Peptides and compositions for improving glucose levels

Abstract

The present invention relates to modified glucagon peptides with increased specificity, high solubility and increased potency to a GCG receptor. In particular, the modified glucagon peptides of the present invention are used in the prevention and / or treatment of hypoglycemia, including acute, sub-acute, chronic hypoglycemia, post-bariatric hypoglycemia, congenital hyperinsulinism, body weight control, liver fat (lipid) metabolism, other lipid parameter control, treatment of Type 2 diabetes, or protein load renoprotection.

Description

ABVA.002WO PATENT PEPTIDES AND COMPOSITIONS FOR IMPROVING GLUCOSE LEVELS FIELD

[0001] Some embodiments described herein relate generally to modified glucagon peptides with enhanced physicochemical properties for medical use, for example in the prevention and / or treatment of hypoglycemia, including acute, sub-acute, chronic hypoglycemia, post- bariatric hypoglycemia, congenital hyperinsulinism, body weight control, liver fat (lipid) metabolism, other lipid parameter control, treatment of Type 2 diabetes, or protein load renal protection. REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “ABVA.002WO.xml” created on September 7, 2025, which is 98,487 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. BACKGROUND

[0003] Glucagon is a 29 amino acid hormone secreted by pancreatic α cells in response to low blood glucose levels. When blood glucose levels are low, glucagon signals the liver to release glucose, thereby elevating blood glucose levels. The most common clinical setting where hypoglycemia is observed is in insulin-treated diabetes, whether the Type 1 or Type 2 variations of the condition. In both Type 1 and Type 2 diabetes, the predilection to insulin-induced hypoglycemia is accentuated in many subjects as endogenous glucagon secretion from the alpha cell of the pancreatic islet is also impaired, rendering subjects especially prone to severe forms of hypoglycemia. In less common instances, hypoglycemia can also occur in the absence of insulin treatment where endogenous levels of insulin are abnormally elevated (e.g. insulinoma, congenital hyperinsulinism) or glucose homeostasis is asynchronous with insulin action through gastro-intestinal abnormality (e.g. post-bariatric hypoglycemia) or where the alpha cell is directly harmed by inflammation (chronic pancreatitis) and glucagon secretion is impaired in inflammation-related pancreatic diabetes. In all of the above clinical instances, exogenously administered glucagon (or its analog form) is able to raise blood glucose to correct hypoglycemia.ABVA.002WO PATENT While the standard of treatment for insulin-induced hypoglycemia is administration of glucagon, there remains a growing need for improved glucagon analogs with enhanced physiochemical properties that are more readily available for treatment. SUMMARY

[0004] In some embodiments, an isolated peptide comprising a sequence of HX1QGTFTSDYSKYLDX2X3RAX4X5FVX6WLX7X8X9X10(SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof is disclosed. In some embodiments, X1 is S or Aib; X2 is Aib, S, E, or A; X3 is R, K*, K** or K***; X4 is Aib, Q, A, or E; X5 is D or E; X6 is Q, E, K, K*, K** or K***; X7is V, E, I or L; X8is D, E, Q or N; X9is T or K, K*, K** or K***; X10is absent, K, K*, K** or K***. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)ndiacid or (L) Lys-decanoyl, wherein AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid and wherein n is 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xa is absent or (AEEAc-AEEAc) and m = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG).

[0005] In some embodiments, the isolated peptide further comprises an additional K, K*, K** or K*** on the carboxy- terminal end of the peptide.

[0006] In some embodiments, the isolated peptide is conjugated to a fatty acid. In some embodiments, the fatty acid is selected from the group consisting of caproic acid (C6), hexanedioic acid (C6 dicarboxylic acid), caprylic acid (C8), octanedioic acid (C8 dicarboxylic acid), capric acid (C10), decanedioic acid (C10 dicarboxylic acid), lauric acid (C12), dodecanedioic acid (C12 dicarboxylic acid), myristic acid (C14), tetradecanedioic acid (C14 dicarboxylic acid), palmitic acid (C16), hexadecanedioic acid (C16 dicarboxylic acid), stearic acid (C18), octadecanedioic acid (C18 dicarboxylic acid), icosanoic acid (C20), icosanedioic acid (C20 dicarboxylic acid), docosanoic acid (C22) or docosanedioic acid (C22 dicarboxylic acid).

[0007] In some embodiments, the isolated peptide is conjugated to PEG. In some embodiments, the PEG is between 0.2-50 kD in size.

[0008] The isolated peptide of claim 3, wherein the fatty acid is conjugated to K, K*, K** or K*** residue of the peptide. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu- (CH2)n dicarboxylic acid or (L) Lys-decanoyl and wherein n is 6, 8, 10, 12, 14, 16, 18, 20 or 22. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-ABVA.002WO PATENT AEEAc) and wherein n is 5, 7, 9, 11, 13, 15, 17. In some embodiments, K*** is L-Lys-PEG and wherein PEG is between 0.2-50 kD in size.

[0009] In some embodiments, the isolated peptide further comprises a carboxy terminal peptide comprising GPSSGAPPPS (SEQ ID NO: 53), GPSSGA (SEQ ID NO: 54), or GPS. In some embodiments, the isolated peptide is configured for increased specificity and increased potency to a GCG receptor as compared to native glucagon. In some embodiments, the isolated peptide is capable of activating a glucagon receptor. In some embodiments, the isolated peptide has high solubility.

[0010] In some embodiments, the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAQDFVK*WLMDT-OH (SEQ ID NO: 3); HSQGTFTSDYSKYLDAibRRAQDFVK*WLLDT-OH (SEQ ID NO: 4); HSQGTFTSDYSKYLDERRAQDFVK*WLLDT-OH (SEQ ID NO: 5); HSQGTFTSDYSKYLDERRAAibDFVK*WLLDT-OH (SEQ ID NO: 6); and HSQGTFTSDYSKYLDARRAAibDFVK*WLMDT-OH (SEQ ID NO: 7).

[0011] In some embodiments, the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT-OH (SEQ ID NO: 8); HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT- NH2(SEQ ID NO: 12) and HSQGTFTSDYSKYLDEK*RAADFVQWLMDT-OH (SEQ ID NO: 16).

[0012] In some embodiments, the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAAEFVQWLMNTK*SEQ ID NO: 20); HSQGTFTSDYSKYLDAibRRAAEFVQWLMNK*(SEQ ID NO: 21); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDTK*(SEQ ID NO: 22); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDK*(SEQ ID NO: 23); HSQGTFTSDYSKYLDAibRRAAEFVKWLMDTK*(SEQ ID NO: 24); and HSQGTFTSDYSKYLDAibRRAAEFVKWLMDK*(SEQ ID NO: 25).

[0013] In some embodiments, the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGAPPPS-NH2(SEQ ID NO: 26); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGAPPPS-NH2 (SEQ ID NO: 27); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGA-NH2(SEQ ID NO: 28);ABVA.002WO PATENT HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGA-NH2(SEQ ID NO: 29); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPS-NH2 (SEQ ID NO: 30); and HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPS-NH2 (SEQ ID NO: 31).

[0014] In some embodiments, the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 3-52. In some embodiments, the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 32-52.

[0015] In some embodiments, the isolated peptide has an increased affinity of binding to albumin. In some embodiments, a pharmaceutical composition comprising the isolated peptide disclosed herein as an active ingredient is disclosed. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a formulation for injection.

[0016] In some embodiments, a method of treating a subject suffering from hypoglycemia is disclosed. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an isolated peptide disclosed herein. In some embodiments, the hypoglycemia is acute, sub-acute and / or chronic hypoglycemia. In some embodiments, the administering is subcutaneous administration.

[0017] In some embodiments, the isolated peptide is administered in an amount ranging from 0.001 µg / kg / min to an amount of 1000 µg / kg / min. In some embodiments, the administering occurs daily, or multiple times a day. In some embodiments, the administering occurs continuously for up to 48 hours. In some embodiments, the subject has diabetes. DETAILED DESCRIPTION

[0018] There is also an increasing appreciation that although glucagon is primarily recognized for its plasma glucose elevating properties (hypoglycemia rescue and / or prevention), glucagon also elicits broader effects that can bring clinical benefit in diabetes and related clinical disease settings. Glucagon exerts favorable effects on lipid metabolism that confers benefit in the setting of fatty liver disease (also known as metabolic dysfunction-associated steatotic liver disease in its broader sense, or its more severe form metabolic dysfunction-associated steatohepatitis). Glucagon effects to impact lipid metabolism, particularly to stimulate fat oxidation (also termed beta oxidation), also translates into overall body weight reductionABVA.002WO PATENT primarily through reduction in fat mass. Furthermore, glucagon causes a lowering of circulating amino acids, particularly after ingesting a meal, and although this does not translate into serious detrimental effects on protein metabolism it is thought to have a potential beneficial effect on reducing the protein load handled by the kidneys. This becomes an important additional glucagon attribute that is highly relevant in the clinical setting of compromised kidney function, particularly relevant in diabetic kidney disease, chronic kidney disease, and other forms of acute and chronic renal failure. Further, glucagon is known to have effects to slow gut motility that may differ from the gut effects of other incretin peptide hormones (e.g. GLP-1, oxyntomodulin, glicentin). This raises the potential for glucagon (and its analogs) to be used instead of certain incretin hormone alternatives where the predominant effect of the latter is to slow gastric emptying and this in turn can be a safety concern (e.g. gastroparesis). It also raises the potential for glucagon (and its analogs) to be used in combination with certain incretin hormone therapeutics where the combined gut effects are advantageous. Glucagon and oxyntomodulin is a particularly interesting combination as the latter exerts its effect through both the glucagon and GLP-1 receptor. Without being limited by theory, glucagon has been used as a standard of treatment for hypoglycemia and may provide additional metabolic benefits such as weight loss and favorable changes in lipids. In addition, glucagon has been used in emergency use to treat severe hypoglycemia. Commercially available glucagon includes a lyophilized powder that is provided with a diluent. Once solubilized by the diluent, the human glucagon must be immediately used or discarded due to its poor stability. Moreover, native human glucagon is also sparingly soluble at physiological pH. Ready to use glucagon formulations that are commercially available are single-use products designed for emergency rescue use and are not considered viable multi-use products for chronic use. Therefore, there remains an increasing need for glucagon analogs with enhanced biophysical, physiochemical and pharmacokinetic (PK) properties for expanded medical use, especially for the prevention, treatment and / or control of hypoglycemia events.

[0019] Accordingly, in some embodiments described herein, glucagon analogs with one or more enhanced properties including stability, solubility, specificity and / or potency for glucagon receptors combined with enhanced PK including absorption, distribution, metabolism, and excretion are provided.ABVA.002WO PATENT Definitions

[0020] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0021] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0022] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0023] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0024] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.ABVA.002WO PATENT

[0025] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules that include amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 amino acids long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N-terminus of a subsequent sequence.

[0026] The term “purified polypeptide” or “isolated peptide” refers to a polypeptide that has been separated from other molecules or compounds including but not limited to nucleic acids, lipids and / or other contaminants normally associated with the polypeptide in its natural environment.

[0027] The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. It includes both D-enantiomeric and L-enantiomeric amino acids. Natural amino acids include those found in nature, such as, e.g., the 25 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L-enantiomeric amino acids, although a few D-enantiomeric amino acids occur in bacterial envelopes and some antibiotics.ABVA.002WO PATENT

[0028] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later post-translationally modified, e.g., hydroxyproline, γ- carboxyglutamate, O- phosphothreonine, O-phosphotyrosine, O-phosphoserine, iso-aspartic acid, iso-glutamic acid and pyroglutamic acid.

[0029] Single letter and three letter abbreviations for 20 naturally occurring amino acids described in some embodiments herein are defined in Table 1. Table 1 Amino acid Three letter code One letter code Al i Al AABVA.002WO PATENT

[0030] Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, for example, an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Typically, the names of naturally occurring and non-naturally occurring amino-acyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC- IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of α-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and amino-acyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader. Some abbreviations useful in describing embodiments set forth herein are defined in Table 2. Table 2 Abbreviation Definition (1-Me)His (1-Meth l)HistidineABVA.002WO PATENT Abbreviation Definition CONH2 CarboxamideABVA.002WO PATENT Abbreviation Definition N-cyclohexyl-L-asparagine (L-asparagine, N- N(Nchx) )

[0031] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R forABVA.002WO PATENT arginine, etc.). Unless otherwise indicated, three-letter and single-letter abbreviations of amino acids refer to the L-isomeric form of the amino acid in question. The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dasp, (d)Asp or D-Asp; Dphe, (d)Phe or D- Phe). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide. D-amino acids may be indicated as customary in lower case when referred to using single-letter abbreviations.

[0032] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four- character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, including, for example, N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,β diphenylalanine), and Ida (Iminodiacetic acid).

[0033] As is clear to the skilled artisan, the peptide sequences disclosed herein are shown proceeding from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide. Among sequences disclosed herein are sequences incorporating a “Hy-” moiety at the amino terminus (N-terminus) of the sequence, and either an “-OH” moiety or an “-NH2” moiety at the carboxy terminus (C-terminus) of the sequence. In such cases, and unless otherwise indicated, a “Hy-” moiety at the N-terminus of the sequence in question indicates a hydrogen atom, corresponding to the presence of a free primary or secondary amino group at the N-terminus, while an “-OH” or an “–NH2” moiety at the C-terminus of the sequence indicates a hydroxy group or an amino group, corresponding to the presence of an amido (CONH2) group at the C-terminus, respectively. In embodiments of the sequences described herein, a C-terminal “–OH” moiety may be substituted for a C-terminal “– NH2” moiety, and vice-versa.

[0034] One of skill in the art will appreciate that certain amino acids and other chemical moieties are modified when bound to another molecule. For example, an amino acid side chain may be modified when it forms an intramolecular bridge with another amino acid side chain, e.g., one or more hydrogen may be removed or replaced by the bond. Accordingly, as used herein,ABVA.002WO PATENT reference to an amino acid or modified amino acid present in embodiments of the peptides described herein is meant to include the form of such amino acid or modified amino acid present in the peptide both before and after forming the intramolecular bond.

[0035] The term “NH2,” as used herein, can refer to a free amino group present at the amino terminus of a polypeptide. The term “OH,” as used herein, can refer to a free carboxy group present at the carboxy terminus of a peptide. Further, the term “Ac,” as used herein, refers to Acetyl protection through acylation of the C- or N-terminus of a polypeptide. In certain peptides shown herein, the NH2 locates at the C-terminus of the peptide indicates an amide group.

[0036] The term “carboxy,” as used herein, refers to –CO2H.

[0037] The term “isostere replacement,” as used herein, refers to any amino acid or other analog moiety having chemical and / or structural properties similar to a specified amino acid. In certain embodiments, an isostere replacement is a conservative substitution or an analog of a specified amino acid.

[0038] The term “cyclized,” as used herein, refers to one part of a peptide molecule being linked to another part of the peptide molecule to form a closed ring, such as by forming a lactam, disulfide bridge or thioether bond.

[0039] The terms “component” or “subunit,” are used interchangeably and refer to one of a pair of peptide monomers that are joined to form a peptide as described herein.

[0040] The term “linker moiety,” as used herein, refers broadly to a chemical structure that is capable of linking or joining together two peptide subunits.

[0041] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill willABVA.002WO PATENT recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0042] As to chemical synthesis, polypeptides can be synthesized by convergent methods such as “native chemical ligation”, and variations thereof, in which two or more peptide fragments with appropriate orthogonally reactive ends are ligated with native amide bond formation. The newly formed peptide can be further ligated to create even longer polypeptides. The individual starting peptides can be derivatized as desired or can be derivatized after a ligation step. Alternately, polypeptides can be manufactured by using chemo-enzymatic peptide synthesis, in which a multiple fragment strategy is used to assemble the full peptide sequence using peptiligases. Thus, short fragments are prepared as unprotected crude peptides for enzymatic ligation by peptiligase enzymes. This fragment condensation approach leads to higher yields of final material, superior impurity profiles and simplification of downstream purification processes.

[0043] As to synthesis of peptides, the peptides may alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor (1989). The Peptides of the Invention produced by recombinant technologies may be expressed from a polynucleotide. One skilled in the art will appreciate that polynucleotides, including DNA and RNA, that encode such glucagon peptides may be obtained from the wild-type cDNA, taking into consideration the degeneracy of codon usage, or may be engineered as desired. These polynucleotide sequences may incorporate codons facilitating transcription and translation of mRNA in microbial hosts. Such manufacturing sequences may readily be constructed according to the methods well known in the art. See, e.g., WO 83 / 04053. The polynucleotides above may also optionally encode an N-terminal methionyl residue. Alternatively, the peptides may be produced by a combination of biosynthetic and synthetic strategies. In some embodiments, the peptides may be produced by a platform that enables site-specific incorporation of synthetic amino acids into proteins, enabling long-acting hormones.

[0044] As such, polynucleotide sequences are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacterial, yeast, and mammalian cells grown in culture), and new and usefulABVA.002WO PATENT methods for cultured growth of such host cells capable of expression of the present glucagon peptides.

[0045] Host cells may be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells. Mammalian host systems for the expression of the recombinant peptide are also well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed peptide or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, C-terminal amidation, acetylation, carboxylation, glycosylation, phosphorylation, myristolation, palmitolation, lipidation and acylation. Post- translational processing, which cleaves a “prepro” form of the peptide, may also be important for correct insertion, folding and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post- translational activities, and may be chosen to ensure the correct modification and processing of the introduced foreign peptide.

[0046] Alternatively, a yeast system may be employed to generate the peptides disclosed herein. The coding region of for example, the glucagon polypeptide cDNA is amplified by PCR. A DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using one primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complementary to nucleotides 255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)). The pre-pro-alpha leader coding sequence and glucagon polypeptide coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature glucagon polypeptide. As taught by Rose and Broach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, Calif. (1990), the vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast “2- micron” replication origin, the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene, and an E. coli origin of replication. The beta-lactamase and leu-2d genes provide for selection in bacteria and yeast, respectively. The leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression. The REP1 and REP2 genes encode proteins involved in regulation of the plasmid copy number.ABVA.002WO PATENT

[0047] The DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Steams et al., Meth. Enz. 185: 280- 97 (1990)). The ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et al., Gene 55: 287 (1987)). The pre-pro-alpha sequence effects secretion of the fusion protein from the cells. Concomitantly, the yeast KEX2 protein cleaves the pre-pro sequence from the mature glucagon polypeptides (Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984)).

[0048] Peptides disclosed herein may also be recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, Calif.), following the manufacturer's instructions. This system also relies on the pre-pro- alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol. The secreted peptide is purified from the yeast growth medium by, e.g., the methods used to purify peptide from bacterial and mammalian cell supernatants.

[0049] Alternatively, the cDNA encoding peptides may be cloned into an expression vector, for example, a baculovirus expression vector pVL1393. This glucagon-compound- encoding vector is then used according to the manufacturer's directions (Sigma Chemical Co., St. Louis, MO) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant protein. The protein is purified and concentrated from the media using a heparin- Sepharose column (Pharmacia, Piscataway, N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass.), and resuspended in PBS. SDS-PAGE analysis is then used to confirm the size of the protein, and Edman sequencing on a Proton 2090 Peptide Sequencer to confirm its N-terminal sequence.

[0050] In another example, the DNA sequence encoding the peptides may be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector is designed to produce a fusion protein comprising glutathione-5-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site. The primers for the PCR may be generated to include, for example, an appropriate cleavage site. The recombinant fusion protein may then be cleaved from the GST portion of the fusion protein. For example, the pGEX-3X / glucagon analog peptide construct is transformed into E. coli XL-1 Blue cells (Agilent, Santa Clara, CA), and individual transformants are isolated and grown at 37°C. in LB medium (supplemented with carbenicillin) to an optical density atABVA.002WO PATENT wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl beta-D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis, Mo.). Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the desired peptide-encoding gene insert in the proper orientation.

[0051] A fusion protein, expected to be produced as an insoluble inclusion body in the bacteria, may be purified as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg / mL lysozyme (Sigma Chemical Co.) for 15 min. at room temperature. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 min. at 12,000×g. The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g. The pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg++and Ca++. The fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If for example a GST / glucagon polypeptide fusion protein is produced in bacteria as a soluble protein, it may be purified using the GST Purification Module (Pharmacia Biotech).

[0052] A fusion protein may be subjected to digestion to cleave the GST from the mature glucagon peptide. The digestion reaction (20-40 μg fusion protein, 20-30 units human thrombin (4000 U / mg (Sigma) in 0.5 mL PBS) is incubated 16-48 hrs. at room temperature and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KCl to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of the peptide may be confirmed by partial amino acid sequence analysis using an Orbitrap Exploris 490 Mass Spectrometer (ThermoFisher Scientific, Waltham, MA).

[0053] It is preferable that the transformed cells are used for long-term, high-yield peptide production and as such stable expression is desirable. Once such cells are transformed with vectors that contain selectable markers along with the desired expression cassette, the cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The selectable marker is designed to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.ABVA.002WO PATENT

[0054] A number of selection systems may be used to recover the cells that have been transformed for recombinant peptide production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside, G418; also, that confers resistance to chlorsulfuron; and hygro, that confers resistance to hygromycin. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a visual indication for identification of transformants include anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

[0055] The peptides disclosed herein may be produced using a combination of both automated peptide synthesis and recombinant techniques. For example, a glucagon peptide may contain a combination of modifications including deletion, substitution, and chemically modified by PEGylation or lipidation. Such a glucagon peptide may be produced in stages. In the first stage, an intermediate glucagon peptide containing the modifications of deletion, substitution, insertion, and any combination thereof, may be produced by recombinant techniques as described. Then after an optional purification step as described herein, the intermediate glucagon peptide is lipidated or PEGylated such as through chemical modification with an appropriate PEGylating reagent (e.g., from NeKtar Transforming Therapeutics, San Carlos, Calif.) to yield the desired glucagon peptide. One skilled in the art will appreciate that the above-described procedure may be generalized to apply to a Peptide of the Invention containing a combination of modifications selected from deletion, substitution, insertion, derivation, and other means of modification well known in the art and contemplated by the invention. In some embodiments, the peptides are produced using a semi-recombinant method that includes recombinant expression techniques and chemical peptide synthesis, for example using any of the methods as described in US Patent No. 9,732,137, which is incorporated by reference in its entirety.

[0056] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequenceABVA.002WO PATENT is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the constructs provided herein.

[0057] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0058] “Percentage of sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (for example, gaps) as compared to the reference sequence (e.g., a polypeptide of the constructs provided herein), which does not include additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0059] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, or over the full length of the reference sequence.ABVA.002WO PATENT With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

[0060] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or embodiment parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0061] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0062] The terms “individual”, “subject”, “host,” or “patient” as used herein have their usual meaning as understood by those skilled in the art and thus includes a human or a non-human mammal. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys), humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0063] As used herein, “treatment” or “therapy” of a disease or condition refers to reducing the severity, frequency, or occurrence of at least one symptom of that disease or condition, compared to a similar but untreated patient. Treatment can also refer to halting, slowing, or reversing the progression of a disease or condition, compared to a similar but untreated patient. Treatment may further include addressing the root cause of the disease and / or one or more symptoms. The term “prevent” does not require the absolute prohibition of the disorder or disease.ABVA.002WO PATENT

[0064] The terms “effective amount” or “effective dose” as used herein have their usual meaning as understood by those skilled in the art and refer to that amount of a recited composition or compound that results in an observable biological effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose- limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0065] A “therapeutically effective amount” or a “therapeutically effective dose” is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of the disorder and / or symptoms, and / or alleviating symptoms associated with the condition, for example for measuring changes in blood glucose level. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic analog (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and / or the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly, given the present disclosure. For additional guidance, see Remington: The Science and Practice of Pharmacy 21.sup.st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.

[0066] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,”ABVA.002WO PATENT “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic or absorption delaying agents, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or carriers, which can be incorporated in any one or more of the compositions described herein, include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, dimethyl sulfoxide (DMSO), or aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers. Suitable pharmaceutical diluents and / or excipients, which can be incorporated in any one or more of the compositions described herein, also include starch, glucose, lactose, sucrose, mannitol, trehalose, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, or ethanol. The physiologically acceptable excipients may also include one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or cyclodextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt- forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), PLURONICS® or preservatives such as methyl paraben, phenol, m-cresol, phenoxyethanol, bronidiol, benzyl alcohol, propyl paraben, or sodium salt of parabens. Preferably, the preservative is phenol, m-cresol, phenoxyethanol, and / or benzyl alcohol. . The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode ofadministration.ABVA.002WO PATENT

[0067] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid (EGTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, poloxamer 188, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, gelatin, esters, ethers, 2-phenoxyethanol, phenol, m-cresol, benzyl alcohol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of the excipient may be found in the composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0068] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs. In some embodiments, the carrier is a plasma protein. Some non-limiting examples of the plasma proteins include, but not limited to albumin, transferrin and fibrinogen.

[0069] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.ABVA.002WO PATENT

[0070] As used herein, the term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal and intracranial. By “co- administer” it is meant that a first composition described herein is administered at the same time, just prior to, or just after the administration of a second composition.

[0071] As used herein, “fibrillation” refers to non-covalent polymerization of glucagon molecules to aggregate and form insoluble fibrils. The type of fibrils formed can be dependent on various parameters including but not limited to pH, ionic strength, temperature and protein concentration.

[0072] As used herein, “conjugated” refers to binding or attachment of two or more moieties. Two or more moieties can be conjugated via a covalent or a non-covalent attachment. In some embodiments, conjugation can comprise a direct conjugation between two moieties. In some embodiments, conjugation can comprise a linker.

[0073] As used herein, “diacid(s)” and “dicarboxylic acid(s)” can be used interchangeably. As used herein, “fatty acids” refer to components of lipid compounds that are composed of a hydrocarbon or acyl chain with a methyl group at one end of the chain and a carboxyl group at the other end of the chain. Fatty acids can also be dicarboxylic acids and composed of a hydrocarbon or acyl chain with a carboxyl group at one end of the chain and a carboxyl group at the other end of the chain. The fatty acids can be short or long chain fatty acids. The fatty acids can comprise 6-22 carbon atoms. Fatty acids can be saturated with no double bonds, monosaturated with one double bond or polysaturated with one or more double bonds.

[0074] As used herein, “PEGylation” is the covalent conjugation of polyethylene glycol (PEG) to the proteins or peptides. PEG can be branched or linear. Monofunctional methoxy-PEG (mPEG) is preferred for peptide modification – CH3O-(CH2CH2O)n-CH2CH2-OH. The molecular weight of PEG can range from 1-50 kilo Dalton (kD) in size. In some embodiments, the molecular weight of PEG used for conjugation is, is about 0.1, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50 kD or any size in between 1 and 50 kD.ABVA.002WO PATENT Some embodiments of isolated glucagon analog peptides and properties thereof

[0075] In some embodiments, the native glucagon peptide includes the amino acid sequence of SEQ ID NO: 1 or HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 2).

[0076] In some embodiments, the isolated glucagon analog peptide includes the amino acid sequence corresponding to any one of the SEQ ID NOs: 3-52. In some embodiments, the isolated glucagon analog peptide includes the amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%, identity to SEQ ID NO: 1. In some embodiments, the isolated glucagon analog peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 70 and 100%, identity to SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the isolated glucagon analog peptide has at least 80% identity to SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the isolated glucagon analog peptide has at least 90% identity to SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0077] In some embodiments, the isolated glucagon analog peptide includes the amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%, identity to any one of SEQ ID NOs: 3-52. In some embodiments, the isolated glucagon analog peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 70 and 100%, identity to any one of SEQ ID NOs: 4- 35. In some embodiments, the isolated glucagon analog peptide has at least 80% identity to any one of SEQ ID NOs: 3-52. In some embodiments, the isolated glucagon analog peptide has at least 90% identity to any one of SEQ ID NOs: 3-52. In some embodiments, the isolated glucagon analog peptide has at least 95% identity to any one of SEQ ID NOs: 3-52.

[0078] In some embodiments, the position of the amino acids in SEQ ID NO: 1 is based on numbering the first amino acid at the amino-terminal end of the peptide as position 1 and counting up sequentially and monotonically to the carboxy-terminal amino acid. In some embodiments, the initial amino acid position is 1. In some embodiments, the initial amino acid position is 0. In some embodiments, the initial amino acid position is a negative number. In some embodiments, the numbering of the amino acid positions does not increase monotonically. In some embodiments, the reference to SEQ ID NO: 1 as native glucagon peptide sequence does not mean that the native glucagon peptide sequence is limited to just SEQ ID NO: 1. In someABVA.002WO PATENT embodiments, the numbering of an amino acid position in the native glucagon peptide sequence can be absolute, for example, starting at the first amino acid of the native glucagon peptide and continuing through the end of the native glucagon peptide, or it can be relative. In some embodiments, an amino acid position may be skipped when numbering a sequence.

[0079] In some embodiments, the isolated glucagon analog peptides of the SEQ ID NOs: 3-52 can differ from native glucagon peptide of SEQ ID NO: 1 and / or SEQ ID NO: 2 by substitution of 1 amino acid, 2, 3, 4, 5-10, 10-20, or 20-29 amino acids. In some embodiments, an isolated glucagon analog peptide can include the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 with 1 residue, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues substituted. In some embodiments, an isolated glucagon analog peptide sequence can have 1 residue, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues substituted with a conservative amino acid. In other embodiments, an isolated glucagon analog peptide sequence can have 1 residue, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues substituted with a non-conservative amino acid.

[0080] In some embodiments, the amino acid residues of the native glucagon peptide sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 that are responsible for lower stability are substituted with amino acids that confer higher stability. In some embodiments, one or more amino acid residues at positions 2, 3, 9, 15, 16, 17, 20, 21, 24, 25, 26, 27, 28 or 29 are substituted. In some embodiments, one or more amino acid residues at positions 2, 3, 9, 15, 16, 17, 20, 21, 24, 25, 26, 27, 28 or 29 are substituted with 2-Aminoisobutyric acid (Aib, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine), E, Q, A, D, R, K, K*, K**, K***, M, L, N, V, I, or T. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)ndiacid or (L) Lys-decanoyl. In some embodiments, AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, K** is L-Lys-Xa- (C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 30, 40 or 50 kD or any size in between 0.2 and 50 kD.ABVA.002WO PATENT

[0081] In some embodiments, substitutions of amino acids in positions 2, 3, 9, 15, 16, 17, 20, 21, 24, 25, 26, 27, 28, and / or 29 confer higher stability to the peptide analogs as compared to stability of native glucagon.

[0082] In some embodiments, substitutions of amino acids in positions 2, 3, 9, 15, 16, 17, 20, 21, 24, 25, 26, 27, 28, and / or 29 confer higher potency at the glucagon receptor to the peptide analogs as compared to native glucagon.

[0083] In some embodiments, substitutions of amino acids in positions 2, 3, 9, 15, 16, 17, 20, 21, 24, 25, 26, 27, 28, and / or 29 confer extended pharmacokinetics and a pharmacodynamic responses, noted by time of increases in blood glucose levels, to the peptide analogs as compared to native glucagon.

[0084] In some embodiments, the amino acid residues of the native glucagon peptide sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 that are responsible for aggregation of the native glucagon peptide are substituted with amino acids that confer reduced aggregation. In some embodiments, the amino acid residues at positions 20-29 are substituted. In some embodiments, one or more amino acid residues at positions 20-29 are substituted with 2-Aminoisobutyric acid (Aib, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine), E, Q, A, D, R, K, K*, K**, K***, M, L, N, V, I, or T. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)ndiacid or (L) Lys-decanoyl. In some embodiments, AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD or any size in between 0.2 and 50 kD.

[0085] In some embodiments, substitutions of one or more amino acids in positions 20-29 confer resistance to fibrillation in the peptide analogs as compared to fibrillation of native glucagon.ABVA.002WO PATENT

[0086] In some embodiments, the peptide analogs include one or more additional amino acid as compared to SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the additional amino acids are on the N-terminal end of the peptide. In some embodiments, the additional amino acids are on the C-terminal end of the peptide. In some embodiments, the additional amino acids are interspersed throughout the peptide. In some embodiments, the one or more additional amino acids are selected from the group including 2-aminoisobutyric acid (Aib, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine), E, Q, A, D, R, K, K*, M, L, N, V, I, or T. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)n diacid or (L) Lys-decanoyl. In some embodiments, AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, K** is L-Lys-Xa- (C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD or any size in between 0.2 and 50 kD.

[0087] In some embodiments, the additional amino acid is K. In some embodiments, the additional amino acid is K is on the N-terminal end of the peptide. In some embodiments, the additional amino acid is K is on the C-terminal end of the peptide. In some embodiments, the additional amino acid is K*. In some embodiments, the additional amino acid is K* is on the N- terminal end of the peptide. In some embodiments, the additional amino acid is K* is on the C- terminal end of the peptide. In some embodiments, the additional amino acid is K** is on the N- terminal end of the peptide. In some embodiments, the additional amino acid is K** is on the C- terminal end of the peptide. In some embodiments, the additional amino acid is K*** is on the N- terminal end of the peptide. In some embodiments, the additional amino acid is K*** is on the C- terminal end of the peptide.

[0088] In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)n diacid. The term “diacid(s)” and “dicarboxylic acid(s)” are used interchangeably herein. In some embodiments, the diacids include but are not limited to oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, brassylic acid,ABVA.002WO PATENT dodecanedioic acid, thapsic acid, japanic acid, phellogenic acid or equisetolic acid. In some embodiments, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0089] In some embodiments, the amino acid in at least one position denoted by X in SEQ ID NO: 2 (HXQGTFTSDYSKYLDXXRAXXFVXWLXXX) can be substituted. In some embodiments, X is Aib, E, Q A, D, E, R, K, K*, K*, K**, M, L, N, V, I, T or OH. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)n diacid or (L) Lys-decanoyl. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-C=0-(CH2)n-R where R is CH3or COOH. In some embodiments, AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD or any size in between 0.2 and 50 kD.

[0090] In some embodiments, the amino acid in at least one position denoted by X1-10in HX1QGTFTSDYSKYLDX2X3RAX4X5FVX6WLX7X8X9X10 (SEQ ID NO: 2) can be substituted. In some embodiments, X1 is S or Aib , X2 is Aib, E, or A; X3 is R, K*, K** or K***; X4 is Aib, Q, A, or E; X5is D or E; X6is Q, K, K*, K** or K***; X7is V, E, I or L; X8is D, E, Q or N; X9is T, K, K*, K** or K*** and X10is absent, K, K*, K** or K***. In some embodiments, K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)n diacid or (L) Lys-decanoyl. In some embodiments, AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc-AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD or any size in between 1 and 50 kD. In some embodiments, the amino acid in at least one position denoted byABVA.002WO PATENT X1-9in HX1QGTFTSDYSKYLDX2X3RAX4X5FVX6WLX7X8X9-OH (SEQ ID NO: 35) can be substituted. In some embodiments, X1 is S or Aib , X2 is Aib, E, or A; X3 is R, K*, K** or K***; X4 is Aib, Q, A, or E; X5 is D or E; X6 is Q, K, K*, K** or K***; X7 is V, E, I or L; X8 is D, E, Q, or N and X9is T, K, K*, K** or K***. In some embodiments, K* is L-Lys(AEEAc-AEEAc- L-γ-Glu-(CH2)n diacid or (L) Lys-decanoyl. In some embodiments, AEEAc is 2-(2-(2- aminoethoxy)ethoxy)acetic acid. In some embodiments, n is 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, K** is L-Lys-Xa-(C=O)-(CH2)m-CH3, wherein Xais absent or (AEEAc- AEEAc). In some embodiments, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In some embodiments, K*** is L-Lys-Poly Ethylene Glycol (PEG) and PEG is between 0.2-50 kD in size. In some embodiments, the size of PEG that is conjugated to the peptide is 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD. In some embodiments, the size of PEG that is conjugated to the peptide is, is about, 0.2, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 kD or any size in between 0.2 and 50 kD.

[0091] In some embodiments, the isolated glucagon analog peptides are selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAQDFVK*WLMDT-OH (SEQ ID NO: 3); HSQGTFTSDYSKYLDAibRRAQDFVK*WLLDT-OH (SEQ ID NO: 4); HSQGTFTSDYSKYLDERRAQDFVK*WLLDT-OH (SEQ ID NO: 5); HSQGTFTSDYSKYLDERRAAibDFVK*WLLDT-OH (SEQ ID NO: 6); HSQGTFTSDYSKYLDARRAAibDFVK*WLMDT-OH (SEQ ID NO: 7); HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT-OH (SEQ ID NO: 8); HSQGTFTSDYSKYLDAibK*RAADFVQWLLDT-OH (SEQ ID NO: 9); HSQGTFTSDYSKYLDAibK*RAADFVQWLMET-OH (SEQ ID NO: 10); HSQGTFTSDYSKYLDAibK*RAADFVQWLLET-OH (SEQ ID NO: 11); HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT-NH2 (SEQ ID NO: 12); HSQGTFTSDYSKYLDAibK*RAADFVQWLLDT- NH2(SEQ ID NO: 13); HSQGTFTSDYSKYLDAibK*RAADFVQWLMET- NH2 (SEQ ID NO: 14); HSQGTFTSDYSKYLDAibK*RAADFVQWLLET- NH2 (SEQ ID NO: 15); HSQGTFTSDYSKYLDEK*RAADFVQWLMDT-OH (SEQ ID NO: 16); HSQGTFTSDYSKYLDEK*RAADFVQWLLDT-OH (SEQ ID NO: 17);ABVA.002WO PATENT HSQGTFTSDYSKYLDEK*RAADFVQWLMET-OH (SEQ ID NO: 18); HSQGTFTSDYSKYLDEK*RAADFVQWLLET-OH (SEQ ID NO: 19); HSQGTFTSDYSKYLDAibRRAAEFVQWLMNTK* (SEQ ID NO: 20); HSQGTFTSDYSKYLDAibRRAAEFVQWLMNK* (SEQ ID NO: 21); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDTK* (SEQ ID NO: 22); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDK* (SEQ ID NO: 23); HSQGTFTSDYSKYLDAibRRAAEFVKWLMDTK* (SEQ ID NO: 24); and HSQGTFTSDYSKYLDAibRRAAEFVKWLMDK* (SEQ ID NO: 25).

[0092] In some embodiments, the isolated peptide further includes a carboxy terminal peptide including GPSSGAPPPS (SEQ ID NO: 53), GPSSGA (SEQ ID NO: 54), or GPS.

[0093] In some embodiments, the isolated glucagon analog peptides are selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGAPPPS-NH2 (SEQ ID NO: 26); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGAPPPS-NH2 (SEQ ID NO: 27); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGA-NH2(SEQ ID NO: 28); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGA-NH2 (SEQ ID NO: 29); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPS-NH2 (SEQ ID NO: 30); and HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPS-NH2(SEQ ID NO: 31).

[0094] In some embodiments, the isolated glucagon analog peptides are selected from the group consisting of any one of SEQ ID NOs: 32-52. In some embodiments, the isolated glucagon analog peptides are selected from the group consisting of any one of SEQ ID NOs: 3-52.

[0095] In some embodiments, the amino acid residues of the native glucagon peptide sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 are conjugated to lipids or fatty acids. In some embodiments, the fatty acids include, but are not limited to caproic acid (C6), hexanedioic acid (C6 dicarboxylic acid), caprylic acid (C8), octanedioic acid (C8 dicarboxylic acid), capric acid (C10), decanedioic acid (C10 dicarboxylic acid), lauric acid (C12), dodecanedioic acid (C12 dicarboxylic acid), myristic acid (C14), tetradecanedioic acid (C14 dicarboxylic acid), palmitic acid (C16), hexadecanedioic acid (C16 dicarboxylic acid), stearic acid (C18), octadecanedioic acid (C18 dicarboxylic acid), icosanoic acid (C20), icosanedioic acid (C20 dicarboxylic acid),ABVA.002WO PATENT docosanoic acid (C22) or docosanedioic acid (C22 dicarboxylic acid).. In some embodiments, the fatty acids are conjugated to the N-terminus of the peptide. In some embodiments, the fatty acids include, but are not limited to palmitoleic acid, oleic acid, elaidic acid, linoleic acid, α-linolenic acid, β-linolenic acid, stearidonic acid, arachidonic acid, docosapentaenoic acid, docosahexaenoic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, brassylic acid, dodecanedioic acid, thapsic acid, japanic acid, phellogenic acid or equisetolic acid.. In some embodiments, the fatty acids are conjugated to the C-terminus of the peptide. In some embodiments, the fatty acids are conjugated to a lysine residue in the peptide. In some embodiments, the fatty acids are conjugated to the peptide by amide linkage. In some embodiments, the fatty acids are conjugated to the peptide by ester linkage.

[0096] In some embodiments, the fatty acids are conjugated to the peptide using coupling agents or linkers. Some non-limiting examples of coupling agents or linkers include carbodiimide (EDC), N-hydroxysuccinimide (NHS), or γ-glutamyl linker.

[0097] In some embodiments, the attachment of fatty acids to the peptide results in enhanced binding of the peptides disclosed herein to plasma proteins, for example to albumin. In some embodiments, the fatty acid is a dialkylamide fatty acid. In some embodiments, the peptide is attached to a protein moiety specific for albumin. In some embodiments, the enhanced binding of peptides to albumin is reversible. In some embodiments, the enhanced binding of peptides to albumin is irreversible.

[0098] In some embodiments, amino acid substitution at the indicated residues results in a change in solubility, half-life, stability, functionality, activity, specificity, affinity or a combination of the aforementioned parameters. In some embodiments, the isolated glucagon analog peptides provided herein confer increased activity and / or stability. In some embodiments, the isolated glucagon analog peptides provided herein can confer an enhanced function, stability, or activity. In some embodiments, the isolated glucagon analog peptides provided herein can enhance the binding of glucagon to glucagon (GCG) receptors. In some embodiments, the change in solubility, half-life, stability, functionality, activity, specificity, affinity or a combination of the aforementioned parameters can be measured by one or more assays including, but not limited to absorption, chromatography, mass spectrometry or cell-based reporter assays,

[0099] In some embodiments, the half-life of isolated glucagon peptide analogs is enhanced by at least 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,ABVA.002WO PATENT 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any integer that is between 1 and 99%, more than the half-life of native glucagon. As used herein, the term half-life may refer to solution half- life of the peptides described herein, or may refer to the in vivo half-life of the peptides described herein. In some embodiments, the half-life of isolated glucagon peptide analogs as measured by liquid chromatography coupled with mass spectrometry (LC-MS) is enhanced by at least 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any integer that is between 1 and 99%, more than the half-life of native glucagon. Native glucagon in solution has poor half-life. Typically, more than 50% of the native peptide is lost within a day as it forms aggregates. Embodiments of the peptides described herein exhibit enhanced half-life, for example such that the peptides include more than 50% half-life for more than one day, for example more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% half-life for more than one day, such as more than 1, 2, 3, 4, 5, 6, or 7 days, or more than 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0100] In some embodiments, the Tmax, the time taken to reach maximum plasma concentration of the glucagon analogs is at least 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 16 hours later than that of native glucagon.

[0101] In some embodiments, the half-life of isolated glucagon peptide analogs is at least 2, 3, 4, 5, 6, 7, 8, 10, 20, 40, 50, 60, 70, 75, 80, 90, 100-fold, or any integer that is between 2 and 100-fold more than the half-life of native glucagon. In some embodiments, the half-life of isolated glucagon peptide analogs as measured by liquid chromatography coupled with mass spectrometry (LC-MS) is at least, 3, 4, 5, 6, 7, 8, 10, 20, 40, 50, 60, 70, 75, 80, 90, 100-fold, or any integer that is between 2 and 100 fold more than the half-life of native glucagon.

[0102] In some embodiments, the stability of isolated glucagon peptide analogs is longer than the stability of native glucagon. In some embodiments, the stability of isolated glucagon analogs is longer than native glucagon by at least 1, 2, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, or 400 days or any integer that is between 1 and 400 days. In some embodiments, the stability of isolated glucagon peptide analogs as measured by liquid chromatography coupled with mass spectrometry (LC-MS) is at least, 1, 2, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, or 400 days or any integer that is between 1 and 400 days more than the stability of native glucagon. Native glucagon in solution is highly unstable.ABVA.002WO PATENT Typically, more than 50% of the native peptide is lost within a day as it forms aggregates. Embodiments of the peptides described herein exhibit enhanced stability, for example such that the peptides include more than 50% stability for more than one day, for example more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% stability for more than one day, such as more than 1, 2, 3, 4, 5, 6, or 7 days, or more than 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0103] In some embodiments, the solubility of the isolated glucagon peptide analogs in physiological buffers is enhanced by at least 2, 3, 4, 5, 6, 8, 10, 20, 40, 50, 60, 80, 90, 100 fold or any integer that is between 2 and 100 fold more than the solubility of native glucagon in physiological buffers. In some embodiments, the physiological buffer comprises tween-20. In some embodiments, the physiological buffer comprises SDS. In some embodiments, the physiological buffer comprises cyclodextrins. In some embodiments, the physiological buffer is phosphate‑buffered saline (PBS). In some embodiments, the buffers used to solubilize the peptide analogs include tween-20 and / or sodium dodecyl sulfate (SDS). In some embodiments, the solubility of the isolated glucagon peptide analogs in physiological buffers is enhanced by at least 2, 3, 4, 5, 6, 8, 10, 20, 40, 50, 60, 80, 90, 100 fold or any integer that is between 2 and 100 fold, more than the solubility of native glucagon in physiological buffers at pH 7. In some embodiments, the solubility of the isolated glucagon peptide analogs in physiological buffers as measured by absorption assays is enhanced by at least 2, 3, 4, 5, 6, 8, 10, 20, 40, 50, 60, 80, 90, 100 fold, or any integer that is between 2 and 100 fold, more than the solubility of native glucagon in physiological buffers at pH 7. In some embodiments, the absorption assay is an ultraviolet (UV) absorption. In some embodiments, the solubility of the isolated glucagon peptide analogs in physiological buffers 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, 28, 29, or 30 mg / ml, or any number in between 1 mg / ml and 30 mg / ml.

[0104] In some embodiments, the isolated glucagon peptide analogs are dissolved in a physiological buffer at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 10 mg / ml or any concentration between 0.1-10 mg / ml. In some embodiments, the concentration of isolated glucagon peptide analogs is 1 mg / ml. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is a bicarbonate buffer. In some embodiments, the buffer is tris buffered saline (TBS).ABVA.002WO PATENT

[0105] In some embodiments, the pH of the buffer in which the isolated glucagon peptide analogs are dissolved is adjusted to 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.8, 9.9, 10.0 or any pH between 2-10. In some embodiments, the pH of the buffer is, is about 5, 6, 7, 8, 9, or any integer that is between 5 and 9. In some embodiments, the pH of the buffer in which the isolated glucagon peptide analogs are dissolved is 7. In some embodiments, the pH of the buffer in which the isolated glucagon peptide analogs are dissolved is 7.2. In some embodiments, the pH of the buffer in which the isolated glucagon peptide analogs are dissolved is 7.4.

[0106] In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 0%, 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, ,100%, 150%, 200% or any integer that is between 1 and 200%, more than the specificity of binding of native glucagon. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 5% to 200%, more than the specificity of binding of native glucagon. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 5% to 100%, more than the specificity of binding of native glucagon. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 5% more than the specificity of binding of native glucagon. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 100% more than the specificity of binding of native glucagon. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to the glucagon receptor is enhanced by at least 125% more than the specificity of binding of native glucagon. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 50 fold, or any number between 2 and 50-fold higher than the ability of native glucagon to stimulate cAMP production.

[0107] In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 0%, 1%, 5%ABVA.002WO PATENT 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any integer that is between 1 and 99%, more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 0%, 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, ,100%, 150%, 200% or any integer that is between 1 and 200%, more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 5% to 200% more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 5% to 100% more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 5% more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 100% more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is enhanced by at least 125% more than the ability of native glucagon to stimulate cAMP production. In some embodiments, the ability of isolated glucagon peptide analogs to stimulate cAMP production through binding to the glucagon receptor is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 50 fold, or any number between 2 and 50 fold higher than the ability of native glucagon to stimulate cAMP production.

[0108] In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to carriers like albumin is enhanced by at least 0%, 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%,100%, 150%, 200% or any integer that is between 1 and 200%, more than the specificity of binding of native glucagon to carriers like albumin. In some embodiments, the specificity of binding or affinity of isolated glucagon peptide analogs to carriers like albumin is enhanced by at least 5% to 200% more than the specificity of binding of native glucagon to carriers like albuminABVA.002WO PATENT

[0109] In some embodiments, the isolated glucagon peptide analogs regulate the glycemic effect of the subject by at least 0%, 1%, 5% 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any integer that is between 1 and 99%, as compared to native glucagon. In some embodiments, the isolated glucagon peptides increase the blood glucose levels by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100%, or by an amount in a range that is defined by any two of the preceding numbers, as compared to native glucagon. Several Embodiments of Methods of Use

[0110] In some embodiments, methods of treating a subject suffering from hypoglycemia are provided. In some embodiments, methods of protecting, preventing and / or improving a subject from hypoglycemia are provided. In some embodiments, the hypoglycemia is acute hypoglycemia. In some embodiments, the hypoglycemia is sub-acute hypoglycemia. In some embodiments, the hypoglycemia is chronic hypoglycemia. In some embodiments, the methods include administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes one or more isolated glucagon peptides disclosed herein.

[0111] In some embodiments, the administration of a pharmaceutical composition includes one or more isolated glucagon peptides treats, prevents, protects and / or improves hypoglycemia in the subject by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100%, or by an amount in a range that is defined by any two of the preceding numbers, as compared to the subject prior to administration of the pharmaceutical composition.

[0112] In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100%, or by an amount in a range that is defined by any two of the preceding numbers, as compared to the subject prior to administration of the pharmaceutical composition.

[0113] In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject in about 60, 80, 90, 120, 180, 200, 240, or 300 minutes or at any amount of time between 60 to 300 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subjectABVA.002WO PATENT in about 60 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject in about 120 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject in about 180 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject in about 240 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides increases the blood glucose levels in the subject in about 300 minutes. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides provides a biphasic effect. In some embodiments, the administration of a pharmaceutical composition including one or more isolated glucagon peptides provides a dual PD effect.

[0114] In some embodiments, the Tmax, the time taken to reach maximum plasma concentration of the glucagon analogs after single administration of the analogs in the subject, is about 60, 80, 90, 120, 180, 200, 240, or 300 minutes or at any amount of time between 60 to 300 minutes. In some embodiments, the Tmax of the glucagon analogs after single administration of the analogs in the subject is about 60 minutes. In some embodiments, the Tmaxof the glucagon analogs after single administration of the analogs in the subject is about 120 minutes. In some embodiments, the Tmax of the glucagon analogs after single administration of the analogs in the subject is about 180 minutes. In some embodiments, the Tmaxof the glucagon analogs after single administration of the analogs in the subject is about 240 minutes. In some embodiments, the Tmaxof the glucagon analogs after single administration of the analogs in the subject is about 300 minutes. In some embodiments, the Tmax of the glucagon analogs is at least 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 16 hours later than that of native glucagon.

[0115] In some embodiments, the composition including one or more isolated glucagon peptides exhibit an early onset pharmacodynamic (PD) profile compared to the native peptide wherein the Tmaxoccurs within 60 minutes post single administration of the glucagon peptides. In some embodiments, the one or more isolated glucagon peptides that exhibit an early onset PD profile are known as short-acting analogs. In some embodiments, one or more isolated glucagon peptides exhibiting an early onset PD profile compared to the native peptide are used in hypoglycemic rescue. In some embodiments, the one or more isolated glucagon peptidesABVA.002WO PATENT exhibiting an early onset PD profile compared to the native peptide are used in the prevention and / or treatment of subjects with type 1 and / or type 2 diabetes. In some embodiments, the subjects are treated with insulin prior to administration of one or more isolated glucagon peptides. In some embodiments, the subjects are treated with insulin after administration of one or more isolated glucagon peptides. In some embodiments, the subjects are treated with insulin in conjunction with administration of one or more isolated glucagon peptides.

[0116] In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid (or intermediate) onset PD profile compared to the native peptide wherein the Tmax occurs between 60 minutes to about 720 minutes post single administration of the glucagon peptides. In some embodiments, the one or more isolated glucagon peptides that exhibit a mid-onset PD profile are known as intermediate or mid-acting analogs. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 60, 120, 150, 180, 200, 240, 300, 350, 360, 420, 480, 540, 600, 660, 720 minutes, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours or any time between 60 minutes to about 24 hours post single administration of the glucagon peptides. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 240 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 300 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 360 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 420 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 480 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid- onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 540 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 600 minutes. In some embodiments, the composition including one or more isolated glucagonABVA.002WO PATENT peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 660 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 720 minutes. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 14 hours. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 16 hours. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 18 hours. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 20 hours. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid-onset PD profile compared to the native peptide wherein the Tmaxoccurs at, at about 22 hours. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a mid- onset PD profile compared to the native peptide wherein the Tmax occurs at, at about 24 hours.

[0117] In some embodiments, the composition including one or more isolated glucagon peptides exhibit a delayed or extended-release PD profile compared to the native peptide wherein the Tmax occurs between about 12 hours to about 30 days post single administration of the glucagon peptides. In some embodiments, the one or more isolated glucagon peptides that exhibit a delayed or extended-release PD profile are known as long or very long-acting analogs. In some embodiments, the composition including one or more isolated glucagon peptides exhibit a delayed or extended-release PD profile compared to the native peptide wherein the Tmax occurs at, at about 12 hours, 24 hours, 36 hours or any time between 12 hours to about 36 hours post single administration of the glucagon peptides.

[0118] In some embodiments, the one or more isolated glucagon analogs disclosed herein will exhibit properties of being hepatopreferential. This means the glucagon analog has a pharmacodynamic and pharmacokinetic profile that favors its major site of action in the liver and not in other relevant peripheral tissues. Glucagon analog properties that bias to hepatopreferential action are driven by a number of factors that include rate of appearance in the circulation from the original site of administration (e.g. subcutaneous bed), first pass uptake by the liver bed, sizeABVA.002WO PATENT and structure of the analog (including acyl and / or PEGylation structure components), charge and hydrophilicity of the analog, and the binding properties to circulating albumin.

[0119] In some embodiments, the one or more isolated glucagon analogs disclosed herein can be fused with an insulin molecule structure or insulin moiety sub-structure to form a unique dual-moiety molecule structure that can exhibit both glucagon and insulin action properties. Respective glucagon and insulin action will be driven, in part, by ambient plasma glucose level with insulin action dominating over glucagon action when plasma glucose level is elevated, and the converse when plasma glucose is low. The plasma glucose gradient or threshold that dictates when the opposing actions of insulin or glucagon dominate over the other can be engineered into the unique properties of the dual-moiety invention. The dual moiety molecule structure can also elicit the balance of the opposing glucagon and insulin actions through the moiety receptor binding affinity to respective endogenous glucagon and insulin receptors (e.g. insulin has a receptor binding affinity 10-fold higher than glucagon binding, 4-fold higher, equivalent).

[0120] In some embodiments, the one or more isolated glucagon analogs disclosed herein can be administered in combination with one or more metabolically active therapeutics. In some embodiments, the relevant therapeutic uses of the one or more metabolically active therapeutics include treatment or prevention of hypoglycemia, including acute, sub-acute, chronic hypoglycemia, post-bariatric hypoglycemia, congenital hyperinsulinism, type 1 diabetes, type 2 diabetes, body weight control or diseases related to overweight or obesity, liver fat (lipid) metabolism, or protein load renoprotection. Some non-limiting examples of metabolically active therapeutics include but not limited to insulin and / or insulin analogues, including rapid and ultra- rapid acting insulin and / or incretin analogs. Some non-limiting examples of metabolically active therapeutics include but not limited to glucagon agonists, insulin and / or insulin analogues, including rapid and ultra-rapid acting insulin and / or incretin analogs. In some embodiments, the incretin analogues are inclusive of all single, combination or multi-moiety therapeutics under the broad incretin umbrella term that have one or more of oxyntomodulin (OXM), Glucagon-like peptide-1(GLP1), Glucose-Dependent Insulinotropic Polypeptide (GIP), amylin, peptide YY (PYY), Cholecystokinin (CCK), Ghrelin, (LEAP2) or Glicentin. In the strictest sense, the incretins are Glucagon-like peptide-1(GLP1) and Glucose-Dependent Insulinotropic Polypeptide (GIP). But the broader incretin term applied here includes the other listed gut-borne peptides or targets. Some non-limiting examples of additional metabolically active gut-related therapeuticsABVA.002WO PATENT where the glucagon analogs herein could confer benefit when administered in combination include but not limited to Insulin-like Growth Factor 1(IGF1), Cannabinoid 1 receptor (CB1), Bile acid receptor targets (FXR, TGR5), Fatty Acid GPCRs (GPR119, GPR40, GPR120), and fibroblast growth factor 21 (FGF21). In some embodiments, the one or more metabolically active therapeutics provide an adjunctive therapeutic action with the one or more glucagon peptide analogues disclosed herein. A non-limiting embodiment illustrating the combination of glucagon peptide analogs with varied PD profiles with other metabolically active therapeutics is provided in Table 3 below. Table 3 GCG PD Clinical attribute Patient Partner therapy Product form profile population in in n n DABVA.002WO PATENT GCG: glucagon Incretin: inclusive of all single, combination or multi-moiety therapeutics under the broad incretin umbrella that have one or more of GLP1, GIP, amylin, PYY, IGF1, CB1, FGF21 moieties that would benefit from additional GCG adjunctive therapeutic action beneficial metabolic effects: inclusive of beneficial liver, kidney and body weight effects T1: type 1 diabetes T2: type 2 diabetes URAI: ultra rapid acting insulin RAI: rapid acting insulin QD – once daily QW – once weekly QM – once monthly

[0121] In some embodiments, the composition comprising one or more isolated glucagon peptides are provided in the form of a kit. In some embodiments, the kit is a rescue kit. In some embodiments, the kit includes written instructions for using the one or more isolated glucagon analogs. In some embodiments, the one or more isolated glucagon analogs disclosed herein can be co-formulated in combination with one or more metabolically active therapeutics and delivered or administered via a pump. In some embodiments, the pump is a continuous pump. In some embodiments, the pump is a programmed infusion pump. In some embodiments, the one or more isolated glucagon analogs disclosed herein can be co-formulated in combination with one or more metabolically active therapeutics and delivered or administered via pre-filled injection pen. In some embodiments, the one or more isolated glucagon analogs disclosed herein can be co- formulated in combination with one or more metabolically active therapeutics and delivered or administered via pre-filled injection pen as a mealtime injectable formulation. In some embodiments, the pre-filled pen is a single use pen. In some embodiments, the pre-filled pen is a multi-dose pen. In some embodiments, the one or more isolated glucagon analogs disclosed herein co-formulated in combination with one or more metabolically active therapeutics can be delivered every single day in a once daily dose. In some embodiments, the composition including one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered more than once to the subject in need. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides inABVA.002WO PATENT combination with one or more metabolically active therapeutics is administered at least once, twice, thrice, four times, five times or six times every day. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered at least once a week, twice every week, thrice every week, four times every week, five times every week, six times every week or seven times every week. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered once every month or once every 6 months. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered for up to about 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, 20, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 hours, or for an amount of time that is in a range defined by any two of the preceding values. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks, or for an amount of time that is in a range defined by any two of the preceding values. For example, in some embodiments, an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered for between about 1 and 20, 1 and 10, 1 and 8, 1 and 6, 1 and 4, 1 and 2, 1 and 14, 2 and 20, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 12, 4 and 10, 4 and 8, 4 and 6, 6 and 12, 6 and 10, 6 and 8, 8 and 12, 8 and 10, or 10 and 20 weeks. In some embodiments, an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered for longer than 20 weeks. In some embodiments, an effective amount of one or more isolated glucagon peptides in combination with one or more metabolically active therapeutics is administered on an ongoing basis after insulin treatment. In some embodiments, the composition is administered to the subject until an improvement in one or more associated symptoms described herein are observed. Optionally, the composition is administered to the subject after an improvement in one or more associated symptoms is observed, for example until regulation of blood glucose levels is observed.

[0122] In some embodiments, the subject has been diagnosed with type 1 diabetes or type 2 diabetes. In some embodiments, the subject is at a predisposed risk of developing type 1 diabetesABVA.002WO PATENT or type 2 diabetes. In some embodiments, the subject in need is identified by a blood test. One of ordinary skill in the art would appreciate that the attending physician would know how to identify a subject in need of prevention and / or treatment disclosed herein. In some embodiments, the subject is treated with insulin prior to administration of the pharmaceutical composition including one or more isolated glucagon peptides.

[0123] In some embodiments, the composition including one or more isolated glucagon peptides is administered more than once to the subject in need. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides is administered at least once, twice, thrice, four times, five times or six times every day. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides is administered at least once a week, twice every week, thrice every week, four times every week, five times every week, six times every week or seven times every week. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides is administered once every month or once every 6 months. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides is administered for up to about 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, 20, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 hours, or for an amount of time that is in a range defined by any two of the preceding values. In some embodiments, the composition including an effective amount of one or more isolated glucagon peptides is administered for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks, or for an amount of time that is in a range defined by any two of the preceding values. For example, in some embodiments, an effective amount of one or more isolated glucagon peptides is administered for between about 1 and 20, 1 and 10, 1 and 8, 1 and 6, 1 and 4, 1 and 2, 1 and 14, 2 and 20, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 12, 4 and 10, 4 and 8, 4 and 6, 6 and 12, 6 and 10, 6 and 8, 8 and 12, 8 and 10, or 10 and 20 weeks. In some embodiments, an effective amount of one or more isolated glucagon peptides is administered for longer than 20 weeks. In some embodiments, an effective amount of one or more isolated glucagon peptides is administered on an ongoing basis after insulin treatment. In some embodiments, the composition is administered to the subject until an improvement in one or more associated symptoms described herein are observed. Optionally, the composition is administered to the subject after anABVA.002WO PATENT improvement in one or more associated symptoms is observed, for example until regulation of blood glucose levels is observed.

[0124] In some embodiments, the effective amount of one or more isolated glucagon peptides is administered as at least one dose at an amount of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, the effective amount of one or more isolated glucagon peptides administered subcutaneously, intramuscularly, or intravenously is 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, the effective amount of one or more isolated glucagon peptides administered enterally, orally, dermally, sub-lingually, in buccal space, lingually, intranasally, rectally, or by inhalation is 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, the effective amount of one or more isolated glucagon peptides is administered varies based on the mode of administration. In some embodiments, the composition is formulated as a single dose or as a multidose formulation.

[0125] In some embodiments, the effective amount of one or more isolated glucagon peptides administered to a hypoglycemic subject for an acute rescue is at least one dose at an amount of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, the effective amount of one or more isolated glucagon peptides administered for a short-term hypoglycemic protection and / or prevention in a subject for sub- acute hypoglycemia protection is at least one dose at an amount of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, the effective amount of one or more isolated glucagon peptides administered to a subject for chronic hypoglycemia protection or prevention is at least one dose at an amount of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 700, 800, 1000 µg / kg / min, or any amount within a range defined by any two of the aforementioned amounts. In some embodiments, 2 µg / kg to 60 µg / kg of one or more isolated glucagon peptides is administered. In someABVA.002WO PATENT embodiments, 1 µg / kg to 100 µg / kg of one or more isolated glucagon peptides is administered. In some embodiments, 10 µg / kg to 500 µg / kg of one or more isolated glucagon peptides is administered. In some embodiments, 0.02 µg / kg to 0.5 µg / kg of one or more isolated glucagon peptides is administered. In some embodiments, 0.005 µg / kg to 0.05 µg / kg of one or more isolated glucagon peptides is administered.

[0126] As applied to any of the methods of prevention and / or treatment disclosed herein, in some embodiments, the effective amount of one or more isolated glucagon peptides is administered enterally, orally, dermally, sub-lingually, in buccal space, lingually, intranasally, rectally, by inhalation, parenterally, intracranially, subcutaneously, intramuscularly, intradermally, or intravenously, or any combination thereof. In some embodiments, the effective amount of one or more isolated glucagon peptides is administered orally. In some embodiments, the effective amount of one or more isolated glucagon peptides is administered by a continuous release delivery system, for example a pump.

[0127] Various criteria can be used to determine the inclusion and / or exclusion of a particular subject in the reference or healthy population, including age of the subject (e.g. the reference subject is younger than the subject in need of prevention and / or treatment) and gender of the subject (e.g. the reference subject can be the same gender or different gender as the subject in need of prevention and / or treatment).

[0128] Some embodiments provided herein are described in the following enumerated alternatives, which are not intended to be limiting.

[0129] 1. An isolated peptide comprising a sequence of HX1QGTFTSDYSKYLDX2X3RAX4X5FVX6WLX7X8X9X10 (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein X1 is S or Aib; wherein X2 is Aib, S, E, or A; wherein X3is R, K*, K** or K***; wherein X4is Aib, Q, A, or E; wherein X5is D or E; wherein X6 is Q, E, K, K*, K** or K***; wherein X7 is V, E, I or L; wherein X8 is D, E, Q or N; wherein X9 is T or K, K*, K** or K***; wherein X10 is absent, K, K*, K** or K***; wherein K* is L- Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)ndiacid or (L) Lys-decanoyl; wherein AEEAc is 2-(2-(2- aminoethoxy)ethoxy)acetic acid; wherein n is 6, 8, 10, 12, 14, 16, 18, or 20; wherein K** is L- Lys-Xa-(C=O)-(CH2)m-CH3; wherein Xa is absent or (AEEAc-AEEAc); wherein m = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; wherein K*** is L-Lys-Poly Ethylene Glycol.ABVA.002WO PATENT

[0130] 2. The isolated peptide of alternative 1, further comprising an additional K, K*, K** or K*** on the carboxy-terminal end of the peptide.

[0131] 3. The isolated peptide of alternative 1 or alternative 2, wherein the peptide is conjugated to a fatty acid.

[0132] 4. The isolated peptide of alternative 3, wherein the fatty acid is selected from the group consisting of caproic acid (C6), hexanedioic acid (C6 dicarboxylic acid), caprylic acid (C8), octanedioic acid (C8 dicarboxylic acid), capric acid (C10), decanedioic acid (C10 dicarboxylic acid), lauric acid (C12), dodecanedioic acid (C12 dicarboxylic acid), myristic acid (C14), tetradecanedioic acid (C14 dicarboxylic acid), palmitic acid (C16), hexadecanedioic acid (C16 dicarboxylic acid), stearic acid (C18), octadecanedioic acid (C18 dicarboxylic acid), icosanoic acid (C20), icosanedioic acid (C20 dicarboxylic acid), docosanoic acid (C22) or docosanedioic acid (C22 dicarboxylic acid).

[0133] 5. The isolated peptide of any one of alternatives 1-4, wherein the peptide is conjugated to poly ethylene glycol (PEG).

[0134] 6. The isolated peptide of alternative 5, wherein the PEG is between 0.2-50 kD in size.

[0135] 7. The isolated peptide of any one of alternatives 3-4, wherein the fatty acid is conjugated to K, K*, K** or K*** residue of the peptide.

[0136] 8. The isolated peptide of alternative 7, wherein K* is L-Lys(AEEAc-AEEAc-L-γ- Glu-(CH2)ndiacid or (L) Lys-decanoyl and wherein n is 6, 8, 10, 12, 14, 16, 18, or 20.

[0137] 9. The isolated peptide of alternative 7, wherein K** is K** is L-Lys-Xa-(C=O)- (CH2)m-CH3, wherein Xa is absent or (AEEAc-AEEAc) and wherein m is 5, 7, 9, 11, 13, 15, 17.

[0138] 10. The isolated peptide of alternative 7, wherein K*** is L-Lys-PEG and wherein PEG is between 0.2-50 kD in size.

[0139] 11. The isolated peptide of any one of alternatives 1-10, further comprising a carboxy terminal peptide comprising GPSSGAPPPS (SEQ ID NO: 2), GPSSGA (SEQ ID NO: 3), or GPS.

[0140] 12. The isolated peptide of any one of alternatives 1-11, configured for increased specificity and increased potency to a GCG receptor as compared to native glucagon.

[0141] 13. The isolated peptide of any one of alternatives 1-12, wherein the peptide is capable of activating a glucagon receptor.ABVA.002WO PATENT

[0142] 14. The isolated peptide of any one of alternatives 1-13, wherein the isolated peptide has high solubility.

[0143] 15. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAQDFVK*WLMDT-OH (SEQ ID NO: 3); HSQGTFTSDYSKYLDAibRRAQDFVK*WLLDT-OH (SEQ ID NO: 4); HSQGTFTSDYSKYLDERRAQDFVK*WLLDT-OH (SEQ ID NO: 5); HSQGTFTSDYSKYLDERRAAibDFVK*WLLDT-OH (SEQ ID NO: 6); and HSQGTFTSDYSKYLDARRAAibDFVK*WLMDT-OH (SEQ ID NO: 7).

[0144] 16. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT-OH (SEQ ID NO: 8); HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT- NH2 (SEQ ID NO: 12); and HSQGTFTSDYSKYLDEK*RAADFVQWLMDT-OH (SEQ ID NO: 16).

[0145] 17. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of:HSQGTFTSDYSKYLDAibRRAAEFVQWLMNTK* (SEQ ID NO: 20); HSQGTFTSDYSKYLDAibRRAAEFVQWLMNK* (SEQ ID NO: 21); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDTK* (SEQ ID NO: 22); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDK* (SEQ ID NO: 23); HSQGTFTSDYSKYLDAibRRAAEFVKWLMDTK* (SEQ ID NO: 24); and HSQGTFTSDYSKYLDAibRRAAEFVKWLMDK* (SEQ ID NO: 25).

[0146] 18. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGAPPPS-NH2 (SEQ ID NO: 26); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGAPPPS-NH2 (SEQ ID NO: 27); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGA-NH2(SEQ ID NO: 28); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGA-NH2 (SEQ ID NO: 29); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPS-NH2 (SEQ ID NO: 30); and HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPS-NH2(SEQ ID NO: 31).ABVA.002WO PATENT

[0147] 19. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 3-52.

[0148] 20. The isolated peptide of any one of alternatives 1-14, wherein the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 32-52.

[0149] 21. The isolated peptide of any one of alternatives 1-20, wherein the isolated peptide has an increased affinity of binding to albumin.

[0150] 22. A pharmaceutical composition comprising the isolated peptide of any one of alternatives 1-21 as an active ingredient.

[0151] 23. The pharmaceutical composition of alternative 22, further comprising a pharmaceutically acceptable carrier.

[0152] 24. The pharmaceutical composition of any one of alternatives 22-23, wherein the pharmaceutical composition is formulation for injection.

[0153] 25. A method of treating a subject suffering from hypoglycemia, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an isolated peptide of any one of alternatives 1-21.

[0154] 26. The method of alternative 25, wherein the hypoglycemia is acute, sub-acute and / or chronic hypoglycemia.

[0155] 27. The method of any one of alternatives 25-26, wherein the administering is subcutaneous administration.

[0156] 28. The method of any one of alternatives 25-27, wherein the isolated peptide is administered in an amount ranging from 0.001 µg / kg / min to an amount of 1000 µg / kg / min.

[0157] 29. The method of any one of alternatives 25-28, wherein the administering occurs daily or multiple times a day.

[0158] 30. The method of any one of alternatives 25-29, wherein the administering occurs continuously for up to 48 hours.

[0159] 31. The method of any one of alternatives 25-30, wherein the subject has diabetes. EXAMPLES

[0160] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the presentABVA.002WO PATENT disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the embodiments described herein, as it is described herein above and in the claims. Example 1: Synthesis and purification of isolated glucagon analog peptides

[0161] Peptides were synthesized on Gyros Protein Technologies Symphony X synthesizers via solid phase peptide chemistry (SPPS) / Fmoc chemistry using polystyrene or PEG based resins. Fmoc chemistry employs Fmoc (fluorenylmethyloxycarbonyl) protecting group at the N-terminus of each amino acids, which were removed by a base (usually 20% piperidine in DMF) before the next coupling cycle. Linear SPPS was performed for the main chain of peptide using dipeptides at selected sites and orthogonal protection on Lys for branch chain synthesis. Upon completion of SPPS, TFA mediated cleavage from the resin along with concomitant protecting group removal was performed, followed by isolation via precipitation and filtration with diethyl ether to yield crude material. Preparative reverse-phase HPLC was performed on a Teledyne Isco ACCQPrep HP150 System with a variable wavelength UV absorbance detector, and the material was lyophilized to purified peptide powder. Purity was determined by analytical reverse-phase HPLC performed on an Dionex UltiMate 3000 system and mass identity was confirmed on a Kratos Axima CFR Plus MALDI-MS. The peptides were further characterized by amino acid analysis to determine peptide content. Example 2: Measurement of biophysical stability and solubility of isolated glucagon analog peptides

[0162] In order to determine physicochemical properties, a defined weight of the lyophilized each peptide is reconstituted in a defined composition in the high throughput UNCLE instrument (https: / / www.unchainedlabs.com / uncle / ). 3-4 rounds of screening DOE studies was conducted. From each round of screening, best conditions were selected and other parameters or levels were modified for the next round. Screening parameters include: 3 buffer types (Acetate, Tris and Phosphate), 5 pH conditions (5, 5.56.5, 7.0, 7.5), various NaCl concentrations, without and with phenol (1.5 mg / mL) or m-cresol (1.7 mg / mL), without and with tonicifiers: glycerin (2 concentrations), mannitol (2 concentrations), sucrose (2 concentrations). Based on the above studies, combination of components within each parameter were tested (e.g., glycerin and mannitol, Tris and Phosphate, phenol and m-cresol, etc.).ABVA.002WO PATENT

[0163] For solubility assessment, 1 or 5 mg of lyophilized glucagon analogs were weighed into Eppendorf tubes and treated with 200-400 µL of the buffers determined from above. Samples were then vortexed and sonicated for 10 min, equilibrated at room temperature for 1 hr, then centrifuged at 10 krpms for 10 min. Concentrations of the peptides in the supernatants were determined by measuring UV absorbance at l = 280 nm. Potential for aggregation into fibrils were assessed by a Thioflavin-T fluorescence assay protocol.

[0164] Best formulation candidates were identified from above studies and were set up for short term stability studies. Stability to agitation / shear (stir model) were conducted by agitation at 100 rpm at ambient condition, then tested at 1d, 2d, 3d, 5d by visual appearance (pictures) and soluble fraction by RP-HPLC, SEC for HMW species. Thermal stress stability (5ºC, 25ºC, 37ºC), in support of injection / vial or pump delivery, was tested at 1d, 3d, 5d, 8d by visual appearance (pictures), SEC for HMW species, purity / degradation and content by RP-HPLC. Example 3: Measurement of functional affinity of isolated glucagon analog peptides with Glucagon receptor

[0165] Peptides were tested in a cAMP cell-based assay to determine their potency with glucagon human receptor. Peptide activation of this receptor results in downstream production of cAMP second messenger which was measured in a functional activity assay. The potency (EC50) of peptides was evaluated using stably transfected CHO-K1 cells overexpressing glucagon receptor (cAMP Hunter™ CHO-K1 GCGR Gs Cell Line, Catalog #: 95-0042C2) from Eurofins DiscoverX (Fremont, CA), and in the presence of casein. It should be noted that the lipidated glucagon analogs do not associate with casein, and hence this protein does not affect the receptor functional activity of the glucagon analogs.

[0166] Cells were seeded in a total volume of 20 µL into white walled, 384-well microplates and incubated at 37°C overnight prior to testing. Prior to testing, cell plating media was exchanged with 10 µL of Assay buffer (HBSS + 10 mM HEPES). All compounds were run in assay buffer containing 0.1% casein. Briefly, intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer. 5 µL of 4X sample was added to cells and incubated at 37°C for 30 minutes. Final assay vehicle concentration was 1%. After appropriate compound incubation, assay signal was generated through incubation with 5ul of Antibody and 20 ul cAMP XS+ED / CL lysis cocktail for one hour followed by incubation with 20ul cAMPABVA.002WO PATENT XS+EA reagent for 2 hours at room temperature. Microplates were read following signal generation with PerkinElmer Envision instrument for chemiluminescent signal detection. Data was normalized to the maximal and minimal response was observed in the presence of control ligand and vehicle. The results as shown in Table 4 below are expressed asEC50 values for functional activity (cAMP production) and maximal responses and are compared with human or native glucagon, the control ligand. Table 4 SEQ ID NO Sequence EC50 (pM) E ID N 1 H TFT DY KYLD RRA DFV WLMNT ti 2 2ABVA.002WO PATENT SEQ ID NO: 40 HSQGTFTSDYSKYLDERRAAibDFVK*WLLDT-OH 15.8 K* or LYS* = L-Lys(AEEAc-AEEAc-L-γ-Glu-ABVA.002WO PATENT SEQ ID NO: 48 HSQGTFTSDYSKYLDAibRRAAEFVQWLMDTK* 6.9 K* or LYS* = L-Lys(AEEAc-AEEAc-L-γ-Glu-Example 4: Measurement of functional affinity of isolated glucagon analog peptides with Glucagon receptor in the presence and absence of albumin

[0167] The functional assays were also performed as above but in the presence of 2% albumin in the assay medium. As the analogs are lipidated, any binding to albumin lowers the effective concentration of free peptide. Thus, an increase in the EC50 values is indicative of albumin binding. The assay results in the presence of albumin is illustrated in Table 5 below. Table 5 SEQ ID NO Sequence EC50 (pM)ABVA.002WO PATENT SEQ ID NO: 35 HSQGTFTSDYSKYLDAibRRAQDFVK*WLLDT-OH 10.781 K* or LYS* = L-Lys(AEEAc-AEEAc-L-γ-Glu-ABVA.002WO PATENT SEQ ID NO: 52 HSQGTFTSDYSKYLDAibRRAAEFVKWLMDTK* 41.118 K* or LYS* = L-Lys-decanoylog peptides

[0168] To evaluate the pharmacodynamic effects of the glucagon analogs, a study was conducted in male Sprague Dawley rats aged 12 to 14 weeks. Animals were fasted at least 3-4 hours prior to dosing. Each analog was administered as a single subcutaneous injection at a dose range of 2nM / kg or 3nM / kg. Control animals were administered dasiglucagon (a well characterized glucagon analog that is equipotent to native human glucagon) at the same concentration as test analogs. Blood samples from the rats were collected at multiple time points post dosing (15, 30, 60, 120, 180 and 240 minutes) and blood glucose concentration was measured using the AlphaTRAK® 3 Blood Glucose Monitoring System. The change in blood glucose concentration (Δ BG) of the analogs from baseline as measured in mg / dl is presented in Table 6 which illustrates that the PD response of the glucagon analogs as measured in mg / dl was right- shifted as compared to the dasiglucagon controls which shows a peak change from baseline at 30 min, is consistent with the PK data provided in the US label for dasiglucagon.ABVA.002WO PATENT Table 6 SEQ ID N SEQ I NO: 3 SEQ I NO: 4 SEQ I NO: 3 SEQ I NO: 4 SEQ I NO: 4 SEQ I NO: 4ABVA.002WO PATENT SEQ I NO: 5 SEQ I NO SEQ I NO: 3 SEQ I NO: 4 SEQ I NO: 3 SEQ I NO: 4 SEQ I NO: 4 SEQ I NO: 4ABVA.002WO PATENT SEQ I NO: 5ABVA.002WO PATENT Example 6: Acute rescue of acute hypoglycemia using isolated glucagon analog peptides

[0169] The ability of the isolated glucagon analog peptides disclosed herein for treating a hypoglycemic patient in an emergency setting would be tested. The isolated glucagon analog peptides would be administered to the patients via systemic subcutaneous administration, intramuscular or intravenous routes at a dosing of 2-60 µg / kg per bolus. The dose range for non- systemic routes of administration which includes oral (includes sub-lingual, buccal space, lingual topical application), intranasal, inhalable, dermal, rectal would be 10-500 µg / kg per bolus. Example 7: Sub-acute hypoglycemia prevention using isolated glucagon analog peptides

[0170] The ability of the isolated glucagon analog peptides disclosed herein for preventing sub-acute hypoglycemia in a subject with high risk of hypoglycemia would be tested. Glucagon analogs disclosed herein would be administered to the subject to allow for a period of short-term hypoglycemic protection. The glucagon analog peptides would be administered to the subject via systemic subcutaneous administration, intramuscular or intravenous routes at a dosing of 5-50 ng / kg per minute for 0-24 hours. The dose range for non-systemic routes of administration which includes oral (includes sub-lingual, buccal space, lingual topical application), intranasal, inhalable, dermal, rectal would be 20-500 ng / kg per minute for up to 24 hours. Example 8: Chronic hypoglycemia prevention using isolated glucagon analog peptides

[0171] The ability of the isolated glucagon analog peptides disclosed herein for preventing chronic hypoglycemia in a subject with high risk of hypoglycemia would be tested. Glucagon analogs disclosed herein would be administered to the subject to allow for a period of chronic hypoglycemic protection. The glucagon analog peptides would be administered to the subject via systemic subcutaneous administration, intramuscular or intravenous routes at a dosing of 5-50 ng / kg per minute. The dose range for non-systemic routes of administration which includes oral (includes sub-lingual, buccal space, lingual topical application), intranasal, inhalable, dermal, rectal would be 20-500 ng / kg per minute.

[0172] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. InABVA.002WO PATENT particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0173] Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise. While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

[0174] The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have”, “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0175] Features, materials, characteristics, or groups described in conjunction with a particular aspect, or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing examples. The protectionABVA.002WO PATENT extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0176] Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.

[0177] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order shown or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described can be incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some examples, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the example, certain of the steps described above may be removed or others may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure.

[0178] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0179] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain examples include, while other examples do not include, certainABVA.002WO PATENT features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular example.

[0180] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0181] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0182] The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

[0183] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the present disclosure, and thus, are not to be limited in scope by the specific embodiments and examples described herein. While the fundamental novel features of the disclosure as applied to various specific embodiments thereof have been shown, described, and pointed out, it will also be understood that various omissions, substitutions, and changes in the details of the compositions and methods that are disclosed, may become apparent and may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those method steps that perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that method steps shown and / or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in anyABVA.002WO PATENT other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations can be made without departing from the spirit or scope of the disclosure as set forth in the following claims both literally and in equivalents recognized in law.

Claims

ABVA.002WO PATENT WHAT IS CLAIMED IS:

1. An isolated peptide comprising a sequence of HX1QGTFTSDYSKYLDX2X3RAX4X5FVX6WLX7X8X9X10 (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein X1 is S or Aib; wherein X2 is Aib, S, E, or A; wherein X3is R, K*, K** or K***; wherein X4 is Aib, Q, A, or E; wherein X5 is D or E; wherein X6is Q, E, K, K*, K** or K***; wherein X7is V, E, I or L; wherein X8 is D, E, Q or N; wherein X9 is T or K, K*, K** or K***; wherein X10is absent, K, K*, K** or K***; wherein K* is L-Lys(AEEAc-AEEAc-L-γ-Glu-(CH2)ndiacid or (L) Lys-decanoyl; wherein AEEAc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid; wherein n is 6, 8, 10, 12, 14, 16, 18, or 20; wherein K** is L-Lys-Xa-(C=O)-(CH2)m-CH3; wherein Xa is absent or (AEEAc-AEEAc); wherein m = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; and wherein K*** is L-Lys-Poly Ethylene Glycol.

2. The isolated peptide of claim 1, further comprising an additional K, K*, K** or K*** on the carboxy-terminal end of the peptide.

3. The isolated peptide of claim 1, wherein the peptide is conjugated to a fatty acid.

4. The isolated peptide of claim 3, wherein the fatty acid is selected from the group consisting of caproic acid (C6), hexanedioic acid (C6 dicarboxylic acid), caprylic acid (C8), octanedioic acid (C8 dicarboxylic acid), capric acid (C10), decanedioic acid (C10 dicarboxylic acid), lauric acid (C12), dodecanedioic acid (C12 dicarboxylic acid), myristic acid (C14), tetradecanedioic acid (C14 dicarboxylic acid), palmitic acid (C16), hexadecanedioic acid (C16 dicarboxylic acid), stearic acid (C18), octadecanedioic acid (C18 dicarboxylic acid), icosanoic acidABVA.002WO PATENT (C20), icosanedioic acid (C20 dicarboxylic acid), docosanoic acid (C22) or docosanedioic acid (C22 dicarboxylic acid).

5. The isolated peptide of claim 3, wherein the peptide is conjugated to poly ethylene glycol (PEG).

6. The isolated peptide of claim 5, wherein the PEG is between 0.2-50 kD in size.

7. The isolated peptide of claim 3, wherein the fatty acid is conjugated to K, K*, K** or K*** residue of the peptide.

8. The isolated peptide of claim 7, wherein K* is L-Lys(AEEAc-AEEAc-L-γ-Glu- (CH2)n diacid or (L) Lys-decanoyl and wherein n is 6, 8, 10, 12, 14, 16, 18, or 20.

9. The isolated peptide of claim 7, wherein K** is K** is L-Lys-Xa-(C=O)-(CH2)m- CH3, wherein Xais absent or (AEEAc-AEEAc) and wherein m is 5, 7, 9, 11, 13, 15, 17.

10. The isolated peptide of claim 7, wherein K*** is L-Lys-PEG and wherein PEG is between 0.2-50 kD in size.

11. The isolated peptide of claim 1, further comprising a carboxy terminal peptide comprising GPSSGAPPPS (SEQ ID NO: 2), GPSSGA (SEQ ID NO: 3), or GPS.

12. The isolated peptide of claim 1, configured for increased specificity and increased potency to a GCG receptor as compared to native glucagon.

13. The isolated peptide of claim 1, wherein the peptide is capable of activating a glucagon receptor.

14. The isolated peptide of claim 1, wherein the isolated peptide has high solubility.

15. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAQDFVK*WLMDT-OH (SEQ ID NO: 3); HSQGTFTSDYSKYLDAibRRAQDFVK*WLLDT-OH (SEQ ID NO: 4); HSQGTFTSDYSKYLDERRAQDFVK*WLLDT-OH (SEQ ID NO: 5); HSQGTFTSDYSKYLDERRAAibDFVK*WLLDT-OH (SEQ ID NO: 6); and HSQGTFTSDYSKYLDARRAAibDFVK*WLMDT-OH (SEQ ID NO: 7).

16. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT-OH (SEQ ID NO: 8); HSQGTFTSDYSKYLDAibK*RAADFVQWLMDT- NH2(SEQ ID NO: 12) andABVA.002WO PATENT HSQGTFTSDYSKYLDEK*RAADFVQWLMDT-OH (SEQ ID NO: 16).

17. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAAEFVQWLMNTK* (SEQ ID NO: 20); HSQGTFTSDYSKYLDAibRRAAEFVQWLMNK* (SEQ ID NO: 21); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDTK* (SEQ ID NO: 22); HSQGTFTSDYSKYLDAibRRAAEFVQWLMDK* (SEQ ID NO: 23); HSQGTFTSDYSKYLDAibRRAAEFVKWLMDTK* (SEQ ID NO: 24); and HSQGTFTSDYSKYLDAibRRAAEFVKWLMDK* (SEQ ID NO: 25).

18. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of: HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGAPPPS-NH2 (SEQ ID NO: 26); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGAPPPS-NH2(SEQ ID NO: 27); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPSSGA-NH2 (SEQ ID NO: 28); HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPSSGA-NH2(SEQ ID NO: 29); HSQGTFTSDYSKYLDAibRRAEDFVK*WLMNTGPS-NH2 (SEQ ID NO: 30); and HSQGTFTSDYSKYLDAibRRAEDFVK*WLLNTGPS-NH2 (SEQ ID NO: 31).

19. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 3-52.

20. The isolated peptide of claim 1, wherein the isolated peptide is selected from the group consisting of any one of SEQ ID NOs: 32-52.

21. The isolated peptide of any one of claims 1-20, wherein the isolated peptide has an increased affinity of binding to albumin.

22. A pharmaceutical composition comprising the isolated peptide of any one of claims 1-21 as an active ingredient.

23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulation for injection.ABVA.002WO PATENT 25. A method of treating a subject suffering from hypoglycemia, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an isolated peptide of any one of claims 1-21.

26. The method of claim 25, wherein the hypoglycemia is acute, sub-acute and / or chronic hypoglycemia.

27. The method of claim 25, wherein the administering is subcutaneous administration.

28. The method of claim 25, wherein the isolated peptide is administered in an amount ranging from 0.001 µg / kg / min to an amount of 1000 µg / kg / min.

29. The method of claim 25, wherein the administering occurs daily or multiple times a day.

30. The method of claim 25, wherein the administering occurs continuously for up to 48 hours.

31. The method of claim 25, wherein the subject has diabetes.

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