WO2025085842A1

WO2025085842A1 - A method to mitigate peptide aggregation

Info

Publication number: WO2025085842A1
Application number: PCT/US2024/052107
Authority: WO
Inventors: Robert P. DOYLE; Ian C. TINSLEY; Amber LILES; Nancy CHAM
Original assignee: Syracuse University
Current assignee: Syracuse University
Priority date: 2023-10-19
Filing date: 2024-10-18
Publication date: 2025-04-24
Anticipated expiration: 2026-04-19

Abstract

Provided herein are modified glucagon molecules with improved stability. The glucagon molecules are part of a compound and include a corrin complex, which is the core structure of Vitamin B12. The corrin complex is conjugated to the glucagon molecule at different sites in the glucagon molecule and provides increased chemical and physical stability to the glucagon molecule. Further provided are formulations and methods of making and using the glucagon compounds.

Description

A METHOD TO MITIGATE PEPTIDE AGGREGATION

RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/591,593, filed October 19, 2023, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Number 1R01DK128443 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (S234170003WO00-SEQ- KUW.xml; Size: 11,239 bytes; and Date of creation: October 17, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Hypoglycemia, or low blood glucose, is a common side effect of using insulin to manage diabetes. Glucagon is a hormone that raises blood glucose levels and is commonly used to treat hypoglycemia. The pharmaceutical development of glucagon for use in treating acute hypoglycemia has proved challenging, due in large part to poor solubility, a short halflife, and a tendency to aggregate. Glucagon (Gcg) is a 29-amino acid pancreatic hormone derived from proglucagon. Gcg is released from pancreatic a-cells in response to postprandial release of insulin from pancreatic P-cells with a view to increase energy expenditure Gcg acts at the glucagon receptor (GcgR), a G_q-coupled G-protein coupled receptor found primarily in liver, adipose, heart, and kidney tissues. Gcg has a short half-life (< 5 minutes) and is highly prone to aggregation, a reason pharmaceutical development of such to treat acute hypoglycemia has proven challenging. Over the past 70 years, there have been only two FDA-approved formulations of standard Gcg: GlucaGen HypoKit® from Novo Nordisk and Glucagon Emergency Kit® from Eli Lilly. Both Gcg products are supplied as lyophilized powder and need to be reconstituted with water or dilute hydrochloric acid prior to injection. Dissolved Gcg has a propensity to fibrillate and form aggregates and thus, must be used immediately after reconstitution, making it inadequate for prolonged use. Due to this complex process, several industrial groups are developing a stable liquid form of Gcg such as Gvoke® from Xeris Pharmaceuticals. The Gvoke HypoPen (TM) is a single-use auto-injector in which Gcg is dissolved in dimethyl sulfoxide (DMSO) for the treatment of severe hypoglycemia. Although DMSO prevents aggregation and fibrillation of the drug, it causes side effects such as irritative and burning skin reactions¹⁰ at the injection site. Since it is delivered subcutaneously, other side effects include nausea and vomiting. Therefore, there is a need to develop a method to improve both the physical and chemical stability of peptides that are often unstable in aqueous solutions, such as Gcg.

BRIEF SUMMARY

[0005] The present invention provides a method of improving peptide stability (e.g., solubility) through corrination of the peptide. In some aspects, provided herein is a physically stable (e.g., soluble) glucagon compound. In some aspects, a compound comprises a molecule of glucagon conjugated to a corrin complex (e.g., a corrin ring). In some aspects, a corrin complex is conjugated to a naturally occurring amino acid on a glucagon molecule. In some aspects, an amino acid (e.g., an azido lysine) is added to the N-terminus and or C- terminus of a glucagon molecule and a corrin complex is conjugated to this additional amino acid. In some aspects, one or more amino acids of the glucagon molecule are substituted (e.g., with azido lysine) and one or more corrin complexes are conjugated to the substituted amino acids. In some embodiments, a corrinated glucagon is, e.g., a compound as shown in Table 2. As an example, glucagon was modified with commercially available dicyanocobinamide at two sites on the peptide backbone, K17 or K30, using copper catalyzed azine-alkyne cycloaddition. The corrination of the exemplary glucagon with a corrin complex (e.g., a corrin ring) containing compound, dicyanocobinamide (Cbi), provided improved protein solubility and peptide stability without interfering with the efficacy of the protein to bind to its receptor.

[0006] In some aspects, provided is a compound comprising a glucagon that comprises a lysine at position 17 (K17) of the wild-type glucagon amino acid sequence. [0007] In some aspects, a compound comprises a glucagon that is extended at a C- terminal end by a lysine (K30).

[0008] In some aspects, a compound comprising a glucagon and a corrin complex (e.g., a corrin ring) comprises the corrin ring conjugated to a lysine at position 17 or position 30.

[0009] In some aspects, the corrin ring is dicyanocobinamide. [0010] In some aspects, provided is a method of providing an improvement to a pharmaceutical development of glucagon, comprising the step of conjugating a molecule of dicyanocobinamide to a molecule of glucagon at a predetermined site of the molecule of glucagon.

[0011] In some aspects, the predetermined site on the glucagon peptide backbone is K17.

[0012] In some aspects, the predetermined site on the glucagon peptide backbone is K30.

[0013] In some aspects, the step of conjugating the molecule of dicyanocobinamide to the molecule of glucagon comprises using copper(I)-mediated alkyne-azide cycloaddition using strain promoted alkyne-azide cycloaddition (SPAAC).

[0014] In some aspects, provided is a compound having an improved pharmaceutical effect, the compound comprising glucagon and a dicyanocobinamide bound to the glucagon. [0015] In some aspects, provided is method of preparing a glucagon therapeutic, the method comprising preparing a compound comprising a glucagon molecule and a corrin complex described herein in a phosphate buffer.

[0016] In some aspects, the method further comprises maintaining the compound comprising a glucagon molecule and a corrin complex in the phosphate buffer for up to 2 weeks prior to use.

[0017] In some aspects, provided is a method of preparing a glucagon therapeutic, the method comprising preparing a compound comprising a glucagon molecule and a corrin complex described herein in a hydrochloric acid buffer.

[0018] In some aspects, the method further comprises maintaining a compound comprising a glucagon molecule and a corrin complex in the hydrochloric acid buffer for up to 48 hours prior to use.

[0019] In some aspects, provided is a method of using a compound described herein to treat hypoglycemia, wherein the method comprises preparing a compound comprising a glucagon molecule and a corrin complex in a phosphate buffer.

[0020] In some aspects, the method comprises preparing the compound comprising a glucagon molecule and a corrin complex in phosphate buffer up to 2 weeks prior to use.

[0021] In some aspects, provided is a method of using a compound described herein to treat obesity, wherein the method comprises preparing a compound comprising a glucagon molecule and a corrin complex in a phosphate buffer. [0022] In some aspects, the method comprises preparing the compound comprising a glucagon molecule and a corrin complex in a phosphate buffer up to 2 weeks prior to use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0024] FIG. 1 is a graph of an assessment of the chemical stability of glucagon.

[0025] FIG. 2 is a graph of an aggregate formation assay in 0.1M HC1 using the THT fluorescence assay.

[0026] FIG. 3 is a series of graphs of differential GcgR agonist properties of synthetic Gcg, 2, or 5, as revealed in FRET assays that monitor intracellular levels of cAMP. FIG. 3A shows FRET assays of wild-type glucagon at 0 hours (Al) and 24 hours (A2) and EC50 at the glucagon receptor at 0 and 24 hours (A3). FIG. 3B shows FRET assays of glucagon having a lysine substitution at position 17 (GlucagonK17) at 0 hours (Bl) and 24 hours (B2) and EC50 at the glucagon receptor at 0 and 24 hours (B3). FIG. 3C shows FRET assays of glucagon having a lysine substitution at position 17 and a corrin complex (cbi-GlucagonK17) at 0 hours (Cl) and 24 hours (C2) and EC50 at the glucagon receptor at 0 and 24 hours (C3).

[0027] FIG. 4 is a series of graphs displaying that the purity of Cbi was confirmed by semi-preparative RP-HPLC. FIG. 4A shows purity tested on an Eclipse XDB-C18 column. FIG. 4B shows a MALDI-TOF-MS analysis. FIG. 4C shows a 1H NMR analysis. FIG. 4D shows a UV-Vis analysis.

[0028] FIG. 5 is a series of graphs displaying that Cbi with an available alkyne reactive group was purified by RP-HPLC. FIG. 5A shows Zorbax SB-C18 column purification analysis. FIG. 5B shows a MALDI-TOF-MS analysis. FIG. 5C shows a 1H NMR analysis. FIG. 5D shows a UV-Vis analysis.

[0029] FIG. 6 is a series of graphs displaying that Gcg was purified by RP-HPLC. FIG. 6A shows an Eclipse XDB-C18 column purification analysis. FIG. 6B shows a MALDI-TOF-MS analysis.

[0030] FIG. 7 is a series of graphs displaying that Gcg 17 was purified by RP-HPLC. FIG. 7A shows an Eclipse XDB-C18 column purification analysis. FIG. 7B shows a MALDI-TOF-MS analysis. [0031] FIG. 8 is a series of graphs displaying that 2 was purified by RP-HPLC. FIG. 8A show an Eclipse XDB-C18 column purification analysis. FIG. 8B shows a MALDI-TOF- MS analysis.

[0032] FIG. 9 is a series of graphs displaying that 3 was purified by RP-HPLC. FIG. 9A shows an Eclipse XDB-C18 column purification analysis. FIG. 9B shows a MALDI- TOF-MS analysis.

[0033] FIG. 10 is a series of graphs displaying that 4 was purified by RP-HLC. FIG. 10A shows an Eclipse XDB-C18 column purification analysis. FIG. 10B shows a MALDI- TOF-MS analysis.

[0034] FIG. 11 shows a MALDI-TOF-MS analysis of Compound 5 purified by Slide- a-Lyzer protein dialysis cassette.

[0035] FIG. 12 shows a MALDI-TOF-MS analysis of Compound 6 purified by Slide- a-Lyzer protein dialysis cassette.

[0036] FIG. 13 shows a MALDI-TOF-MS analysis of Compound 7 purified by Slide- a-Lyzer protein dialysis cassette.

[0037] FIG. 14 is a graph of fluorescence data of Cbi and the corrinated compounds (Compounds 5, 6, and 7).

DETAILED DESCRIPTION

[0038] In some aspects, provided are peptide compounds that are stable (e.g., soluble) through corrination of the peptide. In some aspects, provided herein is a physically stable (e.g., soluble) glucagon compound. In some aspects, a compound comprises a molecule of glucagon conjugated to a corrin complex (e.g., a corrin ring). In some aspects, a corrin complex is conjugated to a naturally occurring amino acid on a glucagon molecule. In some aspects, an amino acid (e.g., an azido lysine) is added to the N-terminus and or C-terminus of a glucagon molecule and a corrin complex is conjugated to this additional amino acid. In some aspects, one or more amino acids of the glucagon molecule are substituted (e.g., with azido lysine) and one or more corrin complexes are conjugated to the substituted amino acids. In some embodiments, a corrinated glucagon is, e.g., a compound as shown in Table 2 [0039] In some aspects, provided are compounds comprising a glucagon molecule and a corrin complex (e.g., corrin ring). In some aspects, provided is a compound comprising a glucagon molecule conjugated to a corrin complex, e.g., a compound as shown in Table 2. In some aspects, provided are compounds comprising a glucagon molecule and more than one corrin complex. [0040] In some aspects, a glucagon molecule comprises a modification at position 17 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises a lysine at position 17 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises an azido-lysine at position 17 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises an amino acid sequence of SEQ ID NO: 2.

[0041] In some aspects, provided is a glucagon molecule comprising an additional amino acid following position 29 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises a lysine added to a C-terminal end of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises an azido-lysine added to a C-terminal end of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a glucagon molecule comprises an amino acid sequence of SEQ ID NO: 3. [0042] In some aspects, provided is a compound comprising a glucagon molecule as described herein conjugated to a corrin complex. In some aspects, a glucagon molecule is conjugated to one corrin complex (e.g., corrin ring). In some aspects, a glucagon molecule is conjugated to more than one corrin complex (e.g., corrin ring).

[0043] ‘Corrin complex” refers to a metal coordination complex of a compound

[0044] In some aspects, the corrin complex is a cobalt corrin complex. In some aspects, the corrin complex is a cobalamin. In some aspects, the corrin complex is a cobinamide. In some aspects, the corrin complex is a dicyanocobinamide (Cbi).

[0045] In some aspects, provided is a compound comprising a glucagon modified at position 17 and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide. In some aspects, provided is a compound comprising a glucagon modified at position 17 and a corrin complex conjugated to position 17 of the glucagon, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide. In some aspects, a compound comprises a glucagon substituted at position 17 with a lysine and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide. In some aspects, a compound comprises a glucagon substituted at position 17 with an azido-lysine and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide. [0046] In some aspects, a compound comprises a glucagon substituted at position 17 with an azido-lysine and a cobalt corrin complex conjugated to the lysine at position 17. In some aspects, a compound comprises a glucagon substituted at position 17 with an azidolysine and a cobalamin conjugated to the lysine at position 17. In some aspects, a compound comprises a glucagon substituted at position 17 with an azido-lysine and a cobinamide conjugated to the lysine at position 17. In some aspects, a compound comprises a glucagon substituted at position 17 with an azido-lysine and a dicyanocobinamide conjugated to the lysine at position 17. In some aspects, a compound comprises a modified glucagon comprising a sequence of SEQ ID NO: 5.

[0047] In some aspects, a compound comprises a glucagon extended at a C-terminal end by one amino acid residue following position 29 of wild-type glucagon of SEQ ID NO: 1 and a corrin complex. In some aspects, a compound comprises a glucagon extended at a C- terminal end by a lysine and the compound further comprising a corrin complex. In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and the compound further comprising a corrin complex.

[0048] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide. In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and a corrin complex conjugated to the lysine at a C-terminal end of the glucagon, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0049] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and a cobalt corrin complex. In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and a cobalamin. In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine and a cobinamide. In some aspects, a compound comprises a glucagon extended at a C- terminal end by an azido-lysine and a dicyanocobinamide. In some aspects, a compound comprises a modified glucagon comprising a sequence of SEQ ID NO: 6. [0050] In some aspects, a compound comprises a glucagon substituted at position 17 with an azido-lysine and a corrin complex conjugated to the lysine at position 17 and the glucagon molecule is further extended at a C-terminal end by an azido-lysine and a corrin complex is conjugated to the C-terminal extended lysine.

[0051] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of an amino acid. In some aspects, a compound comprises a glucagon comprising a D-isomer of an arginine. In some aspects, a compound comprises a glucagon comprising a D- isomer of an arginine at position 17. In some aspects, a compound comprises a glucagon comprising an amino acid sequence of SEQ ID NO: 8.

[0052] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of serine at position 2 of a wild-type glucagon of SEQ ID NO: 1.

[0053] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of glycine at position 4 of a wild-type glucagon of SEQ ID NO: 1.

[0054] In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine substitution at any position of positions 1-29 of the wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine substitution at more than one position of a wild-type human glucagon of SEQ ID NO: 1. For example, a compound comprising a glucagon molecule can have 1 or more azidolysine substitutions from 1-29, e.g., at positions 2 and 17 compared to a wild-type human glucagon of SEQ ID NO: 1 so that more than one corrin complex can be linked to the modified glucagon molecule. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine addition to a C-terminal and/or a N -terminal end of a wild-type human glucagon of SEQ ID NO: 1.

[0055] In some aspects, provided are compounds comprising a glucagon molecule conjugated at more than one position of positions 1-29 of the wild-type human glucagon of SEQ ID NO: 1 to a corrin complex. In some aspects, provided are compounds comprising a glucagon molecule conjugated to more than one corrin complex.

[0056] In some aspects, provided are compounds comprising a glucagon molecule conjugated to a corrin complex at a C-terminal azido-lysine of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine extension at a N-terminal end of a wild-type human glucagon of SEQ ID NO: 1.

[0057] In some aspects, provided are compounds comprising a glucagon molecule conjugated to a corrin complex at a C-terminal azido-lysine of a wild-type human glucagon of SEQ ID NO: 1 and at a N-terminal azido-lysine of a wild-type human glucagon of SEQ ID

NO: 1.

[0058] In some aspects, provided are compounds comprising a glucagon molecule comprising an azido-lysine at position 2 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, provided are compounds comprising a glucagon molecule comprising an azidolysine at position 4 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine at position 2 and position 4 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine at position 2 and at position 17 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine at position 4 and at position 17 of a wild-type human glucagon of SEQ ID NO: 1.

[0059] In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine at position 2, at position 17 and a C-terminal extension of an azido-lysine of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprising a glucagon molecule comprises an azido-lysine at position 4, at position 17 and a C-terminal extension of an azido-lysine of a wild-type human glucagon of SEQ ID NO: 1.

[0060] In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 2 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 4 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 2 and a corrin complex conjugated at position 17 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 4 and a corrin complex conjugated at position 17 of a wild-type human glucagon of SEQ ID NO: 1.

[0061] In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 2 and a corrin complex conjugated to a lysine extension at position 30 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 4 and a corrin complex conjugated at a lysine extension at position 30 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 2, a corrin complex conjugated at position 17, and a corrin complex conjugated at a lysine extension at position 30 of a wild-type human glucagon of SEQ ID NO: 1. In some aspects, a compound comprises a glucagon molecule conjugated to a corrin complex at position 4, a corrin complex conjugated at position 17, and a corrin complex conjugated at a lysine extension at position 30 of a wild-type human glucagon of SEQ ID NO: 1.

[0062] In some aspects, a compound comprises a glucagon extended at a C-terminal end by one amino acid residue following position 29 of a wild-type glucagon of SEQ ID NO: 1, a D-isomer of an amino acid, and a corrin complex.

[0063] In some aspects, a compound comprises a glucagon extended at a C-terminal end by one amino acid residue, a D-isomer of an arginine, and a corrin complex.

[0064] In some aspects, a compound comprises a glucagon extended at a C-terminal end by a lysine, a D-isomer of an arginine, and a corrin complex. In some aspects, a compound comprises a glucagon extended at a C-terminal end by a lysine, a D-isomer of an arginine, and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0065] In some aspects, a compound comprises a glucagon extended by one azidolysine following position 29, a D-isomer of an arginine, and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0066] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex. In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azidolysine, a D-isomer of an arginine at position 17, and a corrin complex selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0067] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a cobalt corrin complex. [0068] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a cobalamin.

[0069] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a cobinamide.

[0070] In some aspects, a compound comprises a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a dicyanocobinamide. [0071] In some aspects, a compound comprises a glucagon comprising an amino acid sequence of SEQ ID NO: 7.

[0072] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of serine at position 2 of a wild-type glucagon of SEQ ID NO: 1 and an azido- lysine at position 17 of the wild-type glucagon of SEQ ID NO: 1 and a corrin complex conjugated to position 17 of the glucagon, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0073] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of serine at position 2 of SEQ ID NO: 2.

[0074] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of glycine at position 4 of a wild-type glucagon of SEQ ID NO: 1 and an azidolysine at position 17 of the wild-type glucagon of SEQ ID NO: 1 and a corrin complex conjugated to position 17 of the glucagon, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0075] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of glycine at position 4 of SEQ ID NO: 2.

[0076] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of serine at position 2 of a wild-type glucagon of SEQ ID NO: 1 and extended at a C-terminal end of the wild-type glucagon of SEQ ID NO: 1 by an azido-lysine and a corrin complex conjugated to the C-terminal lysine, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0077] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of serine at position 2 of SEQ ID NO: 6.

[0078] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of glycine at position 4 of a wild-type glucagon of SEQ ID NO: 1 and extended at a C-terminal end of the wild-type glucagon of SEQ ID NO: 1 by an azido-lysine and a corrin complex conjugated to the C-terminal lysine, wherein the corrin complex is selected from a cobalt corrin complex, a cobalamin, a cobinamide and a dihydrocobinamide.

[0079] In some aspects, provided is a compound comprising a glucagon comprising a D-isomer of glycine at position 4 of SEQ ID NO: 6.

[0080] In some aspects, provided is a glucagon described herein as a therapeutic. In some aspects, provided is a compound comprising a glucagon molecule and a corrin complex as a therapeutic. In some aspects, a compound comprising a glucagon molecule and a corrin complex is developed as a pharmaceutical.

[0081] In some aspects, provided is a glucagon therapeutic described herein in a formulation. In some aspects, a glucagon therapeutic described herein is formulated for enteral administration to a subject in need thereof. In some aspects, a glucagon therapeutic described herein is formulated for parenteral administration to a subject in need thereof. In some aspects, the parenteral administration is intravenous, subcutaneous, intramuscular, or intraperitoneal administration, administration via inhalation, topical administration or administration by direct injection to one or more cells, tissues, or organs.

[0082] In some aspects, a glucagon therapeutic described herein is present in a formulation further comprising carrier. The term “carrier” refers to a diluent, adjuvant, or excipient. In some aspects, a carrier is a pharmaceutically acceptable carrier. In some aspects, a carrier is a liquid carrier. In some aspects, a liquid carrier is, e.g., a saline solution, aqueous dextrose, or glycerol solution. In some aspects, a liquid carrier is a phosphate buffered saline, HEPES -buffered saline, or water for injection. In some aspects, a liquid carrier may be combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. In some aspects, a liquid carrier comprises a phosphate buffer. In some aspects, a phosphate buffer comprises sodium phosphate. In some aspects, a phosphate buffer comprises about 50 mM sodium phosphate. [0083] In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising a phosphate buffer for storage of the formulated glucagon therapeutic for up to 2 weeks prior to use. In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising phosphate buffer for storage of the formulated glucagon therapeutic at up to 37°C for up to 2 weeks prior to use.

[0084] In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising a standard extracellular saline solution (SES).

[0085] In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising hydrochloric acid. In some aspects, a glucagon therapeutic described herein is formulated in a hydrochloric acid buffer. In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising a hydrochloric acid buffer having a pH of between about 1 and about 4. In some aspects, a glucagon therapeutic described herein is formulated in a liquid comprising a hydrochloric acid buffer for storage of the formulated glucagon therapeutic for up to 48 hours prior to use.

[0086] In some aspects, a glucagon therapeutic described herein is formulated as a liquid as described herein for storage for about 2 weeks at temperatures of up to 37°C. In some aspects, a glucagon therapeutic described herein is formulated as a liquid as described for storage for more than about 2 weeks. In some aspects, a glucagon therapeutic described herein is formulated as a liquid as described herein without lyophilization of the glucagon therapeutic. In some aspects, a glucagon therapeutic described herein is formulated in a liquid as described without the need for reconstitution of the glucagon therapeutic from a lyophilized form.

[0087] In some aspects, a glucagon therapeutic described herein formulated as a liquid comprises a single amino acid substitution, e.g., a replacement of an arginine (R) at position 17 of wild-type glucagon of SEQ ID NO: 1 with a lysine (K). In some aspects, an R- to-K substituted glucagon further comprises a corrin complex conjugated to K17. In some aspects, a glucagon therapeutic as described herein formulated as a liquid comprises a single amino acid addition at a C-terminal end of wild-type glucagon of SEQ ID NO: 1, e.g., addition of a lysine (K). In some aspects, a glucagon molecule extended at a C-terminus by a lysine (K30) further comprises a corrin complex conjugated to K30. Without wanting to be bound by a particular theory it is hypothesized that the glucagon compounds described herein have a low likelihood of eliciting a strong immune response in a subject to whom they are administered because the glucagon compounds described contain a single amino acid substitution or a single amino acid addition compared to a wild-type glucagon of SEQ ID NO: 1 and the conjugated corrin complex comprises a corrin ring structure that is present in Vitamin B12. Therefore, it is hypothesized that the glucagon compounds have a low antigenicity when administered to a subject in need thereof.

[0088] In some aspects, provided are pharmaceuticals comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex. Ther term “pharmaceutical,” as used herein refers to a composition comprising a compound as described herein for use in a method of treating a condition or disease, e.g., hypoglycemia and/or obesity.

[0089] In some aspects, provided are pharmaceuticals comprising a compound comprising a glucagon comprising a R-to-K substitution at position 17 and a corrin complex conjugated to K17.

[0090] In some aspects, provided are pharmaceuticals comprising a compound comprising a glucagon extended at a C-terminal end by an azido-lysine (K30), a D-isomer of an arginine at position 17, and a corrin complex conjugated to K30.

[0091] In some aspects, a pharmaceutical comprises a glucagon that is substituted at position 17 with an azido-lysine and a corrin complex and the pharmaceutical further comprises a buffer. In some aspects, a pharmaceutical is a liquid pharmaceutical and comprises a glucagon that is substituted at position 17 with an azido-lysine and a corrin complex and the pharmaceutical further comprises a buffer. In some aspects, a pharmaceutical comprises a glucagon that is extended at a C-terminal end by an azido-lysine and a corrin complex and the pharmaceutical further comprises a buffer. In some aspects, a pharmaceutical is a liquid pharmaceutical and comprises a glucagon that is extended at a C- terminal end by an azido-lysine and a corrin complex and the pharmaceutical further comprises a buffer.

[0092] In some aspects, the buffer has a pH of between about 6.6 and about 7.8, or about 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, or 7.8. In some aspects, the buffer has a pH of about 7.

[0093] In some aspects, a pharmaceutical comprises a glucagon that is substituted at position 17 with an azido-lysine and comprises a corrin complex and the pharmaceutical further comprises a phosphate buffer. In some aspects, a pharmaceutical comprises a glucagon extended at a C-terminal end by an azido-lysine and comprises a corrin complex and the pharmaceutical further comprises a phosphate buffer. In some aspects, a phosphate buffer comprises sodium phosphate. In some aspects, a phosphate buffer comprises about 50 mM sodium phosphate.

[0094] In some aspects, a pharmaceutical comprises a glucagon that is substituted at position 17 with an azido-lysine and comprises a corrin complex and the pharmaceutical further comprises hydrochloric acid buffer. In some aspects, provided are pharmaceuticals comprising a compound comprising a glucagon extended at a C-terminal end by an azidolysine, a D-isomer of an arginine at position 17, and a corrin complex and the pharmaceutical further comprises a hydrochloric acid buffer. In some aspects, the buffer has a pH of between about 1 and about 4, or about 1, 2, 3, or 4.

[0095] In some aspects, a pharmaceutical comprises a glucagon extended at a C- terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex and the pharmaceutical comprises a hydrochloric acid buffer. In some aspects, the buffer has a pH of between about 1 and about 4.

[0096] In some aspects, a pharmaceutical comprises a glucagon that is substituted at position 17 with an azido-lysine and comprises a corrin complex and the pharmaceutical comprises a standard extracellular saline solution (SES).

[0097] In some aspects, a compound comprising a glucagon and a corrin complex is used to treat hypoglycemia in a subject in need thereof. In some aspects, a compound comprising a glucagon and a corrin complex is used to treat acute hypoglycemia in a subject in need thereof. In some aspects, a compound comprising a glucagon and a corrin complex is used to treat acute obesity in a subject in need thereof.

[0098] In some aspects, a subject in need of a glucagon compound as described herein includes a subject (e.g., a human subject) with hypoglycemia and/or obesity, a subject (e.g. a human subject) having diabetes and/or obesity and/or a subject experiencing hypoglycemia following insulin administration, a subject (e.g. a human subject) having diabetes and experiencing hypoglycemia following insulin administration who is unable to ingest glucose, a subject (e.g. a human subject) experiencing acute hypoglycemia, a subject (e.g. a human subject) experiencing severe hypoglycemia.

[0099] In some aspects, a subject in need of a glucagon compound as described herein includes a subject (e.g., a human subject) being overweight. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is used to treat obesity in a subject in need thereof. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered to a subject in need thereof prior to a meal. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered to a subject in need thereof prior to each meal.

[00100] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered between about 1 hour and about 5 minutes prior to a meal. [00101] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered between about 1 hour and about 5 minutes prior to a meal and an incretin is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered between about 1 hour and about 5 minutes prior to a meal and a GLP-1R agonist is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered about 1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 15 minutes, about 12 minutes, about 10 minutes, or about 5 minutes prior to a meal.

[00102] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered between about twice a day to about once a month, or about twice a day, once a day, once every other day, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, or once every 31 days. [00103] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered at a dose of about 0.1 units to about 10 units, e.g., about 0.1 mg to about 10 mg, or about 0.1 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 1.5 mg, about 1.5 mg to about 2 mg, about 2 mg to about 2.5 mg, about 2.5 mg to about 3 mg, about 3 mg to about 3.5 mg, about 3.5 mg to about 4 mg, about 4 mg to about 4.5 mg, about 4.5 mg to about 5 mg, about 5 mg to about 5.5 mg, about 5.5 mg to about 6 mg, about 6 mg to about 6.5 mg, about 6.5 mg to about 7 mg, about 7 mg to about 7.5 mg, about 7.5 mg to about 8 mg, about 8 mg to about 8.5 mg, about 8.5 mg to about 9 mg, about 9 mg to about 9.5 mg, or about 9.5 mg to about 10 mg.

[00104] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered with an additional therapeutic agent. In some aspects, an additional therapeutic agent is an incretin. In some aspects, an additional therapeutic agent is a GLP-1 receptor agonist. In some aspects, an additional therapeutic agent is a GLP-1 receptor agonist. In some aspects, an additional therapeutic agent is a multi-agonist peptide comprising a GLP-1 receptor agonist amino acid sequence.

[00105] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is present in a multi-agonist peptide. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is present in a multi-agonist peptide comprising an incretin agonist amino acid sequence. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is present in a multi-agonist peptide comprising a GLP-1 receptor agonist amino acid sequence.

[00106] In some aspects, a multi-agonist peptide compound comprising a glucagon and a corrin complex as described herein and a GLP- 1 receptor agonist amino acid sequence is administered between about twice a day to about once a month, or about twice a day, once a day, once every other day, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, or once every 31 days.

[00107] In some aspects, a multi-agonist peptide compound comprising a glucagon and a corrin complex as described herein and a GLP- 1 receptor agonist amino acid sequence is administered at a dose of about 0.1 units to about 10 units, e.g., about 0.1 mg to about 10 mg, or about 0.1 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 1.5 mg, about 1.5 mg to about 2 mg, about 2 mg to about 2.5 mg, about 2.5 mg to about 3 mg, about 3 mg to about 3.5 mg, about 3.5 mg to about 4 mg, about 4 mg to about 4.5 mg, about 4.5 mg to about 5 mg, about 5 mg to about 5.5 mg, about 5.5 mg to about 6 mg, about 6 mg to about 6.5 mg, about 6.5 mg to about 7 mg, about 7 mg to about 7.5 mg, about 7.5 mg to about 8 mg, about 8 mg to about 8.5 mg, about 8.5 mg to about 9 mg, about 9 mg to about 9.5 mg, or about 9.5 mg to about 10 mg.

[00108] In some aspects, a compound comprising a glucagon and a corrin complex as described herein and an additional therapeutic agent are administered to a subject in need thereof to treat hypoglycemia. In some aspects, a compound comprising a glucagon and a corrin complex as described herein and the additional therapeutic agent are administered to a subject in need thereof to treat obesity. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered together with an incretin. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered together with an incretin in a single formulation. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered together with an incretin in separate formulations. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered together with an incretin in separate formulations at the same time. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered together with an incretin in separate formulations at different times.

[00109] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is used in combination with an incretin to induce weight loss in a subject in need thereof. In some aspects, provided is a method of treating obesity, the method comprising administering to a subject in need thereof a compound comprising a glucagon and a corrin complex as described herein and an incretin to induce weight loss in a subject. In some aspects, provided is a method of treating obesity, the method comprising administering to a subject in need thereof a multi-agonist peptide compound comprising a glucagon and a corrin complex as described herein and an incretin amino acid sequence. In some aspects, the incretin is a GLP-1R agonist. In some aspects, provided is a method of treating obesity, the method comprising administering to a subject in need thereof a multi-agonist peptide compound comprising a glucagon and a corrin complex as described herein and a GLP-1R agonist amino acid sequence. [00110] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is used in combination with a GLP-1R agonist to induce weight loss in a subject in need thereof. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is used in combination with a GLP-1R agonist in a formulation that is administered to a subject in need thereof once a day. In some aspects, provided is a method of treating obesity in a subject in need thereof, the method comprising administering a formulation comprising a compound comprising a glucagon and a corrin complex as described herein and a GLP-1R agonist to the subject twice a day. In some aspects, provided is a method of treating obesity in a subject in need thereof, the method comprising administering a formulation comprising a compound comprising a glucagon and a corrin complex as described herein and a GLP-1R agonist to the subject once a day

[00111] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered with a GLP-1R agonist in separate formulations to a subject in need thereof. In some aspects, a compound comprising a glucagon and a corrin complex as described herein and a GLP-1R agonist are administered in different formulations at the same time. In some aspects, a compound comprising a glucagon and a corrin complex as described herein and a GLP-1R agonist are administered in different formulations at different times. For example, a compound comprising a glucagon and a corrin complex as described herein can be administered more than once a day and a GLP-1R agonist can be administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered prior to a meal and a GLP-1R agonist is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered prior to each meal, e.g., three times a day and a GLP-1R agonist is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered between about 1 hour and about 5 minutes prior to a meal and a GLP-1R agonist is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered about 1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 15 minutes, about 12 minutes, about 10 minutes, or about 5 minutes prior to a meal and a GLP-1R agonist is administered once a day. In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered about 1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 15 minutes, about 12 minutes, about 10 minutes, or about 5 minutes prior to a meal and a GLP-1R agonist is administered once every other day, every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, or once every 31 days.

[00112] In some aspects, a compound comprising a glucagon and a corrin complex as described herein is administered once a day, once every other day, every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, or once every 31 days and a GLP-1R agonist is administered once a day, once every other day, every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week.

[00113] In some aspects, a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex is used to treat hypoglycemia. In some aspects, a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex is used to treat acute hypoglycemia. In some aspects, a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex is used to treat obesity.

[00114] In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex to treat hypoglycemia. In some aspects, methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex treat acute hypoglycemia. In some aspects, methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and comprising a corrin complex treat obesity. [00115] In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a cobalt corrin complex to treat hypoglycemia. In some aspects, methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a cobalt corrin complex comprise treating acute hypoglycemia. In some aspects, methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a cobalt corrin complex comprise treating obesity. In some aspects, the cobalt corrin complex is cobalamin, cobinamide or dicyanocobinamide.

[00116] In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine and comprising a corrin complex to treat hypoglycemia. In some aspects, methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine and a corrin complex comprise treating acute hypoglycemia. In some aspects, methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine and a corrin complex comprise treating obesity.

[00117] In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex. In some aspects, methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex comprise treating acute hypoglycemia. In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a cobalt corrin complex. In some aspects, methods of using a compound comprising a glucagon extended at a C- terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a cobalt corrin complex comprise treating acute hypoglycemia. In some aspects, methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D- isomer of an arginine at position 17, and a cobalt corrin complex comprise treating obesity. In some aspects, the cobalt corrin complex is cobalamin, cobinamide or dicyanocobinamide. [00118] In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex to treat hypoglycemia, wherein the method comprises preparing a pharmaceutical comprising the compound up to 2 weeks prior to use. In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex to treat obesity, wherein the method comprises preparing a pharmaceutical comprising the compound up to 2 weeks prior to use.

[00119] In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex to treat hypoglycemia, wherein the method comprises preparing the compound up to 2 weeks prior to use. In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, a D-isomer of an arginine at position 17, and a corrin complex to treat obesity, wherein the method comprises preparing the compound up to 2 weeks prior to use.

[00120] In some aspects, a compound comprising a glucagon molecule and a corrin complex is prepared about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 48 hours, or about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days prior to use.

[00121] In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex to treat hypoglycemia, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer for up to 2 weeks prior to use.

[00122] In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex to treat hypoglycemia, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer at up to 37°C for up to 2 weeks prior to use. In some aspects, provided are methods of using a compound comprising a glucagon substituted at position 17 with an azido-lysine and a corrin complex to treat obesity, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer at up to 37°C for up to 2 weeks prior to use.

[00123] In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, comprising a D-isomer of an arginine at position 17, and a corrin complex to treat hypoglycemia, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer for up to 2 weeks prior to use. [00124] In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, comprising a D-isomer of an arginine at position 17, and a corrin complex to treat hypoglycemia, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer at up to 37°C for up to 2 weeks prior to use. In some aspects, provided are methods of using a compound comprising a glucagon extended at a C-terminal end by an azido-lysine, comprising a D-isomer of an arginine at position 17, and a corrin complex to treat obesity, wherein the method comprises preparing the compound in a phosphate buffer and maintaining the compound in phosphate buffer at up to 37°C for up to 2 weeks prior to use.

[00125] In some aspects, the compound described herein in phosphate buffer is maintained at about 4°C, about 8°C, about 10 °C, about 12°C, about 14°C, about 16 °C, about 18°C, about 20°C, about 22 °C, about 24°C, about 26°C, about 28 °C, about 30°C, about 32°C, about 34 °C, about 36°C, or about 37°C prior to use.

EXAMPLES

[00126] Example JCbi was produced from commercia B12 (cyanocobalamin) starting material using a microwave reaction as described by Gryko et al. Briefly, a 10 mL microwave reaction vessel was charged with B12 (100.8 mg, 0.075 mmol) and NaCN (14.2 mg, 0.273 mmol) in 5 mL of EtOH producing a red coloured solution. The reaction vial was sealed, and micro wave heated to 120 °C for 10 minutes at 300 W. After reaction, the now deep purple solution was diluted with H2O (total volume 25 mL), flash frozen in liquid N2 and lyophilized. Cbi was purified on an Isolera One Biotage system using a 50 g SNAP cartridge with an isocratic flow rate of 12 ml/min of 100% MeOH over 10 minutes, tracked at 360 nm. The isolated product was precipitated with Et2O, flash frozen as before and lyophilized. Purity was confirmed by RP-HPLC on a C18 column (FIG. 7). MALDLTOF-MS expected m/z = 1042, observed m/z = [M-2CN+H]⁺ = 991; ^JH NMR (400 MHz, D₂O): 5 5.891 (s, 1H), 3.907 (m, 7=5.95 Hz, 1H), 3.839 (d, 7=8.23, 1H), 3.748 (d, 7=10.44, 1H), 3.654 (q, 7=7.11, 2H), 3.572 (q, 7=7.08 Hz, 1H), 3.404 (dd, 7=7.00,4.78, 1H), 3.312-3.151 (3H, overlapped), 2.914-2.857 (m, 1H), 2.748-2.712 (m, 4H), 2.600-2.380 (6H, overlapped), 2.316-2.231 (14H, overlapped), 2.148-2.069 (m, 4H), 2.030-1.943 (m, 1H), 1.915 (s, 3H), 1.891-1.744 (m, 3H), 1.683 (s, 3H), 1.532 (s, 3H), 1.489 (s, 3H), 1.430 (s, 3H), 1.312 (s, 3H), 1.184 (t, 7=7.12 Hz, 7H), 1.151 (d, 7=6.36 Hz, 3H); UV-vis (H₂O): k_max 277, 313, 368, 540, 580 nm. Yield was 65% based on B12. [00127] In preparation for conjugation to Gcg, a linker moiety in the form of 1-amino- 5-hexyne (AH) was reacted with Cbi at the 5’-OH position to prepare Cbi with an available alkyne reactive group. Cbi with an available alkyne reactive group was prepared by combining Cbi (42.5 mg, 0.041 mmol) with l,l’-Carbonyl-di-(l,2,4-triazole) (CDT) (167.3 mg, 1.019 mmol) in 5 mL of N-Methyl-2-pyrrolidone (NMP) and stirring for one hour under argon at 40°C, at which time AH (123 pL, 98 mg, 1.012 mmol) and Triethylamine (TEA) (50 pF) were added to the solution. After an additional hour, a second equivalent of AH and TEA were added, and the reaction was allowed to stir overnight at 40°C. Cbi with an available alkyne reactive group was purified by RP-HPLC on a C8 column to produce the Cbi with an available alkyne reactive group at 80% purity, with unreacted Cbi reclaimed during purification. MALDI-TOF-MS expected m/z = 1157, observed m/z = [M-H20]⁺: 1139; ’ H NMR (400 MHz, D2O): characteristic signals of P- (6.43) and a- (6.36) aquo-isomers; UV-vis (H₂O): k_max 355, 405, 495, 528 nm.

[00128] Azido-modified Gcg analogues as seen in Table 2 below were designed to test the hypothesis that corrination of Gcg could improve stability over time, measured as reduced aggregation and maintained functional agonism at the GcgR, relative to non-corrinated Gcg (Compounds 2-4).

Table 2 - the peptide sequence and coding for glucagon peptides where X = £-azidolysine, r = D-arginine, and Z = Cbi-lysine

[00129] The synthesis of Compounds 2-4 was accomplished by Fmoc/tBu solid-phase synthesis on a CEM Liberty Blue synthesizer using non-preloaded resin and repetitive DIC/Oxyma activation. Upon completion, the peptides were cleaved from the resin and deprotected using a cocktail containing 95% TFA, 2.5% TIPS, 2.5% H2O at 40°C for 40 mins using a Liberty Blue Razor. Compounds 2-4 were precipitated from solution using cold ET2O and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded and peptide pellet resolubilized in water and lyophilized in vacuo prior to purification (See FIGs. 10-14). To help stabilize the peptides and increase their bioavailability, all peptides were made with an amidated C-terminal end.

[00130] Corrination of 2-4 was facilitated by a copper(I)-mediated alkyne-azide cycloaddition (CuAAC). Cui (6.5 mg, 0.034 mmol) was added to a solution of tris[(l-benzyl- lH-l,2,3-triazol-4-yl)methyl]amine (TBTA) (22 mg, 0.042 mmol) in 4:1 DMF: water and allowed to stir for approximately 30 minutes at room temperature producing a faint yellow colour. Cbi with an available alkyne reactive group (15.3 mg, 0.013 mmol) and Compounds 2-4 (5 mg, 0.0014 mmol) were then dissolved in 4:1 DMF: H2O, producing a final reaction volume of 2 mL. with the reaction gently stirred at room temperature overnight to produce Compounds 5-7, which were purified by semi-preparative RP-HPLC (Solvent A: MeCN B: H2O, 0.1% TFA, from 1% to 70% over 20 minutes) on a Cs column (Zorbax 300SB Cs 5pm, 9.4 x 250 mm) to produce Compounds 5-7 at > 95% purity(confirmed by LC; FIGs. 15-17). [00131] GgcR agonist properties of Gcg, Compound 2, and Compound 5 were evaluated in a FRET assay using suspensions of HEK293 cells that stably express both the human GcgR (hGcgR) and the genetically encoded cAMP biosensor H188. These HEK293 cells were generated by O.G. Chepumy in the Holz laboratory, and they were designated as HEK293-hGcgR-H188 clone 10. The HEK293-hGcgR-H188 cells were plated at 80% confluence on 96-well clear-bottom assay plates (Costar 3904, Corning, NY) coated with rat tail collagen (Collaborative Biomedical Products, Bedford, MA). On the day of the experiment, the cell culture media was removed and replaced by 200 pl/well of a standard extracellular saline (SES) solution supplemented with 11 mM glucose and 0.1% BSA. The composition of the SES was (in mM): 138 NaCl, 5.6 KC1, 2.6 CaCh, 1.2 MgCh, 11.1 glucose, and lOHepes (295 mOsmol, pH 7.4). Real-time kinetic assays of FRET were performed using a FlexStation 3 microplate reader equipped with dual excitation and emission light monochromators (Molecular Devices, Sunnyvale, CA). Excitation light was delivered at 435/9 nm (455 nm cut-off), and emitted light was detected at 485/15 nm (CFP) or 535/15 nm (YFP) . The emission intensities were the averages of 12 excitation flashes for each time point per well. Test solutions dissolved in SES were placed in V-bottom 96-well plates (Greiner Bio-One, Monroe, NC), and an automated pipetting procedure was used to transfer 50 pl of each test solution to each well of the assay plate containing monolayers of these cells. The 485/535 emission ratio was calculated for each well and the mean +/- s.d. values for 12 wells were averaged. These FRET ratio values were normalized using baseline subtraction so that a y-axis value of 0 corresponds to the initial baseline FRET ratio, whereas a value of 100 corresponds to a 100% increase (i.e., doubling) of the FRET ratio. The time course of the A FRET ratio was plotted after exporting data to Origin 8.0 (OriginLab, Northampton, MA). Origin 8.0 was also used for non-linear regression analysis to quantify dose-response relationships.

[00132] Referring to FIG. 4, the purity of Cbi was confirmed by semi-preparative RP- HPLC using solvent system A: MeOH B: H2O, 0.1% TFA, from 1% to 70% A over 15 minutes) on an Agilent Eclipse XDB-C18 (5 pm, 4.6 x 150 mm) column at 360 nm (A). Purity 90%. Cbi was analyzed and characterized by MALDI-TOF-MS expected m/z = 1042, observed m/z = [M-2CN-+H+]+: 991 (B). 1H NMR ( 400 MHz, D2O): 5 5.891 (s, 1H), 3.907 (m, J=5.95 Hz, 1H), 3.839 (d, J=8.23, 1H), 3.748 (d, J=10.44, 1H), 3.654 (q, J=7.11, 2H), 3.572 (q, J=7.08 Hz, 1H), 3.404 (dd, J=7.00,4.78, 1H), 3.312-3.151 (3H, overlapped), 2.914- 2.857 (m, 1H), 2.748-2.712 (m, 4H), 2.600-2.380 (6H, overlapped), 2.316-2.231 (14H, overlapped), 2.148-2.069 (m, 4H), 2.030-1.943 (m, 1H), 1.915 (s, 3H), 1.891-1.744 (m, 3H), 1.683 (s, 3H), 1.532 (s, 3H), 1.489 (s, 3H), 1.430 (s, 3H), 1.312 (s, 3H), 1.184 (t, J=7.12 Hz, 7H), 1.151 (d, J=6.36 Hz, 3H) (C) UV-Vis (H2O): Xmax 277, 313, 368, 540, 580 nm (D). [00133] Referring to FIG. 5, Cbi with an available alkyne reactive group was purified by RP-HLC using solvent system A: MeOH B: H2O, 0.1% TFA, from 1% to 70% A over 25 minutes using a C18 column (Zorbax SB-C18 5pm, 4.6 x 150 mm) at 360 nm. Tr 12.4, 12.9. Purity 90% by LC (A). 1 was analyzed and characterized by MALDI-TOF-MS expected m/z = 1165, observed m/z = [M-CN]+: 1116 (B) 1H NMR ( 400 MHz, D2O). Characteristic signals of P- (6.43) and a- (6.36) aquo-isomers. (C). UV-Vis (H2O): Am ax 355, 405, 495, 528 nm (D).

[00134] Referring to FIG. 6, Gcg was purified by RP-HLC using solvent system A: ACN B: H2O, 0.1% TFA, from 1% to 70% A over 20 minutes using a C18 column (Eclipse XDB-C18 5 pm, 4.6x150 mm) at 280 nm. Tr 12.4. Purity = >90% by LC (A). Gcg was analyzed and characterized by MALDI-TOF-MS expected m/z = 3482, observed m/z = [M+2H+]+: 3480 (B).

[00135] Referring to FIG. 7, Gcgl7 was purified by RP-HLC using solvent system A: ACN B: H2O, 0.1% TFA, from 1% to 70% A over 20 minutes using a C18 column (Eclipse XDB-C18 5 pm, 4.6x150 mm) at 280 nm. Tr 19.7. Purity = >90% by LC (A). Gcgl7 was analyzed and characterized by MALDI-TOF-MS expected m/z = 3482, observed m/z = [M]: 3482 (B).

[00136] Referring to FIG. 8, Compound 2 was purified by RP-HPLC using solvent system A: ACN B: H2O, 0.1% TFA, from 1% to 70% A over 20 minutes using a C18 column (Eclipse XDB-C18 5 pm, 4.6x150 mm) at 280 nm. Tr 11.1. Purity = >95% by LC (A). 2 was analyzed and characterized by MALDI-TOF-MS expected m/z = 3480, observed m/z = [M- 3H+]+: 3477 (B).

[00137] Referring to FIG. 9, Compound 3 was purified by RP-HPLC using solvent system A: AN B: H2O, 0.1% TFA, from 1% to 70% A over 20 minutes using a C18 column (Eclipse XDB-C18 5 pm, 4.6x150 mm) at 280 nm. Tr 18.7. Purity = >95% by LC (A). 3 was analyzed and characterized by MALDI-TOF-MS expected m/z = 3636, observed m/z = [M- H+]+: 3635 (B).

[00138] Referring to Fig. 10, Compound 4 was purified by RP-HPLC using solvent system A: AN B: H2O, 0.1% TFA, from 1% to 70% A over 20 minutes) using a Cis column (Eclipse XDB-Cis 5 pm, 4.6x150 mm) at 280 nm. Tr 18.9. Purity = >95% by LC (A). Compound 4 was analyzed and characterized by MALDI-TOF-MS expected m/z = 3636, observed m/z = [M-H⁺]⁺: 3635 (B).

[00139] Referring to FIG. 11, Compound 5 was purified by Slide-a-Lyzer protein dialysis cassette, MW cut off 3500 until there was no longer presence of unreacted starting material by MALDI. Compound 5 was analyzed and characterized by MALDI-TOF-MS expected m/z = 4522, observed m/z = [M+Cu⁺]⁺: 4585

[00140] Referring to FIG. 12, Compound 6 was purified by Slide-a-Lyzer protein dialysis cassette, MW cut off 3500 until there was no longer presence of unreacted starting material by MALDI. Compound 6 was analyzed and characterized by MALDI-TOF-MS expected m/z = 4752, observed m/z = [M+2H⁺]⁺: 4754.

[00141] Referring to FIG. 13, Compound 7 was purified by Slide-a-Lyzer protein dialysis cassette, MW cut off 3500 until there was no longer presence of unreacted starting material by MALDI. Compound 7 was analyzed and characterized by MALDI-TOF-MS expected m/z = 4752, observed m/z = [M+H⁺]⁺: 4753.7. [00142] Referring to FIG. 14, fluorescence data of a Cbi control and all three corrinated compounds were assessed at an excitation of 440 and an emission of 484, to which negligible fluorescence response was detected. This was done to ensure the fluorescence capabilities of Cbi did not interfere or quench the assay being run.

Example 2

[00143] Referring to the figures, wherein like numerals refer to like parts throughout, the present invention comprises a method to improve the solubility of glucagon in physiological solutions. As described previously, glucagon has very poor solubility in physiological solutions. An aim to increase this solubility was then sought out through a combination of D-amino acids and corrination.

[00144] The solubility of all peptides was recorded at 1 hr, 48 hr, and at 2-week time points. Peptides were dissolved in IX PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KC1) (pH 7.4) solution lower than the volume needed to fully dissolve the peptide to ensure maximal solubility. Solutions were centrifuged at 3000 rpm for 5 minutes to pellet any undissolved solid and solubility of the supernatant was then measured by UV absorption at 280 nm on a NanoDrop OneC UV-Vis Spectrophotometer. All analogues had only a fractional loss of solubility as tracked over the 2-week mark at 4°C and the reduction of maximal solubility was consistent across the board.

[00145] Compounds 2-7 were tested in a chemical stability study in 50 mM sodium phosphate buffer at a pH of 7 and incubated at 37 °C. Samples were prepared at 150 pM. All peptides and peptide conjugates were assessed for evidence of aggregation utilizing MALDI- TOF MS over a 2-week time span.

[00146] After 2 weeks, a final sample was assayed to reveal how much of the original [150 pM] remained in solution, as seen in FIG. 1. Substitution of a D-arginine at position 17 of wild-type glucagon (Compound Glurl7; SEQ ID NO: 8) or substitution of an azido-lysine at position 17 of wild-type glucagon (Compound GlucK17; SEQ ID NO: 2) both increased the chemical stability compared to wild-type glucagon (FIG. 1). Corrination of the azidolysine substituted glucagon at position 17 of wild-type glucagon (Compound 5) further increased its chemical stability (FIG. 1). Further, all corrinated compounds (Compounds 5-7) retained their original concentration to a higher degree than unmodified wild-type glucagon (Gcg, SEQ ID NO: 1).

[00147] Referring to FIG. 2, the impact of corrination (Compounds 5-7) on aggregation and functional agonism at the glucagon receptor (GcgR) was assayed next. 1 Aggregate formation assays were performed in triplicate in 0.1M HC1 at a concentration of 25 pM. Compounds were agitated with a stir bar at 37°C for 48 hours prior to assay with a Thioflavin-T (THT) fluorescence kit. Substitution of a D-arginine at position 17 of wild-type glucagon (Compound Glurl7; SEQ ID NO: 8) or substitution of an azido-lysine at position 17 of wild-type glucagon (Compound GlucK17; SEQ ID NO: 2) both did not result in a reduction in physical aggregation compared to wild-type glucagon (FIG. 2). In contrast, all corrinated compounds (Compounds 5-7) showed markedly decreased aggregation compared to wild- type glucagon (Gcg) and non-corrinated glucagon (Compounds 2-4) (FIG. 2). [00148] Referring to FIG. 3, GcgR agonist properties of the different compounds were determined in FRET assays that monitor intracellular levels of cAMP. The glucagon concentration-response relationship was determined under conditions in which 0.03 - 30 nM of the peptide was tested immediately after reconstitution in SES buffer (Al), or after a 24-hr incubation of the reconstituted peptide in SES maintained at 37°C (A2). Linear regression analysis (A3) was used to determine EC50 values. Same experimental designs as in A1-A3 were used for Compound 2 (B1-B3), and Compound 5 (Cl-C3)For these live-cell FRET assays using HEK293-hGcgR-H188 clone 10 cells, the percent increase of the 485/535 nm FRET emission ratio (%AFRET) indicates increased levels of cAMP, as detected by the H188 biosensor. Standard error bars indicate the mean + s.d. A FRET measured in N=12 wells of a 96-well plate.

[00149] While glucagon showed a substantial reduction in cAMP levels after 24 hrs (compare Al and A2), Compounds 2 and 5 did not (Bl and B2, and Cl and C2). The EC50 values of glucagon increased from 0.22 nM to 36.3 nM after 24 hrs (A3), while the EC50 values of Compound 2 increased from 0.17 nM to 9.6 nM (B3) and the EC50 values of Compound 5 increased only from 1.1 nM to 5.5 nM during the 24 hours (C3).

[00150] Table 1 below includes a summary of the stability of glucagon and Compounds 2 and 5 after 24 hr at 37°C as tracked by FRET assays measuring cAMP elevation upon agonism of the glucagon receptor.

Table 1

[00151] Specifically, as described above, glucagon and Compounds 2 and 5 activities at the GcgR were measured in a FRET assay using suspensions of HEK293 cells in which GcgR stimulation leads to increased levels of intracellular cAMP. This assay utilizes HEK293-hGcgR-H188 clone 10 cells that stably co-express the human GcgR (hGcgR) and the genetically encoded biosensor H188. When levels of intracellular cAMP rise, this biosensor exhibits reduced FRET that is measurable as an increase of the 485/535 nm FRET emission ratio. The primary goal was to determine whether Compounds 2 and 5 substitutions preserved the in-solution stability and agonist properties of these peptides relative to glucagon, itself. If so, the characteristic loss of GcgR agonist action that is reported for solubilized glucagon would be reduced. Importantly, the FRET assay used for this analysis allowed live-cell, real-time assessment of the kinetics of agonist- stimulated cAMP production, while also providing an estimate of agonist efficacy and potency in a 96-well format.

[00152] GcgR agonist action was monitored using 25 pM stock solutions diluted in a standard extracellular saline solution (SES). Each peptide was tested on day-1 immediately after solubilization and serial dilution (0.03 - 30 nM) in SES. For comparison, the same 25 pM stock solution was incubated for 24 hr at 37°C in a cell culture incubator, after which serial dilutions were performed on day 2. The FRET assay was then repeated using the same protocol as for day 1. Initially, the dose-response (DR) relationship for peptide-stimulated cAMP production was determined on day-1 for Gcg (Al), Compound 2 (Bl), and Compound 5 (Cl). These DR relationships were then compared to those obtained for Gcg (A2), Compound 2 (B2), and Compound 5 (C2) on day-2 after the 24-hr incubation at 37°C. Day-1 and day-2 data sets were then processed by linear regression analysis so that Hill plot overlays could be created for each of the peptides tested (A3, B3, C3).

[00153] Gcg agonist action at the GcgR to raise levels of cAMP was markedly diminished after a 24-hr incubation in SES at 37°C (FIG. 3A). This was measurable as reduced agonist efficacy, as evidence by a reduced maximal % change of FRET ratio (%AFRET) when testing a 30 nM saturating concentration of Gcg (c.f., Al, A2). Simultaneously, Hill plot analysis revealed that Gcg agonist potency was also reduced. The EC50 value obtained on day-1 was 0.22 nM, in comparison to a value of 36.3 nM on day-2 after a 24-hr incubation at 37°C (A3). Thus, the Gcg DR relationship was right-shifted by 165-fold after incubation for 24 hr at 37 °C. [00154] Compound 2 acted with similar efficacy when compared to Gcg when it was tested after solubilization on day-1 (c.f., Al, Bl). It also exhibited similar potency so that the EC50 value for Compound 2 measured on day-1 was 0.17 nM, whereas that of Gcg was 0.22 nM (c.f., A3, B3). Notably, measurements of agonist efficacy after a 24-hr incubation at 37°C revealed that Compound 2 retained its ability to stimulate a maximal response (FIG. 3B, B2). However, its potency was reduced so that a 56-fold right-shift of the DR relationship was measured (B3). Thus, EC50 values for assays run on day-1 and day-2 were 0.17 nM and 9.6 nM, respectively.

[00155] A consequence of cbi-conjugation of Compound 2 to generate Compound 5 was also measurable in these FRET assays. For Compound 5, a 24-hr incubation at 37°C led to a 5-fold right-shift of the DR relationship. Thus, the right-shift measured for Compound 5 was 11-fold less than that measured for Compound 2 (c.f., FIG. 3B, B3, and FIG. 3C, C3). Furthermore, the potency of Compound 5 was improved relative to that of Compound 2 after a 24-hr incubation at 37°C. EC50 values measured on day-2 were 5.5 nM and 9.6 nM for Compound 5 and Compound 2, respectively (c.f., FIG. 3B, B3, and FIG. 3C, C3). Remarkably, when comparing data obtained in assays using peptide solutions equilibrated for 24 hr at 37°C, Compound 5 exhibited a 6.6-fold increased potency relative to Gcg (c.f., FIG. 3A, A3, and FIG. 3C, C3).

[00156] The development of peptide therapeutics is frequently limited by both chemical/physical properties and their stabilities. Glucagon as a therapeutic is problematic due to its propensity to aggregate and form amyloid fibrils. Its formation of aggregates and fibrils leads to a loss in activity and can lead to toxicity and immunogenicity. Herein we successfully synthesized eight glucagon analogues, see Table 2, and demonstrated improved chemical and physical properties of the novel compounds.

[00157] In a chemical stability assay, it was found that the use of D-isomer amino acids improved the solubility of the amidated native sequence, consistent with previously reported results. To prepare the glucagon peptide for ‘corrination’, an amino acid was selectively substituted with 8-azidolysinc at position 17 or an 8-azidolysinc was added at position 30 of glucagon. These sequence modifications were shown to have improved solubility and chemical stability compared to that of Gcg, as seen in Table 1 and FIG. 1. The 8-azidolysinc modified sequences (Compounds 2-4) were subsequently conjugated to dicyanocobinamide (Cbi) to produce three new corrinated compounds (Compounds 5-7). The corrinated compounds were shown to have maintained solubility compared to that of their non-corrinated counterparts but had improved solubility relative to that of the native glucagon sequence, as seen in FIG. 1.

[00158] While glucagon is limited by its solubility and chemical stability, it is further limited in action by its tendency to form amyloid fibrils. To investigate the physical properties of the modified Gcg constructs a thioflavin T (ThT) assay was performed to monitor the formation of fibrils. While the utilization of D-isomer amino acids and 8- azidolysine substitution in Gcg sequence (Compounds 2-4) were shown to have a positive effect on chemical stability (FIG. 1), these modifications had little to no effect on physical stability and aggregate formation (FIG. 2). However, the corrination of Gcg (Compounds 5- 7) unexpectedly led to near elimination of aggregate formation, as seen in FIG. 2.

[00159] While improving the physical and chemical properties of Gcg is of utmost importance; the modifications performed on the sequence must maintain function. In a realtime in vitro assay Gcg, Compound 2, and Compound 5 were monitored for their functional activity. It was shown that the 8-azidolysinc modified Compound 2 maintained equipotent activity in comparison to unmodified Gcg when both peptides were freshly solubilized. In contrast, corrination to yield Compound 5 led to slightly reduced potency, while retaining full efficacy as judged by the maximal response.

[00160] Following a 24-hour incubation, Gcg, Compound 2 and Compound 5 were assayed again to determine how chemical and physical stability affected function. Gcg was shown to have a 165-fold reduction in potency, while Compound 2 had a 56-fold reduction in potency (Table 1). In contrast, Compound 5 had a marginal 5 -fold reduction in potency while retaining full efficacy, as seen in FIG. 3 and Table 1.

[00161] The data indicates that the corrination of Gcg not only improves solubility and mitigates aggregation, but it also preserves GcgR agonism after 24-hr incubation at 37 °C to a higher degree than that of native Gcg. Thus, use of Cbi conjugation of an unstable glucagon peptide in aqueous solution, resulted in a compound with prolonged stability and agonism.

[00162]

Claims

CLAIMS What is claimed is:

1. A compound comprising a molecule of glucagon conjugated to a corrin complex.

2. The compound of claim 1, wherein the glucagon comprises a lysine in position 17 of the wild-type glucagon amino acid sequence.

3. The compound of claim 1, wherein the glucagon is extended at a C-terminal end by a lysine.

4. The compound of claim 2 or 3, wherein the lysine is azido-lysine.

5. The compound of claim 4, wherein the corrin complex is conjugated to an azido-lysine at position 17 or position 30.

6. The compound of any one of claims 1-5, wherein the corrin complex is dicyanocobinamide.

7. A method of providing an improvement to a pharmaceutical development of glucagon, comprising the step of conjugating a molecule of dicyanocobinamide to a molecule of glucagon at a predetermined site of the molecule of glucagon.

8. The method of claim 7, wherein the predetermined site on the glucagon backbone is K17.

9. The method of claim 7, wherein the predetermined site on the glucagon backbone is a K30 added to a C-terminal end of the glucagon molecule.

10. The method of claim 7, wherein the step of conjugating the molecule of dicyanocobinamide to the molecule of glucagon comprises using copper(I)- mediated alkyne-azide cycloaddition using strain promoted alkyne-azide cycloaddition (SPAAC).

11. A compound having an improved pharmaceutical effect, comprising glucagon and a dicyanocobinamide bound to the glucagon.

12. A method of preparing a glucagon therapeutic, the method comprising preparing a compound comprising a compound of claim 1 in a phosphate buffer.

13. The method of claim 12 further comprising maintaining the compound in the phosphate buffer for up to 2 weeks prior to use.

14. A method of preparing a glucagon therapeutic, the method comprising preparing a compound of claim 1 in a hydrochloric acid buffer.

15. The method of claim 14 further comprising maintaining the compound in the hydrochloric acid buffer for up to 48 hours prior to use.

16. A method of using a compound of claim 1 to treat hypoglycemia, wherein the method comprises preparing the compound in a phosphate buffer.

17. The method of claim 16, wherein the method comprises preparing the compound in the phosphate buffer up to 2 weeks prior to use.

18. A method of using a compound of claim 1 to treat hypoglycemia, wherein the method comprises preparing the compound in a hydrochloric acid buffer.

19. The method of claim 18, wherein the method comprises preparing the compound in the hydrochloric acid buffer up to 48 hours prior to use.