NO: 1 or analog thereof.
  • the extension of about 1 to about 21 amino acids may be C-terminal to the amino acid at position 27 or 28 of the GIP agonist peptide or analog thereof.
  • the acylated or alkylated amino acid within the C- terminal extension can be, for example, any of the amino acids at position 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the C-terminally extended GIP agonist peptide.
  • the C-terminal extension in some embodiments comprises the amino acid sequence of SEQ ID NO: 3 or 4.
  • the GIP agonist peptide comprises a C-terminal extension comprising the amino acid sequence of SEQ ID NO: 3 and 1 to 11 additional amino acids at the C-terminus of SEQ ID NO: 3, which additional amino acid(s) is/are acylated or alkylated, as described herein.
  • the acylated or alkylated amino acid is a Dab, Orn, Lys, or homoLys residue and is located at position 40 of the C-terminally extended GIP agonist peptide or analog thereof.
  • the GIP agonist peptide is modified to comprise an acyl group which is attached to the GIP agonist 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.
  • the GIP agonist 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 GIP agonist peptide.
  • the GIP agonist peptide is directly acylated through the side chain amine, hydroxyl, or thiol of an amino acid.
  • acylation is at position 10, 20, 24, 29, or 40.
  • the acylated GIP agonist 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, 29, and 40 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the direct acylation of the GIP agonist peptide occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position 10 or 40.
  • 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 II 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 GIP agonist peptide comprises a spacer between the peptide and the acyl group.
  • the GIP agonist 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 or doubly a-substituted 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 is suitable.
  • the acylated GIP agonist 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, 29, and 40 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 spacer amino acid can be any amino acid.
  • the spacer amino acid 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, and 8- aminooctanoic acid.
  • the spacer amino acid can be an acidic residue, e.g., Asp and Glu.
  • the spacer amino acid is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • an amino acid of Formula I e.g., Lys or Orn
  • both the alpha amine and the side chain amine of the spacer amino acid to be acylated, such that the GIP agonist peptide is diacylated.
  • Embodiments of the present disclosures 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. In other embodiments, the hydrophilic bifunctional spacer comprises a thiol group and a carboxylate. In specific embodiments, the spacer comprises an amino poly(alkyloxy)carboxylate.
  • 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 a hydroxyl group and a carboxylate.
  • hydrophobic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydropholic 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 bifunctional, 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, lie, 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- -methyl- alanine (Me-Ala), aminobutyric acid (Abu), ⁇ -aminobutyric acid ( ⁇ -Abu), aminohexanoic acid ( ⁇ -Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid, amino
  • DMTA dimethylthiazolidine
  • ⁇ -Glutamic acid ⁇ -Glu
  • homoserine Hse
  • hydroxyproline Hyp
  • isoleucine N-methoxy-N-methyl amide methyl-isoleucine (Melle), isonipecotic acid (Isn), methyl-leucine (MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,
  • methanoproline methionine- sulfoxide (Met(O)), methionine- sulf one (Met(0 2 )), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline (Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4- Cl)), 4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-N0 2 )), 4- cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg), piperidinylalanine,
  • piperidinylglycine 3,4-dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta), 4-amino- 5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), l,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
  • the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids, e.g., one or two acidic residues.
  • 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 GIP agonist 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, 29, or 40, or at the C-terminal amino acid of the GIP agonist peptide.
  • the acyl group is attached to the amino acid at position 10 or 40 of the GIP agonist peptide and, optionally, the length of the spacer and acyl group is 14 to 28 atoms.
  • the amino acid at position 10 or 40 in some aspects, is an amino acid of Formula I, e.g., Lys, or a disubstituted amino acid related to Formula I.
  • the GIP agonist peptide lacks an intramolecular bridge, e.g., a covalent intramolecular bridge.
  • the GIP agonist 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 40 exhibit enhanced potency at the GIP, GLP-1, and glucagon receptors.
  • Peptides comprising the same structure except further comprising an amino acid modification which selectively reduces activity at the glucagon receptor (e.g., substitution of Gin 3 for Glu) are further contemplated herein.
  • Suitable methods of peptide acylation via amines, hydroxyls, and thiols are known in the art. See, for example, Example 19 (for methods of acylating through an amine), Miller, Biochem Biophys Res Commun 218: 377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972) (for methods of acylating through a hydroxyl); and San and Silvius, J Pept Res 66: 169-180 (2005) (for methods of acylating through a thiol); Bioconjugate Chem. "Chemical
  • the acyl group of the acylated GIP agonist 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 GIP agonist peptide is modified to comprise an acyl group by acylation of a long chain alkane by the GIP agonist 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 GIP agonist peptide.
  • the carboxyl group, or activated form thereof, of the GIP agonist peptide can be part of a side chain of an amino acid (e.g., glutamic acid, aspartic acid) of the GIP agonist peptide or can be part of the peptide backbone.
  • an amino acid e.g., glutamic acid, aspartic acid
  • the GIP agonist peptide is modified to comprise an acyl group by acylation of the long chain alkane by a spacer which is attached to the GIP agonist 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, bifunctional spacers, e.g., 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 GIP agonist 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, C18 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 GIP agonist peptide is acylated with a cholesterol acid.
  • the GIP agonist peptide is linked to the cholesterol acid through a modified Cys spacer.
  • the acylated GIP agonist 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 GIP agonist 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, 29, and 40 comprise an acyl group and at least one of the amino acids at position 16, 17, 21, 24, 29, or 40, 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 or 40, 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 GIP agonist 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, Ac-Cys, Lys, Orn, homo-Cys, and Ac-Phe.
  • the acylated GIP agonist peptide comprises the amino acid sequence of any of SEQ ID NOs:201-206, 213-215, 217-219, 223- 225, 228-230, 232-234, 236-238, 241-245, 248, 251, 252, 254, 256, 258, 260, 262, 263, 265, 266, 331, 334-339, 357, and 358, and optionally, further comprises an amino acid modification which selectively reduces activity at the glucagon receptor, e.g., substitution of Gin 3 with Glu.
  • the GIP agonist peptide is modified to comprise an alkyl group, e.g., an alkyl group which is not naturally-occuring 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-occuring on an amino acid (e.g., an alkyl group which is non-native to a naturally-occurring amino acid).
  • alkylation of the GIP agonist peptide of the present disclosures achieves similar, if not the same, effects as acylation of the GIP agonist 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, GIP, and glucagon receptors.
  • Alkylation can be carried out at any positions within the GIP agonist peptide, including any of positions 1-29, a position within a C-terminal extension, or the N- or C- terminal amino acid, provided that the GIP activity (and optionally GLP-1 and/or glucagon activity) is retained, if not enhanced. Alkylation may occur, for example, at any amino acid which is added to the amino acid sequence (SEQ ID NO: 1), e.g., at the N- or C-terminus.
  • Nonlimiting examples include positions 1, 5, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the alkyl group can be covalently linked directly to an amino acid of the GIP agonist peptide, or indirectly to an amino acid of the GIP agonist peptide via a spacer, wherein the spacer is positioned between the amino acid of the GIP agonist peptide and the alkyl group.
  • GIP agonist 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 or 40 and pegylation at one or more positions in the C-terminal portion of the GIP agonist peptide, e.g., position 24, 28 29, or 40, within a C-terminal extension, or at the C-terminus (e.g., through adding a C-terminal Cys).
  • the GIP agonist 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 GIP agonist peptide.
  • the GIP agonist peptide is directly alkylated through the side chain amine, hydroxyl, or thiol of an amino acid.
  • alkylation is at position 10, 20, 24, 29, or 40.
  • the alkylated GIP agonist 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, 29, and 40 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the direct alkylation of the GIP agonist 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 GIP agonist peptide comprises a spacer between the peptide and the alkyl group.
  • the GIP agonist peptide is covalently bound to the spacer, which is covalently bound to the alkyl group.
  • the GIP agonist 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, 29, or 40 of the GIP agonist 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 is suitable.
  • the alkylated GIP agonist 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, 29, and 40 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 the amino acid or a side chain amine.
  • the spacer amino acid can be any amino acid.
  • the spacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5 -amino valeric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid.
  • the spacer amino acid can be an acidic residue, e.g., Asp and Glu, provided that the alkylation occurs on the alpha amine of the acidic residue.
  • the spacer amino acid is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • the alpha amine and the side chain amine of the spacer amino acid it is possible for both the alpha amine and the side chain amine of the spacer amino acid to be alkylated, such that the GIP agonist peptide is dialkylated.
  • Embodiments of the present disclosures 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 poly(alkyloxy)carboxylate.
  • 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.
  • the hydropholic bifunctional spacer comprises an amine group and a carboxylate.
  • the hydropholic 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 bifunctional, 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, e.g., one or two acidic residues.
  • 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 GIP agonist 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 GIP agonist 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 GIP agonist peptide is modified to comprise an alkyl group by reacting a nucleophilic, long chain alkane with the GIP agonist peptide, wherein the GIP agonist 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 GIP agonist 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 GIP agonist peptide is modified to comprise an alkyl group by reacting the nucleophilic, long chain alkane with a spacer which is attached to the GIP agonist 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 GIP agonist 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, C18 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 GIP agonist peptide and a cholesterol moiety.
  • the hydroxyl group of cholesterol can displace a leaving group on the long chain alkane to form a cholesterol-GIP agonist peptide product.
  • the alkylated GIP agonist 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 GIP agonist 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, 29, and 40 comprise an alkyl group and at least one of the amino acids at position 16, 17, 21, 24, 29, and 40, 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 or 40, 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 GIP agonist 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 GIP agonist peptide comprises one or two modifications at position 1 and/or 2 which increase the peptide's resistance to dipeptidyl peptidase IV (DPP IV) cleavage.
  • the amino acid at position 2 of the GIP agonist peptide is substituted with one of: D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, or amino isobutyric acid (AIB).
  • the amino acid at position 1 of the GIP agonist peptide is substituted with one of: D-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine, N- methyl histidine, alpha-methyl histidine, imidazole acetic acid, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA).
  • the GIP agonist peptide comprising an amino acid modification which increases resistance to DPP ⁇ further comprises an intramolecular bridge or alpha, alpha di- substituted amino acid, and optionally an amino acid modification which selectively reduces the activity at the glucagon receptor, such as, for example, a substitution of Gln3 with Glu.
  • any of the GIP agonist peptides of the present disclosures can be further modified to improve stability of the peptide by modifying the amino acid at position 15 and/or 16 of SEQ ID NO: 1 to reduce degradation of the peptide over time, especially in acidic or alkaline buffers. Such modifications reduce cleavage of the Aspl5-Serl6 peptide bond.
  • the amino acid modification at position 15 is a deletion or substitution of Asp with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid.
  • the amino acid modification at position 16 is a deletion or substitution of Ser with Thr or AIB.
  • Ser at position 16 is substituted with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or homocysteic acid.
  • Such modifications can reduce degradation or cleavage at a pH within the range of 5.5 to 8, for example, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, up to 100% of the original peptide after 24 hours at 25 °C.
  • Such modifications reduce cleavage of the peptide bond between Aspl5-Serl6.
  • the GIP agonist peptide comprises a modification which prevents oxidative degradation of the peptide.
  • the methionine residue at position 27 of the native glucagon peptide is modified, e.g. by deletion or substitution.
  • the Met at position 27 is substituted with leucine, isoleucine or norleucine. In some specific embodiments, Met at position 27 is substituted with leucine or norleucine.
  • the GIP agonist peptide comprises one or more
  • the Gin at position 20 and/or 24 of the GIP agonist peptide is modified, e.g. by deletion or substitution.
  • the Gin at position 20 and/or 24 of the GIP agonist peptide is substituted with Ser, Thr, Ala or AIB.
  • the Gin at position 20 and/or 24 of the GIP agonist peptide is substituted with Lys, Arg, Orn, or Citrulline.
  • the GIP agonist peptide comprises an amino acid modification which reduces degradation through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to iso-aspartate. Accordingly, in some aspects, the Asp at position 21 of the GIP agonist peptide is modified, e.g. by deletion or substitution. In some embodiments, position 21 of the GIP agonist peptide is substituted with Glu, homoglutamic acid or homocysteic acid. In some specific embodiments, position 21 is substituted with Glu.
  • the solubility of any of the GIP agonist peptides is improved by one or more amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide, preferably at a position C-terminal to position 27 of SEQ ID NO: 1.
  • one, two or three charged amino acids are introduced within the C-terminal portion, preferably C-terminal to position 27.
  • the native amino acid(s) at positions 28 and/or 29 are substituted with one or two charged amino acids, and/or in further embodiments one to three charged amino acids are also added to the C-terminus of the GIP agonist peptide.
  • one, two or all of the charged amino acids are negative-charged or acidic amino acids.
  • the negative-charged or acidic amino acids are aspartic acid or glutamic acid.
  • one, two, three or all of the charged amino acids are positively charged.
  • Such modifications increase solubility, e.g. provide at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-fold or greater solubility relative to native glucagon at a given pH between about 5.5 and 8, e.g., pH 7, when measured after 24 hours at 25°C.
  • hydrophilic moieties and conjugation thereof to peptides is further described herein. See, "Conjugates.”
  • the GIP agonist peptide is conjugated to a hydrophilic moiety, e.g., polyethylene glycol, at position 16, 17, 20, 21, 24 or 29 of the GIP agonist peptide, within a C-terminal extension, and/or at the C-terminal amino acid of the peptide.
  • a hydrophilic moiety e.g., polyethylene glycol, at position 16, 17, 20, 21, 24 or 29 of the GIP agonist peptide, within a C-terminal extension, and/or at the C-terminal amino acid of the peptide.
  • GIP agonist peptide may be made to the GIP agonist peptide that still allow it to retain GIP activity (and optionally GLP-1 activity and/or glucagon activity).
  • exemplary modifications include but are not limited to the following: [00308] Non-conservative or conservative substitutions, additions or deletions that do not substantially affect activity, for example, conservative substitutions at one or more of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; substitution of one or more of these positions with Ala; deletion of amino acids at one or more of positions 27, 28 or 29; or deletion of amino acid 29 optionally combined with a C-terminal amide or ester in place of the C-terminal carboxylic acid group; substitution of Lys at position 12 with Arg; substitution of Tyr at position 10 with Val or Phe;
  • GPSSGAPPPS SEQ ID NO: 3 to the C-terminus.
  • position 18 is substituted with an amino acid selected from the group consisting of Ala, Ser, or Thr.
  • amino acid at position 20 is substituted with Ser, Thr, Lys, Arg, Orn, Citrulline or AIB.
  • position 21 is substituted with Glu, homoglutamic acid or homocysteic acid.
  • the GIP agonist peptide comprises 1 to 10 amino acid modifications selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28 and 29.
  • the modifications are one or more amino acid substitutions selected from the group consisting of Glnl7, Alal8, Glu21, Ile23, Ala24, Val27 and Gly29.
  • 1 to 5 amino acids selected from positions 17-26 differ from the parent peptide. In other embodiments, 1 to 5 amino acids selected from positions 17-24 differ from the parent peptide. In yet other embodiments, the modifications are Glnl7, Alal8, Glu21, Ile23 and Ala24.
  • one or more amino acids is added to the carboxy terminus of the GIP agonist peptide.
  • the amino acid is typically selected from one of the 20 common amino acids, and in some embodiments the amino acid has an amide group in place of the carboxylic acid of the native amino acid.
  • the added amino acid is selected from the group consisting of glutamic acid and aspartic acid and glycine.
  • the GIP agonist peptides disclosed herein are modified by truncation of the C-terminus by one or two amino acid residues yet retain similar activity and potency at the glucagon, GLP-1 and/or GIP receptors.
  • the amino acid at position 29 and/or 28 can be deleted.
  • the glucagon antagonist peptide exhibits at least or about 60% inhibition of the maximum response of native glucagon at the glucagon receptor. In exemplary embodiments, the glucagon antagonist peptide exhibits at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or at least or about 100% inhibition of the maximum response of native glucagon at the glucagon receptor.
  • the glucagon antagonist peptide binds to the glucagon receptor and counteracts glucagon activity or prevents glucagon function.
  • the glucagon antagonist peptide has an IC50 at the glucagon receptor which is in the micromolar range.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than 1000 ⁇ , less than 900 ⁇ , less than 800 ⁇ , less than 700 ⁇ , less than 600 ⁇ , less than 500 ⁇ , less than 400 ⁇ , less than 300 ⁇ , less than 200 ⁇ .
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is about 100 ⁇ or less, e.g., about 75 ⁇ or less, about 50 ⁇ or less, about 25 ⁇ or less, about 10 ⁇ or less, about 8 ⁇ or less, about 6 ⁇ or less, about 5 ⁇ or less, about 4 ⁇ or less, about 3 ⁇ or less, about 2 ⁇ or less, or about 1 ⁇ or less.
  • the glucagon antagonist peptide has an IC50 at the glucagon receptor which is in the nanomolar range.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than 1000 nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is about 100 nM or less, e.g., about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • the IC50 of the glucagon antagonist at the glucagon receptor is between 0.1 nM and 500 nM. In some aspects, the IC50 is about 0.1 nM or about 500 nM.
  • the glucagon antagonist peptide exhibits an IC50 for glucagon receptor activation which is in the picomolar range. In exemplary
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is about 100 pM or less, e.g., about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 8 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1 pM or less.
  • the glucagon antagonist peptide is a glucagon antagonist which, at a concentration of about 1 ⁇ , exhibits less than or about 20% of the maximum agonist activity achieved by glucagon at the glucagon receptor. In some embodiments, the glucagon antagonist peptide is a glucagon antagonist which exhibits less than or about 15%, less than or about 10%, less than or about 5%, less than or about 1%, or about 0% of the maximum agonist activity achieved by glucagon at the glucagon receptor, when the peptide present at a concentration of about 1 ⁇ .
  • the glucagon antagonist peptide is a "full antagonist” at the glucagon receptor and in other aspects, the glucagon antagonist peptide is a "partial antagonist” at the glucagon receptor
  • full antagonist as used herein is meant an antagonist that binds to the receptor and does not exhibit any agonist activity at the receptor it antagonizes.
  • partial antagonist as used herein is synonomous with "partial agonist” which is a compound that exhibits a lower amount of agonist activity at a receptor as compared to a full agonist, but the partial agonist serves as an antagonist since its occupation of a receptor prevents the full agonist from binding, thereby producing a net decrease in the receptor activation as compared to the level of receptor activation if all receptors were bound by full agonists.
  • the glucagon antagonist peptide exhibits activity (agonist or antagonist) at only one receptor. Accordingly, the glucagon antagonist peptide is some aspects is a "pure glucagon antagonist" and does not produce any detected stimulation of the glucagon receptor or any other receptor, including, e.g., the GLP-1 receptor, the GIP receptor, as measured by cAMP production using a validated in vitro model assay, such as that described in Example 2.
  • a pure glucagon antagonist exhibits less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0%) of the maximum agonist activity achieved by glucagon at the glucagon receptor and exhibits less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, and about 0%) of the maximum agonist activity achieved by GLP-1 at the GLP-1 receptor and/or exhibits less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, and about 0%) of the maximum agonist activity achieved by GIP at the GIP receptor.
  • the glucagon antagonist peptide exhibits activity (agonist or antagonist) at more than one receptor.
  • the glucagon antagonist peptide has lost selectivity for one receptor over another.
  • the glucagon antagonist peptide in some embodiments is a glucagon receptor antagonist and an antagonist or agonist at another receptor, e.g., GLP-1 receptor and/or GIP receptor.
  • the glucagon antagonist peptide in some embodiments exhibits mixed properties insofar as it exhibits antagonist activity at the glucagon receptor and agonist activity at another receptor, e.g., the GLP-1 receptor, the GIP receptor.
  • the glucagon antagonist peptide in some aspects exhibits both antagonist activity at the glucagon receptor and agonist activity at the GLP-1 receptor ("Glucagon receptor antagonist/GLP-1 receptor agonists").
  • the glucagon antagonist peptide has any of the IC50s at the glucagon receptor described herein and has any of the EC50s at the GLP-1 receptor described herein.
  • the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than or about 50-fold, less than or about 40-fold, less than or about 30-fold, or less than or about 20-fold different (higher or lower) from its EC50 at the GLP-1 receptor.
  • the ratio of the IC50 of the glucagon antagonist peptide at the glucagon receptor divided by the EC50 of the glucagon antagonist peptide at the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the EC50 of the glucagon antagonist peptide at the GLP-1 receptor divided by the IC50 of the glucagon antagonist peptide at the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the glucagon antagonist peptides described herein exhibit inhibitory activity at the glucagon receptor and/or agonist activity at the GLP- 1 receptor as described above and, when the glucagon antagonist peptide is part of a conjugate (e.g., is conjugated to a heterologous moiety, e.g., a hydrophilic moiety, e.g., a polyethylene glycol), the glucagon antagonist peptide exhibits an activity that is lower (i.e. lower inhibitory potency or higher IC50) than when the glucagon antagonist peptide is not part of the conjugate.
  • a conjugate e.g., is conjugated to a heterologous moiety, e.g., a hydrophilic moiety, e.g., a polyethylene glycol
  • the glucagon antagonist peptide exhibits an activity that is lower (i.e. lower inhibitory potency or higher IC50) than when the glucagon antagonist peptide is not part of
  • the glucagon antagonist peptide when not part of conjugate exhibits an inhibitory potency at the glucagon receptor that is about 10-fold or greater than the potency of the glucagon antagonist peptide when part of a conjugate.
  • the glucagon antagonist peptide when unconjugated exhibits an inhibitory 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 even greater-fold the potency of the glucagon antagonist peptide when conjugated.
  • the glucagon antagonist peptide is a glucagon antagonist, which exhibits any of the activities (potency or EC50) at the indicated receptor as described above, and is structurally similar to native human glucagon (SEQ ID NO: 1), e.g., is an analog of native human glucagon (or a glucagon analog).
  • native human glucagon SEQ ID NO: 1
  • Such analogs of glucagon exhibiting glucagon receptor antagonist activity are known in the art.
  • glucagon antagonists in which one or more amino acids of the native human glucagon amino acid sequence were deleted or substituted include: [des His 1 ] [Glu 9 ]- glucagon amide (Unson et al., (1989) Peptides 10, 1171; Post et al., (1993) Proc. Natl. Acad. Sci. USA 90, 1662), des His 1 , Phe 6 [Glu 9 ]-glucagon amide (Azizh et al., (1995) Bioorg. & Med. Chem. Lett. 16, 1849) and Nle 9 , Ala u ' 16 -glucagon amide (Unson et al. (1994) J. Biol. Chem.
  • the glucagon antagonist peptide is an analog of native human glucagon (SEQ ID NO: 1) which comprises an amino acid sequence based on SEQ ID NO: 1 but is modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.) amino acid modifications.
  • the glucagon antagonist peptide comprises 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 amino acid modifications relative to the native human glucagon sequence (SEQ ID NO: 1).
  • the modifications are any of those described herein, e.g., truncation at the N- terminus, formation into depsipeptide, substitution at position 9, acylation, alkylation, pegylation, truncation at C-terminus, substitution of the amino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.
  • the glucagon antagonist peptide of the present disclosures comprises an amino acid sequence which has at least 25% sequence identity to the amino acid sequence of native human glucagon (SEQ ID NO: 1).
  • the glucagon antagonist peptide comprises an amino acid sequence which is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to SEQ ID NO: 1.
  • the amino acid sequence of the glucagon antagonist peptide which has the above -referenced % sequence identity is the full-length amino acid sequence of the glucagon antagonist peptide.
  • the amino acid sequence of the glucagon antagonist peptide which has the above-referenced % sequence identity is only a portion of the amino acid sequence of the glucagon antagonist peptide.
  • the glucagon antagonist peptide comprises an amino acid sequence which has about A% or greater sequence identity to a reference amino acid sequence of at least 5 contiguous amino acids (e.g., at least 6, at least 7, at least 8, at least 9, at least 10 amino acids) of SEQ ID NO: 1, wherein the reference amino acid sequence begins with the amino acid at position C of SEQ ID NO: 1 and ends with the amino acid at position D of SEQ ID NO: 1, wherein A is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29. Any and all possible combinations of the foregoing parameters are envisioned, including but not limited to, e.g.
  • the GIP agonist peptides which are analogs of native human glucagon (SEQ ID NO: 1) described herein may comprise a peptide backbone of any number of amino acids, i.e., can be of any peptide length.
  • the GIP agonist peptides described herein are the same length as SEQ ID NO: 1, i.e., are 29 amino acids in length.
  • the GIP agonist peptide is longer than 29 amino acids in length, e.g., the GIP agonist peptide comprises a C-terminal extension of 1-21 amino acids, as further described herein.
  • the GIP agonist peptide in some embodiments, is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
  • the GIP agonist peptide is longer than 29 amino acids in length (e.g., greater than 50 amino acids, (e.g., at least or about 60, at least or about 70, at least or about 80, at least or about 90, at least or about 100, at least or about 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, at least or about 400, at least or about 450, at least or about 500 amino acids in length) due to fusion with another peptide.
  • the GIP agonist peptide is less than 29 amino acids in length, e.g., 28, 27, 26, 25, 24, 23, amino acids.
  • the glucagon antagonist peptide of the present disclosures is an analog of native human glucagon (SEQ ID NO: 1) comprising SEQ ID NO: 1 modified with one or more amino acid modifications which reduce or destroy glucagon activity, which increase or enhance GLP-1 activity or GIP activity, enhance stability, e.g., by reducing degradation of the peptide (e.g., by improving resistance to DPP-IV proteases), enhance solubility, increase half-life, delay the onset of action, extend the duration of action at the GIP, glucagon, or GLP- 1 receptor, or a
  • native human glucagon activates the glucagon receptor in the human body. Described herein are modifications of the native human glucagon amino acid sequence (SEQ ID NO: 1) which alter this hormone, such that is antagonizes (e.g., binds to but does not activate downstream signaling through) the glucagon receptor.
  • the glucagon antagonist peptide comprises an amino acid sequence based on the sequence of native human glucagon (SEQ ID NO: 1) but is modified by the deletion of the first two to five amino acid residues from the N- terminus and substitution of the aspartic acid residue at position nine of the native protein (SEQ ID NO: 1) with a glutamic acid, homoglutamic acid, ⁇ -homo glutamic acid, a sulfonic acid derivative of cysteine, or an alkylcarboxylate derivative of cysteine having the structure of:
  • OOH wherein X5 is C C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl.
  • the glucagon antagonist peptide exhibiting glucagon antagonist activity and comprising the deletion of two to five amino acid residues from the N-terminus and substitution of the Asp at position 9 of the native glucagon is further modified by up to three amino acid modifications.
  • the glucagon antagonist peptide in some aspects comprise one, two, or three conservative amino acid modifications.
  • the glucagon antagonist peptide in some aspects comprises one or more amino acid modifications selected from the group consisting of:
  • SEQ ID NO: 1 SEQ ID NO: 1 with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid;
  • the glucagon antagonist peptide comprises an amino acid modification of A, B, or C, as described above, or a combination thereof.
  • the glucagon antagonist peptide further comprises an amino acid modification of any of D to K as described above, or a combination thereof, in addition to the amino acid modification(s) of A, B, and/or C.
  • the glucagon antagonist peptide comprises the amino acid sequence of native human glucagon in which the first 5 amino acids have been removed from the N-terminus, and the remaining N-terminal alpha amino group has been replaced with a hydroxyl group.
  • the N-terminal residue of these embodiments is phenyl lactic acid (PLA).
  • the amino acid at position 9 (according to the numbering of SEQ ID NO: 1) is modified by substituting the aspartic acid residue at position four (position 9 of the native glucagon) with an amino acid of the general structure:
  • X 6 is Q-C 3 alkyl, C 2 -C 3 alkenyl or C 2 -C 3 alkynyl.
  • X is C C ? alkyl, and in other embodiments, X is C 2 alkyl.
  • the glucagon antagonist peptide comprises SEQ ID NO: 1 in which the first 5 amino acids have been deleted from the N-terminus, and the aspartic acid residue at position four (position 9 of the native glucagon) has been substituted with cysteic acid or homocysteic acid.
  • substitution at position 9 is considered optional in embodiments in which PLA is the N-terminal residue, since the modification at position 9 is not required for antagonist activity at the glucagon receptor.
  • the glucagon antagonist peptide comprises SEQ ID NO: 1 in which the first five amino acids of the N-terminus has been deleted and the 6 th residue of SEQ ID NO: 1 (which is the 1 st amino acid of the glucagon antagonist peptide) is PLA or other phenylalanine analog, including 3,4-2F-phenylalanine (3,4-2F-Phe), 2-naphthyalanine (2-Nal), N-acyl-phenylalanine (Ac-Phe), alpha- methylhydrocinnamic acid (MCA) and benzylmalonic acid (BMA), for example.
  • substitution with PLA at position 6 provides a more potent glucagon antagonist.
  • the glucagon antagonist peptide comprises the general structure of A-B-C, wherein A is selected from the group consisting of:
  • B represents amino acids i to 26 of SEQ ID NO: 1, wherein i is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:
  • Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 1) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, ⁇ -homo glutamic acid, or alkylcarboxylate derivative of cysteine having the structure
  • X5 is C C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl.
  • substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 1) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety;
  • cysteic acid glutamic acid, homoglutamic acid, and homocysteic acid
  • cysteic acid glutamic acid, homoglutamic acid, and homocysteic acid
  • X is Met, Leu, or Nle
  • Y is Asn or a charged amino acid
  • Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid
  • RIO is selected from a group consisting of SEQ ID NOs: 1119-1121 and 1153;
  • the glucagon antagonist peptide comprises the general structure A-B-C as described herein and exhibits agonist activity at the GLP-1 receptor. Accordingly, in some aspects, the glucagon antagonist peptide comprises (1) a stabilized alpha helix through means described herein (e.g., through an intramolecular bridge, or incorporation of one or more alpha, alpha-di-substituted amino acids, or an acidic amino acid at position 16 (according to the numbering of SEQ ID NO : 1), or a combination thereof; (2) a C-terminal amide or ester in place of a C-terminal carboxylate, and (3) a general structure of
  • A is selected from the group consisting of
  • B represents amino acids p to 26 of SEQ ID NO: 1, wherein p is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of:
  • Asp at position 9 (according to the amino acid numbering of SEQ ID NO: 1) is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, ⁇ -homo glutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of:
  • OOH wherein X5 is C C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl;
  • substitution of one or two amino acids at positions 16, 17, 20, 21, and 24 (according to the amino acid numbering of SEQ ID NO: 1) with an amino acid selected from the group consisting of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently attached to a hydrophilic moiety;
  • Asp at position 15 (according to the numbering of SEQ ID NO: 1) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid;
  • Ser at position 16 (according to the numbering of SEQ ID NO: 1) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid;
  • Arg at position 17 is replaced with Gin
  • Arg at position 18 is replaced with Ala
  • Asp at position 21 is replaced with Glu
  • Val at position 23 is replaced with He
  • Gin at position 24 is replaced with Ala
  • C is selected from the group consisting of:
  • X is Met, Leu, or Nle
  • Y is Asn or a charged amino acid
  • Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid
  • R10 is selected from a group consisting of SEQ ID NOs: 1221, 1226, 1227, and 1250.
  • the glucagon antagonist peptide comprises the general structure A-B-C
  • the glucagon antagonist peptide comprises an oxy derivative of PLA.
  • oxy derivative of PLA refers to a compound comprising a modified structure of PLA in which the hydroxyl group has been replaced with O-Rn, wherein Rn is a chemical moiety.
  • the oxy derivative of PLA can be, for example, an ester of PLA or an ether of PLA.
  • the ester may be formed by upon reaction of the hydroxyl of PLA with a carbonyl bearing a nucleophile.
  • the nucleophile can be any suitable nucleophile, including, but not limited to an amine or hydroxyl. Accordingly, the ester of PLA can comprise the structure of Formula V:
  • R7 is an ester formed upon reaction of the hydroxyl of PLA with a carbonyl bearing a nucleophile.
  • the carbonyl bearing a nucleophile (which reacts with the hydroxyl of PLA to form an ester) can be, for example, a carboxylic acid, a carboxylic acid derivative, or an activated ester of a carboxylic acid.
  • the carboxylic acid derivative can be, but is not limited to, an acyl chloride, an acid anhydride, an amide, an ester, or a nitrile.
  • the activated ester of a carboxylic acid can be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), a carbodiimide, or a hexafluorophosphate.
  • the carbodiimide is 1,3- dicyclohexylcarbodiimide (DCC), ⁇ , ⁇ -carbonyldiimidazole (CDI), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD).
  • DCC 1,3- dicyclohexylcarbodiimide
  • CDI ⁇ , ⁇ -carbonyldiimidazole
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride
  • DICD 1,3-diisopropylcarbodiimide
  • the hexafluorophosphate is selected from a group consisting of hexafluorophosphate benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium
  • ethers from reaction with a hydroxyl group e.g., the hydroxyl of PLA
  • Methods of making ethers from reaction with a hydroxyl group also are known in the art.
  • the hydroxyl group of PLA may be reacted with a halogenated alkyl or tosylated alkyl alcohol to form an ether bond.
  • the chemical moiety of Rn is one which does not decrease the activity of the glucagon antagonist peptide. In some embodiments, the chemical moiety enhances the activity, stability, and/or solubility of the glucagon antagonist peptide.
  • the chemical moiety bound to PLA via an oxygen- containing bond is a polymer (e.g., a polyalkylene glycol), a carbohydrate, an amino acid, a peptide, or a lipid, e.g., a fatty acid or a steroid.
  • a polymer e.g., a polyalkylene glycol
  • carbohydrate e.g., an amino acid, a peptide, or a lipid, e.g., a fatty acid or a steroid.
  • the chemical moiety is an amino acid, which, optionally, is a part of a peptide, such that Formula V is a depsipeptide.
  • PLA may be at a position other than the N-terminal amino acid residue of the glucagon antagonist peptide, such that the glucagon antagonist peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) amino acids N-terminal to the PLA residue.
  • the glucagon antagonist peptide can comprise PLA at position n, wherein n is 2, 3, 4, 5, or 6 of the glucagon antagonist peptide.
  • the amino acids N-terminal to the PLA residue may be synthetic or naturally- occurring.
  • the amino acids which are N-terminal to PLA are naturally- occurring amino acids.
  • the amino acids which are N-terminal to PLA are the N-terminal amino acids of native glucagon.
  • the glucagon antagonist peptide can comprise at the N-terminus the amino acid sequence of any of SEQ ID NOs: 1154-1158, wherein PLA is linked to threonine via an ester bond:
  • one or more of the N-terminal amino acids may be substituted with an amino acid other than the amino acid of native glucagon.
  • the glucagon antagonist comprises PLA as the amino acid at position 5 or 6
  • the amino acid at position 1 and/or position 2 may be an amino acid which reduces susceptibility to cleavage by dipeptidyl peptidase IV.
  • position 1 of the glucagon antagonist peptide is 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.
  • position 2 of the glucagon antagonist peptide is an amino acid selected from the group consisting of D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB).
  • the glucagon antagonist peptide comprises PLA as the amino acid at position 4, 5, or 6, the amino acid at position 3 of the glucagon antagonist peptide may be glutamic acid, as opposed to the native glutamine residue of native glucagon.
  • the glucagon antagonist comprises at the N-terminus the amino acid sequence of any of SEQ ID NOs: 1159-1161.
  • the polymer which is the chemical moiety bound to PLA may be any polymer, provided that it can react with the hydroxyl group of PLA.
  • the polymer may be one that naturally or normally comprises a carbonyl bearing a nucleophile.
  • the polymer may be one which was derivatized to comprise the carbonyl bearing the carbonyl.
  • the polymer may be a derivatized polymer of any of: polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl
  • polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycoli
  • the polymer can be a biodegradable polymer, including a synthetic biodegradable polymer (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)), and a natural biodegradable polymer (e.g., alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), as well as any copolymer or mixture thereof.
  • these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion
  • the polymer can be a bioadhesive polymer, such as a bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl
  • the polymer is a water-soluble polymer.
  • Suitable water-soluble polymers include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylic acid copolymers, polymethacrylic acid, polymethylmethacrylate, maleic anhydride
  • the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the chemical moiety bound to PLA is a carbohydrate.
  • the carbohydrate may be any carbohydrate provided that it comprises or is made to comprise a carbonyl with an alpha leaving group.
  • the carbohydrate for example, may be one which has been derivatized to comprise a carbonyl with an alpha leaving group.
  • the carbohydrate may be a derivatized form of a
  • a monosaccharide e.g., glucose, galactose, fructose
  • a disaccharide e.g., sucrose, lactose, maltose
  • an oligosaccharide e.g., raffinose, stachyose
  • a polysaccharide a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.
  • the chemical moiety bound to PLA can be a lipid.
  • the lipid may be any lipid comprising a carbonyl with an alpha leaving group.
  • the lipid for example, may be one which is derivatized to comprise the carbonyl.
  • the lipid may be a derivative of a fatty acid (e.g., a C4-C30 fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine),
  • glycerolipid e.g., mono-, di-, tri-substituted glycerols
  • glycerophospholipid e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine
  • sphingolipid e.g., sphingosine, ceramide
  • sterol lipid e.g., steroid, cholesterol
  • prenol lipid saccharolipid, or a polyketide oil, wax, cholesterol, sterol, fat- soluble vitamin,
  • R7 has a molecular weight of about 100 kDa or less, e.g., about 90 kDa or less, about 80 kDa or less, about 70 kDa or less, about 60 kDa or less, about 50 kDa or less, about 40 kDa or less. Accordingly, R7 can have a molecular weight of about 35 kDa or less, about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 5 kDa or less, or about 1 kDa.
  • the glucagon antagonist peptide of structure A-B-C comprises, as A, a peptide of 2 to 6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond.
  • the ester or ether bond may be, e.g., between amino acids 2 and 3, 3 and 4, 4 and 5, or 5 and 6.
  • the peptide may be further modified by covalent linkage to another chemical moiety including linkage to a polymer (e.g. a hydrophilic polymer), alkylation, or acylation.
  • the peptide may comprise any amino acids, synthetic or naturally occurring, provided that at least two consecutive amino acids of the peptide are linked via an ester or ether bond.
  • the peptide comprises amino acids of native glucagon.
  • the peptide can comprise j to 6 of native glucagon (SEQ ID NO: 1), wherein j is 1, 2, 3, 4, or 5.
  • the peptide can comprise an amino acid sequence based on the N-terminus of SEQ ID NO: 1 with one or more amino acid modifications.
  • the amino acid at position 1 and/or position 2 may be an amino acid which reduces susceptibility to cleavage by dipeptidyl peptidase IV.
  • the peptide can comprise at position 1 of the glucagon antagonist peptide (glucagon antagonist) 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.
  • D-histidine alpha
  • alpha-dimethyl imidiazole acetic acid DAIA
  • N-methyl histidine alpha-methyl histidine
  • imidazole acetic acid imidazole acetic acid
  • desaminohistidine hydroxyl- histidine
  • acetyl-histidine acetyl-histidine and homo-histidine.
  • position 2 of the antagonist peptide is an amino acid selected from the group consisting of D- serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB).
  • amino acid at position 3 of the glucagon antagonist may be glutamic acid, as opposed to the native glutamine residue of native glucagon.
  • the glucagon antagonist can comprise an amino acid sequence of:
  • Xaai is selected from a group consisting of: His, 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;
  • Xaa 2 is selected from a group consisting of: Ser, D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB); and
  • Xaa is Gin or Glu; wherein at least one bond between the amino acids of SEQ ID NO: 1168, 1169, or 1170is an ester or ether bond.
  • B represents amino acids of native glucagon, e.g., i to 26 of SEQ ID NO: 1, wherein i is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications.
  • B represents amino acids 7 to 26 of SEQ ID NO: 1, optionally further modified.
  • B is modified by up to three amino acid modifications.
  • B which represents native amino acid sequence of SEQ ID NO: 1 is modified by one or more conservative amino acid modifications.
  • B comprises one or more amino acid modifications selected from the group consisting of (iv) to (ix), as described herein.
  • B comprises one or both of the amino acid modifications (v) and (vi).
  • B comprises one or a combination of amino acid modifications selected from the group consisting of (iv), (vii), (viii), and (ix), in addition to (v) and (vi).
  • the peptide comprises (1) a stabilized alpha helix through means described herein (e.g., through an intramolecular bridge, or incorporation of one or more alpha, alpha-di-substituted amino acids, or an acidic amino acid at position 16 (according to the numbering of SEQ ID NO : 1), or a combination thereof; (2) a C-terminal amide or ester in place of a C-terminal carboxylate, and (3) a general structure of A-B-C, B comprises one or a combination of amino acid modifications selected from the group consisting of (iv), (vii), (viii), (ix), and (x), in addition to (v) and (vi).
  • the glucagon antagonist peptide comprises one or more charged amino acids at the C-terminus.
  • Y and/or Z can be a charged amino acid, e.g., Lys, Arg, His, Asp, and Glu.
  • the glucagon antagonist peptide comprises one to two charged amino acids (e.g., Lys, Arg, His, Asp, and Glu) C-terminal to Z.
  • Z followed by one to two charged amino acids does not comprise R10.
  • Y is Asp.
  • the glucagon antagonist peptide in some embodiments comprises a hydrophilic moiety covalently bound to an amino acid residue of the glucagon antagonist, as described herein.
  • the glucagon antagonist can comprise a hydrophilic moiety covalently attached to an amino acid at position 1, 16, 20, 21, or 24 according to the numbering of SEQ ID NO: 1 or to the the N- or C-terminal amino acid of the glucagon antagonist peptide.
  • the hydrophilic moiety is attached to the C-terminal amino acid of the glucagon antagonist peptide, which in some cases, is 1 or 11 amino acids C-terminal to Z.
  • the hydrophilic moiety is attached to PLA, when A is PLA, PLA- Phe, or PLA-Thr-Phe, wherein PLA is modified to comprise the hydrophilic moiety.
  • an amino acid comprising a hydrophilic moiety is added to the N- or C- terminus of the glucagon antagonist.
  • the hydrophilic moiety is attached to a Cys residue of the glucagon antagonist peptide comprising the general structure A-B-C.
  • the amino acid at position 16, 21, 24, or 29 (according to the numbering of native glucagon or the N- or C-terminal amino acid may be substituted with a Cys residue.
  • a Cys residue comprising a hydrophilic moiety may be added to the C-terminus of the peptide comprising the general structure A-B-C as position 30 or as position 40, e.g., when the peptide comprising the general structure A-B-C comprises a C-terminal extension (positions according to the amino acid numbering of SEQ ID NO: 1).
  • the hydrophilic moiety may be attached to the PLA of the peptide comprising the general structure A-B-C via the hydroxyl moiety of PLA.
  • the hydrophilic moiety can be any of those described herein, including, for example, polyethylene glycol.
  • the glucagon antagonist peptide comprising the general structure A-B-C comprises a stabilized alpha helix by virtue of comprising modifications as taught herein under "Stabilization of the Alpha Helix Structure.”
  • the glucagon antagonist peptide in some aspects, comprises an intramolecular bridge and/or one or more alpha, alpha di-substituted amino acids within the C-terminal portion of the peptide (residues 12-29 according to the numbering of SEQ ID NO: 1).
  • a stabilized alpha helix is provided by incorporation of an intramolecular bridge into the glucagon antagonist peptide.
  • the intramolecular bridge is a lactam bridge.
  • the lactam bridge may be between the amino acids at positions 9 and 12, the amino acids at positions 12 and 16, the amino acids at positions 16 and 20, the amino acids at positions 20 and 24, or the amino acids at positions 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1).
  • the amino acids at positions 12 and 16 or at positions 16 and 20 may be between the amino acids at positions 9 and 12, the amino acids at positions 12 and 16, the amino acids at positions 16 and 20, the amino acids at positions 20 and 24, or the amino acids at positions 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1).
  • the amino acids at positions 12 and 16 or at positions 16 and 20 may be between the amino acids at positions 9 and 12, the amino acids at positions 12 and 16, the amino acids at positions 16 and 20, the amino acids at positions 20 and 24, or the amino acids at positions 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1).
  • the amino acids at positions 12 and 16 or at positions 16 and 20 may be
  • lactam bridge (according to the amino acid numbering of SEQ ID NO: 1) are linked via a lactam bridge. Other positions of the lactam bridge are contemplated.
  • the peptide comprising the general structure A-B-C can comprise a stabilized alpha helix by virtue of comprising an alpha, alpha di-substituted amino acid at, for example, any of positions 16, 20, 21, or 24 (according to the amino acid numbering of SEQ ID NO: 1).
  • the alpha, alpha di-substituted amino acid is AIB.
  • the AIB is located at position 16 (according to the numbering of SEQ ID NO: 1).
  • the glucagon antagonist peptide comprising the general structure A-B-C may be modified to comprise an acidic amino acid at position 16 (according to the numbering of SEQ ID NO: 1), which modification enhances the stability of the alpha helix.
  • the acidic amino acid in one embodiment, is an amino acid comprising a side chain sulfonic acid or a side chain carboxylic acid.
  • the acidic amino acid is selected from the group consisting of Glu, Asp, homoglutamic acid, a sulfonic acid derivative of Cys, cysteic acid, homocysteic acid, Asp, and an alkylated derivative of Cys having the structure of
  • X5 is C C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl.
  • the glucagon antagonist peptide which is a glucagon antagonist/GLP- 1 agonist may comprise the amino acid sequence of any of SEQ ID NOs: 1260-1270, 1273-1278, 1280-1288, 1290-1296, 1303, 1304, 1306, and 1314-1318, or comprising the amino acid sequence of any of Peptides 2-6 of Table A, Peptides 1-8 of Table B, and Peptides 2-6, 8, and 9 of Table C:
  • the glucagon antagonist peptide comprising the general structure A-B-C is a glucagon antagonist/GLP- 1 agonist which exhibits at least about 50% of the maximum agonism achieved by native GLP- 1 at the GLP- 1 receptor and at least about 50% inhibition of the maximum response achieved by native glucagon at the glucagon receptor.
  • the glucagon antagonist peptide exhibits at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the maximum agonism achieved by native GLP-1 at the GLP-1 receptor.
  • the glucagon antagonist peptide may exhibit at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% inhibition of the maximum response achieved by native glucagon at the glucagon receptor.
  • the glucagon antagonist peptide comprises an acyl group or alkyl group as described herein.
  • the acylation or alkylation can occur off the side chain of the amino acid at position 10, 20, or 24, according to the numbering of SEQ ID NO: 1.
  • the acylation or alkylation occurs off the side chain of the C-terminal amino acid of the glucagon antagonist, which in some cases, is 1 or 11 amino acids C-terminal to Z.
  • A is PLA, PLA-Phe, or PLA-Thr- Phe
  • the PLA is modified to comprise an acyl or alkyl group.
  • the glucagon antagonist comprises the amino acid sequence of any of SEQ ID NOs: 1162, 1164-1167, and 1171 or structures of any of the peptides in Tables D-L.
  • Glu 9 is glutamic acid at position 9 according to the numbering of native glucagon.
  • hGlu homoglutamic acid
  • h pA 2 the negative logarithm of the concentration of the antagonist that reduce the response to lunit of the agonist to the response obtained from 0.5 unit of agonist.
  • Data are average +STD for at least two duplicate experiments.
  • the glucagon antagonist peptide exhibits both glucagon antagonist activity and GLP-1 agonist activity (e.g., a glucagon antagonist, GLP-1 agonist) and the glucagon antagonist peptide comprises:
  • deletion of 2 to 5 amino acids from the N-terminus of wild type glucagon optionally with substitution of Asp at position 9 of wild type glucagon with glutamic acid, homoglutamic acid or a sulfonic acid derivative of cysteine (according to amino acid numbering of wild type glucagon);
  • hydrophilic moiety such as polyethylene glycol, e.g. at the N-terminus, or at position 6, 16, 17, 20, 21, 24, 29, 40 or at the C-terminal amino acid; and/or
  • non-conservative substitutions conservative substitutions, additions or deletions while retaining desired 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, homo glutamic 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
  • any of the modifications within the same class may be combined together and/or modifications of different classes are combined.
  • the modifications of (l)(a) may be combined with (2)(a) and (3);
  • (l)(a) may be combined with (2)(b), e.g. lactam bridge or salt bridge, and (3);
  • (l)(a) may be combined with (2)(c) and (3);
  • (l)(b) may be combined with (2)(a) and (3);
  • (l)(b) may be combined with (2)(b), e.g.
  • lactam bridge or salt bridge and (3); (l)(b) may be combined with (2)(c) and (3); any of the foregoing may be combined with (4) (a) and/or (4)(b); and any of the foregoing may be combined with any of (5)(a) through (5)(k).
  • the a, a-disubstituted amino acid AIB is substituted at one, two, three or all of positions 16, 20, 21, or 24 (according to the amino acid numbering of wild type glucagon).
  • the intramolecular bridge is a salt bridge.
  • the intramolecular bridge is a covalent bond, e.g. a lactam bridge.
  • the lactam bridge is between the amino acids at positions 9 and 12, the amino acids at positions 12 and 16, the amino acids at positions 16 and 20, the amino acids at positions 20 and 24, or the amino acids at positions 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1).
  • acylation or alkylation is at position 6, 10, 20 or 24 or the N-terminus or C-terminus (according to the amino acid numbering of wild type glucagon) SEQ ID NO: 1).
  • modifications include:
  • the glucagon antagonist peptide of any of the foregoing embodiments comprises one or more further amino acid modifications (as compared to SEQ ID NO: 1), such as any of the amino acid modifications taught herein within the sections entitled
  • the glucagon antagonist peptide comprises an amino acid modification which reduces degradation.
  • the glucagon antagonist peptide comprises modifications which provide glucagon antagonist activity and further comprises one or two amino acid modifications at position 15 and/or position 16, as described herein with respect to the GIP agonist peptide. See, "Modifications that reduce degradation.”
  • the glucagon antagonist peptide comprises any of the modifications which confer glucagon antagonist activity and further comprises a substitution of the aspartic acid located at position 15 of the native glucagon peptide with an amino acid selected from the group consisting of cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid.
  • the glucagon antagonist peptide comprises any of the modifications which confer glucagon antagonist activity and further comprises a substitution of the serine at position 16 (according to the numbering of native glucagon) with glutamic acid, cysteic acid, homo-glutamic acid,or homo-cysteic acid.
  • the serine at position 16 (according to the native glucagon sequence numbering) is replaced with glutamic acid.
  • the glucagon antagonist comprising such a modification comprises a C-terminal carboxylate and is not amidated.
  • the glucagon antagonist peptide comprises any of the
  • modifications which confer glucagon antagonist activity and further comprises a substitution of the Met at position 27 (according to the numbering of SEQ ID NO: 1) with a Leu or norleucine to prevent oxidative degradation of the peptide.
  • the glucagon antagonist peptide comprises any of the
  • the amino acid at position 20 and/or 24 (according to the numbering of SEQ ID NO: 1) is substituted with Ser, Thr, Ala, or AIB.
  • the amino acid at position 20 and/or 24 (according to the numbering of SEQ ID NO: 1) is substituted with Lys, Arg, Orn, or citrulline.
  • the amino acid at position 21 (according to the numbering of SEQ ID NO: 1) is substituted with Glu.
  • the glucagon antagonist peptide is modified for enhanced solubility.
  • the glucagon antagonist peptide can be modified in accordance with the teachings under "Modifications that enhance solubility" taught herein.
  • the glucagon antagonist peptide comprises one or two charged amino acids at positions 28 and 29 (according to the numbering of SEQ ID NO: 1) and/or comprises additional charged amino acids C-terminal to position 29 (according to the numbering of SEQ ID NO: 1).
  • the glucagon antagonist peptide in additional aspects comprises any of the modifications taught herein under "Other modifications .”
  • the glucagon antagonist peptide in some aspects comprises a substitution of Lys at position 12 (according to the numbering of SEQ ID NO: 1) with Arg.
  • the glucagon antagonist peptide comprises a charge neutral group in place of the alpha carboxylate of the C-terminal residue.
  • the glucagon antagonist peptide which exhibits glucagon antagonist activity is acylated or alkylated in accordance with the teachings found herein under "Acylation and alkylation.”
  • the present disclosures additionally provide any of the glucagon analog peptides described herein (e.g., the GIP agonist peptides, glucagon antagonist peptides) in free form (e.g., not in combination with a different type of peptide, not conjugated to another peptide), provided that they are not disclosed in any of the references cited herein, including any of International Patent Application No. PCT/US2009/47447, International Patent Application Publication Nos. WO2009/058662 and WO2009/058734, or U.S. Patent Application Nos.
  • the glucagon analog peptide is a GIP agonist peptide not in combination with (e.g., not conjugated to) a glucagon antagonist peptide.
  • the glucagon analog peptide is a glucagon antagonist peptide, not in combination with (e.g., not conjugated to) a GIP agonist peptide.
  • the peptide of the present disclosures is either a GIP agonist peptide or a glucagon antagonist peptide, according to the descriptions herein, is an analog of glucagon (SEQ ID NO: 1), and furthermore is an analog of a peptide disclosed in any of the references cited herein, including any of International Patent Application No. PCT/US2009/47447, International Patent Application Publication Nos. WO2009/058662 and WO2009/058734, or U.S. Patent Application Nos.
  • the glucagon analog peptide of the present disclosures may be an analog of an amino acid sequence found within Sequence Listing 2 or Sequence Listing 3 in which the sequence begins with a Tyr, wherein the analog comprises an amino acid modification which reduces GIP activity as described herein.
  • the analog may be identical in sequence to a sequence of Sequence Listing 2 or 3, but comprises instead of the Tyr at position 1, a small aliphatic residue, e.g., Ala, Gly, or has the amino acid(s) at position 1 or at positions 1 and 2 deleted.
  • a small aliphatic residue e.g., Ala, Gly, or has the amino acid(s) at position 1 or at positions 1 and 2 deleted.
  • the glucagon analog peptide in which the glucagon analog peptide is a GIP agonist peptide, can exhibit activity at the glucagon receptor in addition to activity at the GIP receptor (and, optionally, the GLP-1 receptor).
  • the GIP agonist peptide exhibits tri-agonism at each of the glucagon, GIP, and GLP-1 receptors or co-agonism at each of the glucagon and GIP receptors.
  • the glucagon analog peptide exhibits at least or about 0.1% activity of native glucagon at the glucagon receptor.
  • the GIP agonist peptide exhibits at least or about 0.2%, at least or about 0.3%, at least or about 0.4%, at least or about 0.5%, at least or about 0.6%, at least or about 0.7%, at least or about 0.8%, at least or about 0.9%, at least or about 1%, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 100% of the activity of native glucagon at the glucagon receptor.
  • the EC50 of the GIP agonist peptide at the GIP receptor is less than or about 50-fold, less than or about 40-fold, less than or about 30-fold, or less than or about 20-fold different (higher or lower) from its EC50 at the glucagon receptor.
  • the GIP potency of the GIP agonist peptide is less than or about 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its glucagon potency.
  • the ratio of the EC50 of the GIP agonist peptide at the GIP receptor divided by the EC50 of the GIP agonist peptide at the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the GIP potency of the GIP agonist peptide compared to the glucagon potency of the GIP agonist peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the ratio of the EC50 of the GIP agonist peptide at the glucagon receptor divided by the EC50 of the GIP agonist peptide at the GIP receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the glucagon potency of the GIP agonist peptide compared to the GIP potency of the GIP agonist peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.
  • the selectivity for the human GLP-1 receptor of the GIP agonist peptide is not at least 100-fold the selectivity of for the human GIP receptor.
  • the selectivity of the GIP agonist peptide for the human GLP-1 receptor versus the GIP receptor is less than 100-fold (e.g., less than or about 90-fold, less than or about 80-fold, less than or about 70- fold, less than or about 60-fold, less than or about 50-fold, less than or about 40-fold, less than or about 30-fold, less than or about 20-fold, less than or about 10-fold, less than or about 5-fold).
  • the peptides described herein are glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into a salt (e.g., an acid addition salt, a basic addition salt), and/or optionally dimerized, multimerized, or polymerized, or conjugated.
  • a salt e.g., an acid addition salt, a basic addition salt
  • peptides of the disclosure can be obtained by methods known in the art.
  • the peptide can be recombinantly produced using a nucleic acid encoding the amino acid sequence of the analog using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
  • the peptides of the disclosure are isolated. In some embodiments, the peptides of the disclosure are purified. . It is recognized that "purity" is a relative term, and not to be necessarily construed as absolute purity or absolute enrichment or absolute selection. In some aspects, the purity is at least or about 50%, is at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% (e.g., at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or is approximately 100%.
  • the peptides described herein are commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).
  • the peptides can be synthetic, recombinant, isolated, and/or purified.
  • compositions comprising the peptide.
  • the teachings under “Conjugates” and “Compositions, Pharmaceutical compositions” are applicable to these embodiments.
  • the present disclosures further provides methods of using the peptides which are glucagon analogs and are either GIP agonist peptides or glucagon antagonist peptides.
  • the peptide may be used in any of the methods of treatment described herein, including methods of treating a metabolic disorder, e.g., diabetes, obesity, and the like.
  • the peptide may be used in a method of treating hypoglycemia, or in any method described in the teachings of
  • the present disclosures further provide conjugates.
  • the conjugate comprises a GIP agonist conjugated to a glucagon antagonist peptide.
  • the conjugate comprises at least one of the GIP agonist peptide and the glucagon antagonist peptide conjugated to a heterologous moiety.
  • the conjugate comprises a GIP agonist conjugated to a glucagon antagonist peptide and at least one of the peptides is conjugated to a heterologous moiety.
  • the conjugation between the two peptides or between the peptide and heterologous moiety may involve covalent bonds, non-covalent bonds, or both types of bonds.
  • the covalent bonds are any of the covalent linkages described herein (e.g., disulfide bonds, lactam bridges, olefin metathesis, and the like).
  • the covalent bonds are peptide bonds.
  • the conjugate may be a fusion peptide comprising either or both of the GIP agonist peptide and the glucagon antagonist peptide and optionally a heterologous moiety, e.g., a Fc receptor, or portion thereof.
  • the GIP agonist peptide is conjugated to the glucagon antagonist peptide through non-covalent linkages, e.g., electrostatic interactions, hydrogen bonds, van der Waals interactions, salt bridges, hydrophobic interactions, and the like.
  • the conjugation of the peptide to the other peptide and/or to the heterologous moiety may be indirect or direct conjugation, the former of which may involve a linker or spacer.
  • Suitable linkers and spacers are known in the art and include, but not limited to, any of the linkers or spacers described herein under the sections "Acylation and alkylation" and "Linkages?"
  • heterologous moiety is synonymous with the term “conjugate moiety” and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the GIP agonist peptide or glucagon antagonist peptide to which it is attached.
  • conjugate moieties that can be linked to any of the analogs described herein include but are not limited to a heterologous peptide or polypeptide
  • a conjugate comprising a peptide of the peptide combination and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferin, fibrinogen and globulins. In some embodiments the plasma protein moiety of the conjugate is albumin or transferin.
  • the conjugate in some embodiments comprises one or more of the peptides of the peptide combinations described herein and one or more of: a peptide (which is distinct from the GIP agonist peptide and glucagon antagonist peptide described herein), a polypeptide, a nucleic acid molecule, an antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagnostic agent, a carbohydrate, an amino acid.
  • the heterologous moiety is a peptide which is distinct from the the GIP agonist peptide and glucagon antagonist peptide described herein and the conjugate is a fusion peptide or a chimeric peptide.
  • the heterologous moiety is a peptide extension of 1-21 amino acids.
  • the extension is attached to the C-terminus of the glucagon analog, e.g., to amino acid at position 29.
  • the extension comprises an amino acid sequence of SEQ ID NO: 3
  • the amino acid sequence is attached through the C- terminal amino acid of the peptide, e.g., amino acid at position 29.
  • the amino acid sequence of SEQ ID NOs: 3, 4, 8, and 9 is bound to amino acid 29 of the peptide through a peptide bond.
  • the amino acid at position 29 of the glucagon analog is a Gly and the Gly is fused to one of the amino acid sequences of SEQ ID NOs: 3, 4, 8, and 9.
  • the heterologous moiety is a polymer.
  • the polymer is selected from the group consisting of: polyamides,
  • the polymer is a biodegradable polymer, including a synthetic biodegradable polymer (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide- cocaprolactone)), and a natural biodegradable polymer (e.g., alginate and other
  • polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), as well as any copolymer or mixture thereof.
  • these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • the polymer is a bioadhesive polymer, such as a bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl
  • a bioadhesive polymer such as a bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly
  • the polymer is a water-soluble polymer or a hydrophilic polymer.
  • Hydrophilic polymers are further described herein under "Hydrophilic Moieties.” Suitable water-soluble polymers are known in the art and include, for example,
  • methylcellulose HPMC; Methocel
  • nitrocellulose hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,
  • ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylic acid copolymers, polymethacrylic acid, polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,
  • polyoxyethyleneglycols polyethylene oxide, and derivatives, salts, and combinations thereof.
  • the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the heterologous moiety is a carbohydrate.
  • the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.
  • a monosaccharide e.g., glucose, galactose, fructose
  • a disaccharide e.g., sucrose, lactose, maltose
  • an oligosaccharide e.g., raffinose, stachyose
  • a polysaccharide a starch,
  • the heterologous moiety is a lipid.
  • the lipid in some embodiments, is a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g., mono-, di-, tri- substituted glycerols), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,
  • sphingolipid e.g., sphingosine, ceramide
  • sterol lipid e.g., steroid, cholesterol
  • prenol lipid saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fat- soluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.
  • the 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 CH2 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
  • Amino acid modifications may be made to the Fc region of an immunoglobulin.
  • 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).
  • FcyRI FcyRIIA, FcyRIIB, and/or FcyRIIIA
  • 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).
  • GIP agonist peptide and/or glucagon antagonist peptide described herein can be further modified to improve its solubility and stability in aqueous solutions at
  • Hydrophilic moieties such as PEG groups can be attached to the analogs 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 chemo selective 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 hydrazino group
  • 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-bromo acetic acid, alpha-chloroacetic acid).
  • alpha-halogenated acyl group e.g., alpha-iodo acetic acid, alpha-bromo acetic 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
  • an amino acid residue of the analog 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 analog com rising the thioether linkage shown below:
  • the thiol is modified with a haloacetyl-activated PEG in a nucleophilic substitution reaction to result in a PEGylated analog 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), polyoxyalkylenes, polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy- polyethylene glycol, mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol,
  • polystyrene resin polyvinyl alcohol
  • PVA polyvinyl pyrrolidone
  • poly-1, 3-dioxolane poly-l,3,6-trioxane
  • ethylene/maleic anhydride copolymer poly (.beta.-amino acids) (either homopolymers or random copolymers)
  • poly(n- vinyl pyrrolidone)polyethylene glycol polypropylene glycol homopolymers (PPG) and other polyakylene oxides
  • PPG propropylene glycol homopolymers
  • polypropylene oxide/ethylene oxide copolymers colonic acids or other polysaccharide polymers, Ficoll or dextran and mixtures thereof.
  • 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 analog.
  • the peptide of the conjugate is conjugated to a hydrophilic moiety via covalent linkage between a side chain of an amino acid of the glucagon analog and the hydrophilic moiety.
  • the glucagon analog is conjugated to a hydrophilic moiety via the side chain of an amino acid at position 16, 17, 21, 24, or 29, a position within a C-terminal extension, or the C-terminal amino acid, or a combination of these positions.
  • the amino acid covalently linked to a hydrophilic moiety is 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).
  • the conjugate of the present disclosures comprises the peptide fused to an accessory analog which is capable of forming an extended conformation similar to chemical PEG (e.g., a recombinant PEG (rPEG) molecule), such as those described in International Patent Application Publication No. WO2009/023270 and U.S. Patent Application Publication No. US20080286808.
  • the rPEG molecule in some aspects is a polypeptide comprising one or more of glycine, serine, glutamic acid, aspartic acid, alanine, or proline.
  • the rPEG is a homopolymer, e.g., poly-glycine, poly-serine, poly-glutamic acid, poly-aspartic acid, poly-alanine, or poly-proline.
  • the rPEG comprises two types of amino acids repeated, e.g., poly(Gly-Ser), poly(Gly-Glu), poly(Gly-Ala), poly(Gly-Asp), poly(Gly-Pro), poly(Ser-Glu), etc.
  • the rPEG comprises three different types of amino acids, e.g., poly(Gly-Ser-Glu).
  • the rPEG increases the half-life of the Glucagon and/or GLP-1 agonist analog.
  • the rPEG comprises a net positive or net negative charge.
  • the rPEG in some aspects lacks secondary structure.
  • the rPEG is greater than or equal to 10 amino acids in length and in some embodiments is about 40 to about 50 amino acids in length.
  • the accessory peptide in some aspects is fused to the N- or C- terminus of the analog of the present disclosure through a peptide bond or a proteinase cleavage site, or is inserted into the loops of the analog of the present disclosure.
  • the rPEG in some aspects comprises an affinity tag or is linked to a PEG that is greater than 5 kDa.
  • the rPEG confers the analog of the present disclosure with an increased hydrodynamic radius, serum half-life, protease resistance, or solubility and in some aspects confers the analog with decreased immunogenicity.

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EP11737598.0A EP2528618A4 (en) 2010-01-27 2011-01-26 GLUCAGON ANTAGONISTE AND GIP AGONISTS CONJUGATES AND COMPOSITIONS FOR THE TREATMENT OF METABOLISM DISEASES AND ADIPOSITAS
KR1020127021672A KR20120123443A (ko) 2010-01-27 2011-01-26 대사 장애 및 비만 치료용 글루카곤 길항제-ｇｉｐ 항진제 콘쥬게이트
BR112012018585A BR112012018585A2 (pt) 2010-01-27 2011-01-26 conjungados e composições de glucagon antagonista-gip agonista para o tratamento de distúrbios metabólicos e de obesidade
IN6437DEN2012 IN2012DN06437A (US07919591-20110405-C00054.png) 2010-01-27 2011-01-26
JP2012551270A JP2013518115A (ja) 2010-01-27 2011-01-26 代謝疾患及び肥満の治療のためのグルカゴンアンタゴニスト‐ｇｉｐアゴニスト複合体及び組成物
CA2788304A CA2788304A1 (en) 2010-01-27 2011-01-26 Glucagon antagonist - gip agonist conjugates and compositions for the treatment of metabolic disorders and obesity
MX2012008603A MX2012008603A (es) 2010-01-27 2011-01-26 Conjugados de antagonista de glucagon-agonista de gip y composiciones para el tratamiento de desordenes metabolicos y obesidad.
CN2011800170897A CN102834108A (zh) 2010-01-27 2011-01-26 用于治疗代谢紊乱和肥胖症的胰高血糖素拮抗剂-gip激动剂偶联物和组合物
US13/575,363 US8551946B2 (en) 2010-01-27 2011-01-26 Glucagon antagonist-GIP agonist conjugates and compositions for the treatment of metabolic disorders and obesity
RU2012136450/10A RU2012136450A (ru) 2010-01-27 2011-01-26 Конъюгаты антагонист глюкагона - агонист gip и композиции для лечения метаболических расстройств и ожирения
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