NZ619447B2 - Glucagon/glp-1 receptor co-agonists - Google Patents

Abstract

Disclosed is a peptide comprising or consisting of the amino acid sequence of HXQGTFTSDKSKYLDXRAAQDFVQWLMNTKX or a pharmaceutically acceptable salt thereof. Also disclosed is the use of such a peptide in the manufacture of a medicament for the treatment of a disease or medical condition selected from the group consisting of: metabolic syndrome, diabetes, obesity, liver steatosis, and a neurodegenerative disease. from the group consisting of: metabolic syndrome, diabetes, obesity, liver steatosis, and a neurodegenerative disease.

Description

GLUCAGON/GLP-l RECEPTOR CO-AGONISTS CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US. Provisional Application No. 61/500,027, filed June 22, 2011, and US. Provisional Application No. 61/547,360, filed October 14, 2011, each of which are incorporated by reference in their entirety.

INCORPORATION BY NCE OF MATERIAL TED ELECTRONICALLY Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence g submitted rently th and identified as follows: 15 kilobytes ACII (Text) file named “07012KL_PCT_Sequence_Listing.txt,” created on May 25, 2012.

BACKGROUND oglucagon is a 158 amino acid sor polypeptide that is processed in ent tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide-l (GLP-l), glucagon—like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are ed in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. Glucagon is a 29-amino acid peptide that corresponds to amino acids 33 through 61 of pre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 h 108 of pre- proglucagon. GLP-l(7-36) amide or GLP—1(7-37) acid are biologically potent forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor.

During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing blood glucose levels to rise toward a normal level. Hypoglycemia is a common side effect of insulin therapy in patients with hyperglycemia (elevated blood glucose levels) due to diabetes.

Thus, glucagon’s most recognized role in glucose regulation is to counteract the action of insulin and in blood e levels.

GLP-l has different biological ties compared to glucagon. Its actions include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake. GLP—l has been shown to reduce hyperglycemia in diabetics. Exendin-4, a peptide from lizard venom that shares about 50% amino acid identity with GLP-1, activates the GLP-1 or and likewise has been shown to reduce hyperglycemia in diabetics.

There is also evidence that GLP-1 and exendin-4 may reduce food intake and promote weight loss, an effect that would be beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of diabetes, hypertension, hyperlipidemia, cardiovascular disease, and oskeletal diseases.

SUMMARY The present disclosures provide es and variant peptides that exhibit activity at the glucagon receptor, activity at the GLP-1 or, or activity at each of the glucagon receptor and the GLP-1 receptor. In exemplary embodiments, the presently disclosed peptides and variant peptides exhibit enhanced activity at the GLP-1 receptor, as compared to native glucagon. In exemplary s, the peptides and variant peptides exhibit at least 100-fold selectivity for the human GLP-1 receptor versus the GIP receptor.

The present disclosures further provide ates sing any of the peptides and variant peptides described herein conjugated to a heterologous moiety. In ary s, the heterologous moiety is a peptide or protein and the conjugate is a fusion peptide or chimeric e. In exemplary aspects, the heterologous moiety is a polymer, e.g., a polyethylene glycol. The present disclosures furthermore e dimers and multimers comprising any of the peptides and variant peptides described herein.

The present disclosures er es pharmaceutical compositions comprising any of the peptides and variant peptides described herein and a pharmaceutically acceptable carrier, as well as a method of treating or preventing a disease or medical condition (e.g., metabolic syndrome, diabetes, obesity, liver steatosis, a neurodegenerative disease, hypoglycemia) in a patient. The method comprises administering to the patient a presently disclosed peptide or peptide variant, optionally formulated into a pharmaceutical composition, in an amount effective to treat the disease or medical condition.

] In an embodiment the present disclosure provides a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. (11193644_1):GGG [0009b] In an embodiment the present disclosure provides a conjugate sing a peptide of the invention conjugated to a heterologous moiety, or a ceutically acceptable salt thereof, wherein the conjugate exhibits enhanced activity at the GLP-1 receptor, as compared to native glucagon, and exhibits at least 100-fold greater selectivity for the human GLP-1 or versus the GIP receptor. [0009c] In an embodiment the present disclosure provides a dimer or multimer comprising a peptide of the invention, or a pharmaceutically acceptable salt thereof. [0009d] In an embodiment the present disclosure provides a pharmaceutical composition comprising the peptide of the invention, or a pharmaceutically acceptable salt thereof; a conjugate of the invention, or a ceutically acceptable salt thereof; a dimer or multimer of the ion, or a pharmaceutically acceptable salt thereof; or a combination f, and a pharmaceutically acceptable carrier. [0009e] In an embodiment the t disclosure provides use of the peptide of the invention, or a pharmaceutically acceptable salt thereof; a conjugate of the invention, or a pharmaceutically acceptable salt thereof; a dimer or multimer of the invention, or a combination thereof in the manufacture of a ment for the treatment of a e or medical condition selected from the group consisting of: metabolic syndrome, diabetes, obesity, liver steatosis, and a neurodegenerative disease.

BRIEF DESCRIPTION OF THE GS Figure 1 represents a graph of the cumulative body weight change (grams) of DIO mice treated with a vehicle control or a dose of a e of SEQ ID NO: 12, 17, 18, or 19, as detailed in Example 7. (11193644_1):GGG Figure 2 represents a graph of the basal e (mg/d1) of DIO mice treated with a vehicle control or a dose of a peptide of SEQ ID NO: 12, 17, 18, or 19, as detailed in Example 7.

Figure 3A represents a graph of the cumulative body weight change (%) of obese rhesus monkeys treated with a vehicle control, Liraglutide (Lira), or a dose of a peptide of SEQ ID NO: 17 or 20, as detailed in Example 8.

Figure 3B represents a graph of the tive food intake (expressed as a percent of food intake on Day 0) of obese rhesus monkeys treated with a e control, Liraglutide, or a dose of a peptide of SEQ ID NO: 17 or 20, as detailed in Example 8.

Figure 4 ents a graph of blood glucose levels ) of diabetic rhesus monkeys treated with a vehicle control or a dose of a e of SEQ ID NO: 17, as detailed in Example 9.

DETAILED DESCRIPTION DEFINITIONS The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to designate any value or range of values to only this broader tion. Each value or range of values preceded by the term " is also intended to encompass the embodiment of the stated absolute value or range of values.

As used herein, the term “pharmaceutically able carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or waterfoil emulsion, and various types of wetting agents.

The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

As used herein the term "pharmaceutically able salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of g acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts d from c bases include, but are not limited to, salts of primary, secondary and tertiary amines.

As used herein, the term "treating" includes laxis of the ic disorder or condition, or alleviation of the symptoms associated with a specific er or condition and/or preventing or eliminating said symptoms. For example, as used herein the term "treating diabetes" will refer in general to altering glucose blood levels in the direction of normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.

As used herein an "effective" amount or a "therapeutically effective amount" of a glucagon peptide refers to a ic but sufficient amount of the peptide to provide the desired effect. For example one desired effect would be the tion or treatment of hyperglycemia, e.g., as measured by a change in blood glucose level closer to normal, or inducing weight loss/preventing weight gain, e.g., as measured by ion in body weight, or preventing or reducing an increase in body weight, or normalizing body fat distribution. The amount that is "effective" will vary from subject to subject, depending on the age and general ion of the individual, mode of administration, and the like.

Thus, it is not always possible to specify an exact "effective ." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term, "parenteral" means not through the alimentary canal but by some other route, e. g., subcutaneous, intramuscular, intraspinal, or intravenous.

As used herein, the term de" asses a chain of 3 or more amino acids and typically less than 100 amino acids, wherein the amino acids are naturally occurring or coded or non-naturally occurring or non-coded amino acids. Non-naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. “Noncoded” as used herein refers to an amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr. “Coded” as used herein refers to an amino acid that is an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr. In some embodiments, the es and variant peptides described herein are about the same length as SEQ ID NO: 1 (which is 29 amino acids in length), e. g. 25-35 amino acids in length. Exemplary lengths include 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

Typically, polypeptides and proteins have a polymer length that is greater than that of "peptides." Throughout the application, all references to a particular amino acid position by number (e.g.,position 28) refer to the amino acid at that position in native glucagon (SEQ ID NO: 1) or the corresponding amino acid on in any analogs thereof. For example, a reference herein to “position 28” would mean the corresponding position 27 for a on analog in which the first amino acid of SEQ ID NO: 1 has been deleted.

Similarly, a reference herein to “position 28” would mean the corresponding position 29 for a glucagon analog in which one amino acid has been added before the N-terminus of SEQ ID NO: 1. As used herein an “amino acid modification” refers to (i) a tution or replacement of an amino acid of SEQ ID NO: 1 with a ent amino acid (naturally- ing or coded or non-coded or non-naturally—occurring amino acid), (ii) an addition of an amino acid (naturally-occurring or coded or ded or non-naturally-occurring amino acid), to SEQ ID NO: 1 or (iii) a deletion of one or more amino acids of SEQ ID NO: 1.

“Percent identity” with respect to two amino acid ces refers to the number of amino acids of the first sequence that match (are identical to) the amino acids in the second reference sequence, divided by the length of the reference sequence, when the two sequences are aligned to achieve maximum correspondence (e.g. gaps can be introduced for optimal alignment).

Amino acid ication” refers to an insertion, deletion or substitution of one amino acid with another. In some embodiments, the amino acid substitution or replacement is a conservative amino acid substitution, e.g., a conservative substitution of the amino acid at one or more of positions 2, 5, 7,10, 11,12, 13,14,16, 17,18, 19, 20, 21, 24, 27, 28 or 29. As used herein, the term "conservative amino acid substitution" is the replacement of one amino acid with another amino acid having similar properties, e. g.,size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includes exchanges within one of the following five groups: I. Small aliphatic, ar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negative- d residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; 111. Polar, positive- charged residues: His, Arg, Lys; Omithine (Orn) IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine V. Large, aromatic residues: Phe, Tyr, Trp, acetyl phenylalanine In some embodiments, the amino acid substitution is not a conservative amino acid tution, e.g., is a non-conservative amino acid substitution.

As used herein the term "charged amino acid" or “charged residue” refers to an amino acid that comprises a side chain that is negative- d (i.e., de-protonated) or positive- charged (i.e., ated) in aqueous solution at physiological pH. For example negative- charged amino acids include aspartic acid, glutamic acid, cysteic acid, steic acid, and homoglutamic acid, whereas positive- charged amino acids include arginine, lysine and histidine. Charged amino acids include the charged amino acids among the 20 coded amino acids, as well as al or non-naturally occurring or non- coded amino acids.

As used herein the term "acidic amino acid" refers to an amino acid that comprises a second acidic moiety (other than the carboxylic acid of the amino acid), including for example, a carboxylic acid or sulfonic acid group.

As used herein, the term “acylated amino acid” refers to an amino acid comprising an acyl group which is non—native to a naturally-occurring amino acid, regardless of the means by which it is produced (e.g. acylation prior to incorporating the amino acid into a peptide, or acylation after incorporation into a peptide).

As used herein the term “alkylated amino acid” refers to an amino acid comprising an alkyl group which is tive to a naturally—occurring amino acid, regardless of the means by which it is produced. Accordingly, the acylated amino acids and alkylated amino acids of the present sures are non-coded amino acids.

As used herein, the term “selectivity” of a molecule for a first receptor relative to a second receptor refers to the following ratio: EC50 of the molecule at the second receptor d by the EC50 of the le at the first receptor. For example, a molecule that has an EC50 of 1 nM at a first receptor and an EC50 of 100 nM at a second receptor has lOO-fold ivity for the first receptor relative to the second receptor.

As used herein the term “native glucagon” refers to a peptide consisting of the sequence of SEQ ID NO: 1 and the term “native GLP-1” is a generic term that designates GLP-l(7-36) amide, GLP-l(7-37) acid or a mixture of those two compounds.

As used , gon potency” or “potency compared to native glucagon” of a molecule refers to the inverse ratio of the EC50 of the molecule at the glucagon or divided by the EC50 of native glucagon at glucagon or.

As used herein, “GLP-l potency” or “potency compared to native GLP—l” of a molecule refers to the inverse ratio of the EC50 of the molecule at GLP-1 receptor d by the EC50 of native GLP-l at GLP-1 receptor.

EMBODIMENTS The present sures provide peptides and variant peptides that exhibit activity at the GLP-1 receptor, at the glucagon or, or at both the GLP-1 or and the glucagon receptor. In this regard, the present disclosures provide GLP-1 receptor agonist peptides, glucagon receptor agonist peptides, and GLP-l/glucagon receptor co- agonist peptides. In exemplary embodiments, the presently disclosed peptides and variant peptides exhibit enhanced activity or greater potency at the GLP-1 receptor, as compared to native human glucagon (SEQ ID NO: 1). In ary embodiments, the peptides and variant peptides of the present disclosures exhibit greater potency at the GLP-1 receptor as compared to native human GLP-l (SEQ ID NO: 2) or one of the active forms thereof (SEQ ID NOs: 5 and 6). In exemplary embodiments, the peptides and variant peptides t greater potency at the glucagon receptor compared to native human GLP-l. In exemplary embodiments, the peptides and variant peptides exhibit greater potency at the on receptor compared to native human glucagon.

In exemplary embodiments, the peptides and variant peptides described herein exhibit other ements in properties relative to native glucagon or native GLP-l, such as r stability, greater lity, a prolonged ife in circulation, a delayed onset of , an extended duration of action, a dampened peak (e.g.,relative1y decreased mean peak plasma concentration), and an improved resistance to proteases, such as DPP-IV.

The peptides and variant peptides described herein are based on the amino acid sequence of native human glucagon (SEQ ID NO: 1), and are described herein as “peptides”, “variant peptides”,“glucagon analogs”, “analogs”, or “glucagon peptides.” It is understood that terms such as “analog” or “variant” or “modifications” encompass es or proteins synthesized de novo and do not require the performance of any particular modification step. In some aspects, the peptides and variant es described herein comprise a modified amino acid sequence of SEQ ID NO: 1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid modifications relative to SEQ ID NO: 1, and in some instances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), amino acid modifications, as further bed herein. The following ption of glucagon analogs and/or glucagon peptides thus applies to any of the presently disclosed peptides and variant peptides, regardless of the degree of similarity between native human glucagon (SEQ ID NO: 1) and the e or variant peptide of the present disclosures.

It is contemplated that any of the peptide sequences bed herein may be further varied by incorporating additional amino acid modifications; for example, by including any of the modifications described herein, e.g., at the positions described herein, or by orating conservative substitutions, or by returning to the native glucagon amino acid (see SEQ ID NO: 1) at that position. In exemplary ments, the modifications include, e. g., acylation, alkylation, pegylation, truncation at C-terminus, substitution of the amino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29. For example, where any of the peptide sequences disclosed herein includes a Cys for purposes of pegylation, a variant peptide may use a different amino acid for tion. As another example, a variant peptide may be pegylated at a different position (e. g., replacing the existing Cys with a different amino acid, inserting a new Cys at the ed pegylation position, and ting the new Cys). As yet a further example, where any of the peptide sequences disclosed herein includes a Lys for purposes of acylation, the Lys may be moved to a different on and the new position acylated. In any of the embodiments described herein, the variant peptides may be, for example, 80%, 85%, 90% or 95% identical to the parent peptides over the length of the parent peptides or over amino acids 1—29 of the parent peptide (e.g., may incorporate 1, 2, 3, 4, or 5 additional modifications compared to the parent peptide).

Conjugates, fusion ns and multimers of any of the peptide sequences disclosed herein are also contemplated.

ACTIVITY OF THE PEPTIDES AND VARIANT ES Agom‘st Activigv at the Glucagon Receptor In exemplary embodiments, the peptides and variant peptides of the present disclosures exhibit an EC50 at the glucagon receptor of about 1000 uM or less (e. g., about 750 uM or less, about 500 uM or less, about 250 pM or less, about 100 uM or less, about 75 uM or less, about 50 uM or less, about 25 pM or less, about 10 uM or less, about 5 uM or less, or about 1 uM or less). In exemplary embodiments, the peptides and variant peptides exhibit an EC50 for glucagon receptor activation which is in the nanomolar range. For example, the presently sed peptides and variant peptides exhibit an EC50 at the glucagon receptor which is about 1000 nM or less (e. g., about 750 nM or less, about 500 nM or less, about 250 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less). In exemplary embodiments, the peptides and variant peptides exhibit an EC50 at the glucagon receptor which is in the picomolar range. ingly, in exemplary aspects, the peptides and t peptides exhibit an EC50 for glucagon or activation of about 1000 pM or less (e.g., about 750 pM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, or about 1 pM or less). It is understood that a lower EC50 indicates higher activity or potency at the I'CCCptOI'.

In some embodiments, the glucagon analogs described herein exhibit an EC50 at the glucagon receptor that is about 0.001 pM or more, about 0.01 pM or more, or about 0.1 pM or more. Glucagon receptor activation (glucagon receptor activity) can be measured by in vitro assays measuring cAMP induction in HEK293 cells xpressing the on receptor, e.g.,assaying HEK293 cells co-transfected with DNA encoding the glucagon receptor and a luciferase gene linked to cAMP responsive element as described in Example 2.

In exemplary embodiments, the presently disclosed peptides and variant peptides exhibit about 0.001% or more, about 0.01% or more, about 0.1% or more, about 0.5% or more, about 1% or more, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 75% or more, about 100% or more, about 125% or more, about 150% or more, about 175% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 400% or more, about 450% or more, or about 500% or higher ty at the on receptor relative to native glucagon (glucagon potency). In some embodiments, the peptides and variant peptides described herein exhibit about 5000% or less or about 10,000% or less activity at the glucagon receptor relative to native glucagon.

A glucagon analog’s activity at a receptor relative to a native ligand of the receptor is calculated as the inverse ratio of EC50s for the glucagon analog vs. the native ligand.

In some embodiments, the peptides and variant es exhibit substantial activity (potency) at only the glucagon receptor and little to no activity at the GLP-1 receptor. Accordingly, in some embodiments, the peptides and variant peptides are considered as “pure on or agonists” or are not considered as a gon/GLP-l receptor co-agonist.” In some embodiments these peptides and variant peptides exhibit any of the levels of activity or potency at the glucagon or described herein but have substantially less activity (potency) at the GLP-1 receptor. In some embodiments, the glucagon analog exhibits an EC50 at the GLP-1 receptor which is 100- fold or greater than the EC50 at the glucagon receptor. t Activigy at the GLP-I Receptor In exemplary ments, the peptides and variant peptides exhibit an EC50 for GLP-1 receptor activation of about 1000 uM or less (e. g., about 750 uM or less, about 500 uM or less, about 250 uM or less, about 100 uM or less, about 75 uM or less, about 50 uM or less, about 25 uM or less, about 10 ”M or less, about 5 uM or less, or about 1 uM or less). In exemplary embodiments, the peptides and variant peptides exhibit an EC50 at the GLP-1 receptor of about 1000 nM or less (e.g., about 750 nM or less, about 500 nM or less, about 250 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less). In exemplary embodiments, the es and variant peptides has an EC50 at the GLP-1 receptor which is in the picomolar range. ingly, in some embodiments, the peptides and variant peptides exhibit an EC50 for GLP-1 receptor activation of about 1000 pM or less (e.g., about 750 pM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, or about 1 pM or less). It is understood that a lower EC50 indicates higher activity or potency at the receptor.

In ary embodiments, the peptides and variant peptides described herein exhibit an EC50 at the GLP-1 receptor that is about 0.001 pM or more, about 0.01 pM or more, or about 0.1 pM or more. GLP-1 receptor activation (GLP-1 receptor activity) can be measured by in vitro assays measuring CAMP induction in HEK293 cells over- expressing the GLP-1 receptor, e. g.,assaying HEK293 cells co-transfected with DNA encoding the GLP-1 receptor and a luciferase gene linked to CAMP sive element as described in Example 2.

In some embodiments, the peptides and t peptides of the present disclosures exhibit about 0.001% or more, about 0.01% or more, about 0.1% or more, about 0.5% or more, about 1% or more, about 5% or more, about 10% or more, about % or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 75% or more, about 100% or more, about 125% or more, about 150% or more, about 175% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 400% or more, about 450% or more, or about 500% or higher activity at the GLP-1 or relative to native GLP-l (GLP-l potency). In some embodiments, the peptides and t peptides described herein exhibit about 5000% or less or about 10,000% or less activity at the GLP—1 receptor relative to native GLP-l (GLP-l potency).

In some embodiments, the peptides and variant peptides t substantial activity (potency) at only the GLP-1 receptor and little to no activity at the glucagon receptor. In some embodiments, the peptides and variant peptides are considered as “pure GLP-1 receptor agonists” or are not considered as “glucagon/GLP-l receptor co- agonists.” In some embodiments these peptides and variant peptides exhibit any of the levels of activity or potency at the GLP-1 receptor described herein but have substantially less activity (potency) at the glucagon receptor. In some embodiments, the peptides and variant peptides exhibit an EC50 at the on receptor which is 100-fold or greater than the EC50 at the GLP-1 receptor.

Agonist Activity at the GLP-I Receptor and the Glucagon or In exemplary embodiments, the peptides and variant peptides exhibit ty at both the GLP-1 receptor and glucagon receptor and may be considered as “glucagon/GLP-l receptor co-agonists”. In exemplary embodiments, the activity (e. g., the EC50 or the ve activity or potency) of the peptides and variant peptides at the glucagon receptor is within about 50-fold, about 40-fold, about 30-fold, about 20-fold, about 10-fold, or about 5 fold different (higher or lower) from its ty (e. g., the EC50 or the relative activity or potency) at the GLP-1 receptor. In exemplary aspects, the glucagon potency of the peptide or t peptide is within about 25-, about 20-, about -, about 10-, or about 5-fold different (higher or lower) from its GLP-l potency. In exemplary s, the glucagon potency of the peptide or variant peptide is within about -, about 20-, about 15-, about 10-, or about 5-fold lower from its GLP-l potency.

In exemplary ments, the co—agonist is approximately equipotent or relatively more potent at the GLP-1 receptor than the glucagon receptor. For example, the ratio of the relative activity or the EC50 or the potency of the e or t peptide at the glucagon receptor divided by the relative activity or the EC50 or potency of the peptide or variant peptide at the GLP—1 receptor is less than, or is about, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In exemplary embodiments, the ratio of the EC50 or potency or relative activity of the peptide or variant peptide at the glucagon or divided by the EC50 or potency or relative activity of the peptide or variant peptide at the GLP-1 receptor is about 1 and less than 5 (e. g., about 4, about 3, about 2, about 1). In exemplary embodiments, the ratio of the EC50 or potency or relative activity of the e or variant peptide at the GLP—1 receptor divided by the EC50 or potency or ve activity of the peptide or variant peptide at the glucagon or is less than 5 (e. g., about 4, about 3, about 2, about 1). In exemplary embodiments, the ratio of the glucagon potency of the peptide or variant peptide compared to the GLP-l y of the e or variant peptide is less than, or is about, Y, wherein Y is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In exemplary embodiments, the ratio of the glucagon potency of the peptide or variant e compared to the GLP-l potency of the peptide or variant peptide is less than 5 (e. g., about 4, about 3, about 2, about 1). In some embodiments, the glucagon analog has an EC50 at the glucagon receptor which is 2- to 10—fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold) greater than the EC50 at the GLP-1 receptor.

In exemplary embodiments, the peptide is primarily a glucagon agonist and is vely more potent at the glucagon receptor than the GLP—1 receptor (e. g. the e is 5 times or more potent at the glucagon receptor compared to the GLP—1 receptor). For example, the ratio of the relative activity or y or the EC50 of the peptide or variant peptide at the GLP-1 receptor divided by the relative activity or potency or the EC50 of the peptide or variant peptide at the glucagon receptor is less than, or is about, V, wherein V is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the GLP-l potency of the peptide or variant peptide compared to the glucagon y of the peptide or variant peptide is less than, or is about, W, wherein W is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In some embodiments, the peptide or variant peptide exhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more, about 5% or more, about 10% or more, or more) of the activity of native GLP-l at the GLP-1 receptor (GLP-l potency) and exhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more, about 5% or more, about 10% or more, or more) of the activity of native glucagon at the glucagon or (glucagon potency).

Activifl at the GIP Receptor In on to being active at the on receptor and/or the GLP-1 receptor, the peptides and variant peptides described herein, in some aspects, exhibit low agonist activity at the GIP receptor. In such aspects, preferably such peptides and variant peptides are at least lOO-fold selective for the GLP-1 receptor relative to the GIP receptor.

In other aspects, r, the peptide or variant peptide exhibits appreciable activity at the GIP receptor, e.g. the EC50 of the analog at the GIP receptor is less than about 50-fold different from its EC50 at the GLP—1 or, optionally, wherein the GIP potency of the analog is within about 50—fold of the GLP—l potency of the analog. In exemplary embodiments, the peptides t an EC50 for GIP receptor activation ty of about 1 uM or less, or 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less. It is understood that a lower EC50 indicates higher activity or potency at the receptor. In some embodiments, the peptides and variant peptides described herein exhibit an EC50 at the GIP receptor that is about 0.001 nM, 0.01 nM, or 0.1 nM. In some embodiments, the peptides and variant peptides described herein exhibit an EC50 at the GIP receptor that is no more than about 100 nM. or activation can be ed by in vitro assays measuring CAMP induction in HEK293 cells over-expressing the receptor, e. g. assaying HEK293 cells co-transfected with DNA encoding the receptor and a luciferase gene linked to CAMP responsive element as described in Example 2.

In some embodiments, the presently disclosed peptides and variant peptides exhibit at least about 0.1%, 1%, 10%, 50%, 100%, 150%, or 200% or higher activity at the GIP receptor relative to native GIP (GIP potency). In some ments, the peptides and variant peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GIP receptor relative to native GIP. A glucagon peptide’s activity (potency) at a receptor relative to a native ligand of the receptor is calculated as the inverse ratio of ECSOS for the e vs. the native ligand.

Thus, one aspect of the present disclosures es peptides and variant peptides that exhibit activity at both the glucagon or and the GIP receptor (“glucagon/GIP nists”). In some embodiments, the EC50 of the peptide at the GIP receptor is less than about 50-fold, 40-fold, 30—fold or 20-fold different (higher or lower) from its EC50 at the glucagon receptor. In some embodiments, the GIP potency of the peptide is less than about 500-, 450-, 400—, 350—, 300—, 250-, 200-, 150-, 100-, 75-, 50-, -, 20-, 15-, lO-, or 5-fold different (higher or lower) from its glucagon y. In some embodiments, GLP-l activity has been significantly reduced or destroyed, e. g., by an amino acid modification at position 7, a deletion of the amino acid(s) C-terminal to the amino acid at position 27 or 28, or a combination thereof.

In alternative aspects of the present disclosures, the peptides and variant peptides of the present disclosures exhibit activity at the GLP-1 and GIP receptors, but do not exhibit icant activity at the glucagon receptor GLP-l co-agonists”), e. g., due to an amino acid modification of Gln at position 3. For example, substitution at this position with an acidic, basic, or a hydrophobic amino acid (glutamic acid, ine, cine) reduces glucagon activity. In other aspects, the peptides and variant es exhibit activity at each of the glucagon, GIP and GLP—1 receptors (“glucagon/GlP/GLP-l tri-agonists”). For example, in either of these latter aspects, the EC50 of the peptide at the GIP receptor is less than about 50-fold, 40—fold, 30—fold or 20-fold different (higher or lower) from its EC50 at the GLP-1 receptor. In some embodiments, the GIP potency of the peptide is less than about 25-, 20-, 15-, 10-, or 5—fold different (higher or lower) from its GLP-l potency. In some embodiments these peptides have about 10% or less of the activity of native glucagon at the glucagon receptor, e.g. about l-lO%, or about 0.1-10%, or greater than about 0.1% but less than about 10%.

Activigy 0t Conjugates In some embodiments, the peptides and variant peptides described herein exhibit activity or potency at the glucagon receptor and/or activity at the GLP-1 or and/or activity at the GIP or, as bed above and, when the peptide or t peptide is part of a ate (e. g., is conjugated to a heterologous moiety, e.g., a hydrophilic moiety, e.g., a polyethylene glycol), the peptide or variant peptide exhibits an activity that is lower (i.e. lower potency or higher EC50) than when the peptide or variant peptide is not part of the conjugate. In some aspects, the peptide or variant peptide when not part of ate exhibits a potency at the glucagon receptor and/or the GLP-1 receptor that is about lO-fold or greater than the potency of the peptide or variant peptide when part of a conjugate. In some s, the peptide or variant peptide when unconjugated exhibits an y at the glucagon receptor and/or GLP-1 receptor that is about lO-fold, about 15-fold, about 20-fold, about 25—fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 100-fold, or even greater-fold the potency of the peptide or variant peptide when conjugated.

STRUCTURE OF THE GLUCAGONANALOGS Acylation In ance with some embodiments, the glucagon analog ses an acylated amino acid (e. g., a non-coded acylated amino acid (e. g., an amino acid comprising an acyl group which is non-native to a naturally-occurring amino acid)). The acylated amino acid in some embodiments causes the glucagon analog to have one or more of (i) a ged half-life in circulation, (ii) a delayed onset of action, (iii) an extended on of action, (iv) an improved resistance to proteases, such as DPP-IV, and (v) increased potency at one or both of the GLP—1 and glucagon receptors. As shown herein, acylated glucagon analogs do not exhibit decreased activity at the glucagon and GLP-1 receptors in ison to the corresponding unacylated glucagon .

Rather, in some instances, acylated glucagon analogs actually exhibit increased activity at the GLP-1 and glucagon receptors. Accordingly, the potency of the acylated glucagon analogs is comparable to the unacylated versions of the on s, if not enhanced.

In accordance with one embodiment, the glucagon analog comprises an acyl group which is attached to the glucagon analog via an ester, thioester, or amide linkage for purposes of prolonging half-life in circulation and/or delaying the onset of and/or extending the duration of action and/or improving ance to proteases such as DPP-IV.

Acylation can be carried out at any position within the glucagon analog, including any of ons 1-29, a position C—terminal to the 29Lh amino acid (e. g., the amino acid at position 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, etc., at a position within a C-terminal extension or at the C—terminus), provided that glucagon and/or GLP-l activity is retained, if not enhanced. Nonlimiting examples e positions 5,7,10, 11, 12, 13, 14,16, 17,18, 19, 20, 21, 24, 27, 28, or 29. In exemplary embodiments, the glucagon analog comprises an ed amino acid at one or more positions selected from the group consisting of: 9, 10, 12, 16, and 20. In exemplary ments, the on analog comprises an acylated amino acid at one or more positions selected from the group consisting of: 10, 12, and 16. In exemplary embodiments, the glucagon analog comprises an acylated amino acid at one or more positions selected from the group consisting of: 9, 10, 12, 16, and 20. In exemplary embodiments, the glucagon analog comprises an acylated amino acid at one or more positions 10 and 12. In exemplary embodiments, the glucagon analog comprises an ed amino acid at position 12. In exemplary embodiments, the glucagon analog comprises a C-terminal extension and an ed amino acid at one or more positions ed from the group consisting of 9, 10, 12, 16, 20, and 37—43 (e. g., 40). In specific embodiments, acylation occurs at position 10 of the glucagon analog and the on analog lacks an intramolecular bridge, e.g., a covalent intramolecular bridge (e. g., a lactam bridge). Such acylated glucagon analogs lacking an olecular bridge demonstrate enhanced activity at the GLP-1 and glucagon receptors as ed to the corresponding non-acylated analogs lacking a covalent intramolecular bridge and in comparison to the corresponding analogs lacking an olecular bridge acylated at a position other than position 10. As shown herein, ion at on 10 can even transform a glucagon analog having little activity at the glucagon receptor to a glucagon analog having activity at both the glucagon and GLP—1 receptors. Accordingly, the position at which acylation occurs can alter the overall activity profile of the glucagon analog.

The glucagon analog in some embodiments are acylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.

Nonlimiting examples include acylation at position 10 and pegylation at one or more positions in the C-terminal portion of the glucagon analog, e. g., position 24, 28 or 29, within a C-terminal extension, or at the C—terminus (e. g., through adding a C-terminal Cys).

The acyl group can be covalently linked directly to an amino acid of the on analog, or indirectly to an amino acid of the glucagon analog via a spacer, wherein the spacer is positioned between the amino acid of the glucagon analog and the acyl group.

In specific s, the glucagon analog is modified to comprise an acyl group by direct acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the glucagon analog. In some embodiments, acylation is at on 10, 20, 24, or 29 of the glucagon analog. In this regard, the acylated on analog can comprise the amino acid sequence of SEQ ID NO: 1, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 of the analog modified to any amino acid comprising a side chain amine, hydroxyl, or thiol. In some specific embodiments, the direct acylation of the glucagon analog occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position 10.

In some embodiments, the amino acid sing a side chain amine is an amino acid of Formula I: H2N—C—COOH (CH2)n wherein n = 1 to 4 [Formula I] In some exemplary ments, the amino acid of a I, is the amino acid wherein n is 4 (Lys) or n is 3 (Cm).

In other embodiments, the amino acid comprising a side chain hydroxyl is an amino acid of Formula II: HZN—C—COOH (CH2)n wherein n = 1 to 4 [Formula II] In some exemplary embodiments, the amino acid of Formula II is the amino acid wherein n is 1 (Ser).

In yet other embodiments, the amino acid sing a side chain thiol is an amino acid of Formula III: H2N—C—COOH (CH2)n wherein n = 1 to 4 [Formula III] In some exemplary embodiments, the amino acid of Formula III is the amino acid wherein n is l (Cys).

In yet other embodiments, the amino acid comprising a side chain amine, hydroxyl, or thiol is a disubstituted amino acid comprising the same structure of Formula I, Formula II, or Formula III, except that the hydrogen bonded to the alpha carbon of the amino acid of Formula I, Formula II, or Formula III is replaced with a second side chain.

In some ments, the acylated glucagon comprises a spacer between the analog and the acyl group. In some embodiments, the glucagon analog is covalently bound to the spacer, which is covalently bound to the acyl group.

In some embodiments, the spacer is an amino acid comprising a side chain amine, yl, or thiol, or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol. The amino acid to which the spacer is attached can be any amino acid (e. g., a singly or doubly a-substituted amino acid) comprising a moiety which permits linkage to the spacer. For example, an amino acid comprising a side chain NHZ, —OH, or —COOH (e.g., Lys, Om, Ser, Asp, or Glu) is suitable. In this respect, the acylated glucagon analog can comprise the amino acid sequence of SEQ ID NO: 1, or a ed amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 ed to any amino acid comprising a side chain amine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid sing a side chain amine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol.

When acylation occurs through an amine group of a spacer, the acylation can occur through the alpha amine of the amino acid or a side chain amine. In the instance in which the alpha amine is acylated, the amino acid of the spacer can be any amino acid.

For e, the amino acid of the spacer can be a hydrophobic amino acid, e. g., Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr, 6-amino ic acid, 5-aminovaleric acid, 7- aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, the amino acid of the spacer can be an acidic residue, e. g., Asp, Glu, homoglutamic acid, homocysteic acid, cysteic acid, gamma-glutamic acid.

In the instance in which the side chain amine of the amino acid of the spacer is acylated, the amino acid of the spacer is an amino acid sing a side chain amine, e. g., an amino acid of Formula I (e.g., Lys or Orn). In this instance, it is possible for both the alpha amine and the side chain amine of the amino acid of the spacer to be acylated, such that the glucagon analog is diacylated. Embodiments of the invention e such diacylated molecules.

When acylation occurs through a hydroxyl group of a spacer, the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula II.

In a specific exemplary embodiment, the amino acid is Ser.

When acylation occurs through a thiol group of a spacer, the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula III. In a specific exemplary embodiment, the amino acid is Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. In certain embodiments, the hydrophilic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations f. In certain embodiments, the hydrophilic bifunctional spacer comprises a hydroxyl group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer ses a thiol group and a carboxylate. In a specific embodiment, the spacer comprises an amino poly(alkyloxy)carboxylate. In this regard, the spacer can comprise, for example, NH2(CH2CHZO)D(CH2)mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12, such as, e. g., o-3,6- dioxaoctanoic acid, which is commercially available from Peptides International, Inc. ville, KY).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.

Hydrophobic tional spacers are known in the art. See, e. g., Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated by reference in its ty. In certain embodiments, the hydrophobic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof. In n embodiments, the hydrophobic bifunctional spacer comprises a yl group and a ylate. In other embodiments, the hydrophobic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydrophobic bifunctional spacer comprises a thiol group and a carboxylate. Suitable hydrophobic bifunctional spacers comprising a carboxylate and a hydroxyl group or a thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and aptooctanoic acid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acid comprising an ched, methylene of 1-7 carbon atoms between the ylate groups. In some ments, the bifunctional spacer is a dicarboxylic acid comprising an unbranched, methylene of l-7 carbon atoms between the ylate groups.

The spacer (e.g., amino acid, ide, tripeptide, hydrophilic tional spacer, or hydrophobic bifunctional spacer) in specific embodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e. g., 6, 7, 8, 9, or 10 atoms) in length. In more specific embodiments, the spacer is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12 to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acyl group, such that the total length of the spacer and acyl group is 14 to 28 atoms, e.g., about 14, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms. In some embodiments, the length of the spacer and acyl group is 1? to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctional spacer can be a synthetic or naturally occurring amino acid (including, but not limited to, any of those described herein) comprising an amino acid backbone that is 3 to 10 atoms in length (e. g., 6-amino hexanoic acid, 5-aminovalen'c acid, 7—aminoheptanoic acid, and 8- aminooctanoic acid). Alternatively, the spacer can be a dipeptide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length. Each amino acid of the ide or tripeptide spacer can be the same as or different from the other amino acid(s) of the dipeptide or tripeptide and can be independently selected from the group ting of: naturally-occurring or coded and/or non-coded or non-naturally occurring amino acids, including, for example, any of the D or L isomers of the naturally- occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or L isomers of the non-naturally occurring or non-coded amino acids selected from the group consisting of: ine (B-Ala), N-oc- methyl-alanine (Me-Ala), aminobutyric acid (Abu), y—aminobutyric acid ), exanoic acid (s-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran carboxylic acid, ne N-methoxy-N-methyl amide, fi-aspartic acid (B-Asp), azetidine carboxylic acid, enzothiazolyl)alanine, oc-tert-butylglycine, 2—aminoureido-n- valeric acid (citrulline, Cit), B-Cyclohexylalanine (Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab), diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA), dimethylthiazolidine (DMTA), y—Glutamic acid (y—Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide, methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine (MeLeu), methyl-lysine, yl-lysine, trimethyl-lysine, methanoproline, methionine—sulfoxide )), methionine-sulfone (Met(02)), norleucine (Nle), methyl—norleucine (Me—Nle), ine (Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4—Cl)), 4-fluorophenylalanine (Phe(4-F)), 4- nitrophenylalanine (Phe(4-N02)), 4-cyanophenylalanine ((Phe(4-CN)), glycine (Phg), piperidinylalanine, piperidinylglycine, 3,4-dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), O-Benzyl-phosphoserine, 4-aminohydroxy methylheptanoic acid (Sta), ocyclohexyl—3—hydroxypentanoic acid (ACHPA), 4- aminohydroxyphenylpentanoic acid (AHPPA), 1,2,3,4,—tetrahydro-isoquinoline carboxylic acid (Tic), tetrahydropyranglycine, thienylalanine (Thi) , O—benzyl- phosphotyrosine, O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine, O-(bis- ylamino-phosphono)-tyrosine, tyrosine e tetrabutylamine, methyl-valine (MeVal), and alkylated 3-mercaptopropionic acid.

In some embodiments, the spacer comprises an overall negative charge, e. g., ses one or two negative- charged amino acids. In some embodiments, the ide is not any of the dipeptides of general structure A—B, wherein A is selected from the group consisting of Gly, Gln, Ala, Arg, Asp, Asn, lle, Leu, Val, Phe, and Pro, wherein B is selected from the group ting of Lys, His, Trp. In some ments, the dipeptide spacer is selected from the group consisting of: Ala-Ala, B-Ala- B-Ala, Leu- Leu, Pro-Pro, y-aminobutyric acid- y—aminobutyric acid, Glu-Glu, and y-Glu- y-Glu.

In some ary embodiments, the glucagon analog is modified to comprise an acyl group by ion of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position 10, 20, 24, or 29, or at the C-terminal amino acid of the glucagon analog.

In yet more specific embodiments, the acyl group is attached to the amino acid at position 10 of the glucagon analog and the length of the spacer and acyl group is 14 to 28 atoms. The amino acid at position 10, in some aspects, is an amino acid of Formula I, e. g., Lys, or a disubstituted amino acid related to Formula I. In more specific ments, the glucagon analog lacks an intramolecular bridge, e. g., a covalent intramolecular bridge. The glucagon , for example, can be a glucagon analog comprising one or more alpha, alpha-disubstituted amino acids, e.g., AIB, for stabilizing the alpha helix of the analog.

Suitable methods of peptide acylation via amines, hydroxyls, and thiols are known in the art. See, for example, Example 19 (for methods of acylating through an , Miller, Biochem Biophys Res Commun 218: 371382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19: 54—62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972) (for methods of acylating through a hydroxyl); and San and s, J Pept Res 66: 169-180 (2005) (for methods of acylating through a thiol); Bioconjugate Chem. "Chemical Modifications of Proteins: History and ations" pages 1, 2-12 (1990); Hashimoto et al., Pharmacuetical Res. "Synthesis of oyl Derivatives of Insulin and their Biological Activity" Vol. 6, No: 2 pp.l7l-l76 (1989).

The acyl group of the acylated amino acid can be of any size, e. g., any length carbon chain, and can be linear or branched. In some specific embodiments, the acyl group is a C4 to C30 fatty acid. For example, the acyl group can be any of a C4 fatty acid, C6 fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid. In some embodiments, the acyl group is a C8 to C20 fatty acid, e.g., a C14 fatty acid or a C16 fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bile acid can be any suitable bile acid, including, but not limited to, cholic acid, eoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, holic acid, and cholesterol acid.

In some embodiments, the glucagon analog comprises an ed amino acid by acylation of a long chain alkane by the glucagon analog. In specific aspects, the long chain alkane comprises an amine, hydroxyl, or thiol group (e.g.,octadecylamine, tetradecanol, and hexadecanethiol) which reacts with a carboxyl group, or activated form thereof, of the glucagon analog. The carboxyl group, or activated form thereof, of the glucagon analog can be part of a side chain of an amino acid (e.g., glutamic acid, aspartic acid) of the glucagon analog or can be part of the analog backbone.

In certain embodiments, the glucagon analog is modified to comprise an acyl group by acylation of the long chain alkane by a spacer which is attached to the glucagon analog. In ic aspects, the long chain alkane comprises an amine, hydroxyl, or thiol group which reacts with a carboxyl group, or activated form thereof, of the spacer. le spacers comprising a carboxyl group, or activated form thereof, are described herein and include, for example, bifunctional spacers, e.g., amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers and hydrophobic tional spacers.

As used herein, the term “activated form of a yl group” refers to a carboxyl group with the general a R(C=O)X, wherein X is a leaving group and R is the glucagon analog or the spacer. For example, activated forms of a carboxyl groups may include, but are not limited to, acyl chlorides, ides, and esters. In some embodiments, the activated carboxyl group is an ester with a N-hydroxysuccinimide ester (NHS) leaving group.

With regard to these aspects, in which a long chain alkane is acylated by the glucagon analog or the spacer, the long chain alkane may be of any size and can comprise any length of carbon chain. The long chain alkane can be linear or branched. In certain aspects, the long chain alkane is a C4 to C30 alkane. For example, the long chain alkane can be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane, C14 alkane, C16 , C18 alkane, C20 alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane. In some embodiments, the long chain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane, C16 , or a C18 alkane.

Also, in some ments, an amine, hydroxyl, or thiol group of the glucagon analog is acylated with a terol acid. In a specific embodiment, the glucagon analog is linked to the cholesterol acid h an alkylated des—amino Cys spacer, i.e., an alkylated 3-mercaptopropionic acid spacer. The alkylated des-amino Cys spacer can be, for example, a des-amino-Cys spacer comprising a dodecaethylene glycol moiety. In one embodiment, the glucagon analog comprises the structure: ,N SJ1 H \H/V EMOWO H The acylated glucagon analogs described herein can be further modified to comprise a hydrophilic moiety. In some specific embodiments the hydrophilic moiety can comprise a polyethylene glycol (PEG) chain. The incorporation of a hydrophilic moiety can be accomplished through any le means, such as any of the methods described herein. In this regard, the acylated glucagon analog can comprise SEQ ID NO: 1, including any of the modifications described herein, in which at least one of the amino acids at position 10, 20, 24, and 29 of the analog comprises an acyl group and at least one of the amino acids at position 16, 17, 21, 24, or 29, a position within a inal ion, or the C-terminal amino acid are modified to a Cys, Lys, Om, homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalently bonded to a hydrophilic moiety (e. g., PEG). In some embodiments, the acyl group is ed to position 10, optionally via a spacer comprising Cys, Lys, Orn, homo—Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cys residue at position 24.

Alternatively, the acylated on analog can comprise a spacer, n the spacer is both acylated and ed to comprise the hydrophilic moiety. Nonlimiting examples of suitable spacers include a spacer comprising one or more amino acids selected from the group consisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

Allfllation In accordance with some embodiments, the on analog comprises an alkylated amino acid (e. g., a non-coded alkylated amino acid (e.g., an amino acid comprising an alkyl group which is non-native to a naturally-occurring amino acid».

Without being held to any particular theory, it is believed that alkylation of on analogs achieve r, if not the same, effects as acylation of the glucagon analogs, e. g., a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved ance to proteases, such as DPP—IV, and increased potency at the GLP-1 and glucagon receptors.

Alkylation can be carried out at any positions within the glucagon analog, including any of the positions described herein as a site for acylation, including but not limited to, any of amino acid positions 1-29, an amino acid position C-terminal to the 29Lh residue, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, etc., at a on within a C-terminal extension, or at the C-terminus, provided that the glucagon activity or GLP-l activity is retained. Nonlimiting examples include positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, or 29. In exemplary embodiments, the glucagon analog ses an alkylated amino acid at one or more positions selected from the group consisting of: 9, 10, 12, 16, and 20. In exemplary embodiments, the glucagon analog comprises an alkylated amino acid at one or more positions selected from the group consisting of: 10, 12, and 16. In exemplary embodiments, the glucagon analog comprises an alkylated amino acid at one or more positions selected from the group consisting of: 9, 10, 12, 16, and 20. In exemplary ments, the glucagon analog comprises an alkylated amino acid at one or more positions 10 and 12. In exemplary embodiments, the glucagon analog ses an alkylated amino acid at position 12. In exemplary embodiments, the on analog ses a C-terminal ion and an alkylated amino acid at one or more positions selected from the group consisting of 9, 10, 12, 16, 20, and 37—43 (e.g., 40). The alkyl group can be covalently linked directly to an amino acid of the glucagon analog, or indirectly to an amino acid of the glucagon analog Via a spacer, wherein the spacer is oned between the amino acid of the glucagon analog and the alkyl group. on analog may be alkylated at the same amino acid on where a hydrophilic moiety is linked, or at a different amino acid position. iting examples include alkylation at position 10 and pegylation at one or more ons in the C-terminal portion of the glucagon analog, e. g., position 24, 28 or 29, within a C-terminal extension, or at the C-terminus (e.g., through adding a C- terminal Cys).

In specific aspects, the glucagon analog is modified to comprise an alkyl group by direct alkylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the glucagon analog. In some embodiments, alkylation is at position 10, 20, 24, or 29 of the glucagon analog. In this regard, the alkylated glucagon analog can comprise the amino acid sequence of SEQ ID NO : 2, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol. In some specific ments, the direct tion of the glucagon analog occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position 10.

In some embodiments, the amino acid comprising a side chain amine is an amino acid of Formula I. In some exemplary embodiments, the amino acid of Formula I, is the amino acid wherein n is 4 (Lys) or n is 3 (Cm).

In other embodiments, the amino acid comprising a side chain hydroxyl is an amino acid of Formula II. In some exemplary embodiments, the amino acid of a II is the amino acid wherein n is 1 (Ser).

In yet other ments, the amino acid comprising a side chain thiol is an amino acid of Formula III. In some exemplary embodiments, the amino acid of Formula III is the amino acid wherein n is l (Cys).

In yet other embodiments, the amino acid comprising a side chain amine, hydroxyl, or thiol is a disubstituted amino acid comprising the same structure of Formula I, Formula II, or Formula III, except that the hydrogen bonded to the alpha carbon of the amino acid of a I, Formula II, or Formula III is replaced with a second side chain.

In some embodiments, the alkylated glucagon analog comprises a spacer between the analog and the alkyl group. In some embodiments, the glucagon analog is covalently bound to the spacer, which is covalently bound to the alkyl group. In some exemplary embodiments, the on analog is modified to comprise an alkyl group by alkylation of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position 10, 20, 24, or 29 of the glucagon analog. The amino acid to which the spacer is attached can be any amino acid comprising a moiety which permits linkage to the . For example, an amino acid comprising a side chain NHZ, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) is suitable. In this respect, the alkylated glucagon analog can comprise a modified amino acid sequence of SEQ ID NO: 1, comprising one or more of the amino acid modifications described herein, with at least one of the amino acids at positions 10, 20, 24, and 29 modified to any amino acid comprising a side chain amine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid sing a side chain amine, hydroxyl, or thiol or a ide or tripeptide comprising an amino acid comprising a side chain amine, yl, or thiol.

When tion occurs through an amine group of a spacer, the alkylation can occur through the alpha amine of an amino acid or a side chain amine. In the instance in which the alpha amine is alkylated, the amino acid of the spacer can be any amino acid.

For example, the amino acid of the spacer can be a hydrophobic amino acid, e. g., Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid, 7- aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, the amino acid of the spacer can be an acidic residue, e. g., Asp and Glu, provided that the alkylation occurs on the alpha amine of the acidic residue. In the instance in which the side chain amine of the amino acid of the spacer is ted, the amino acid of the spacer is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Om). In this instance, it is possible for both the alpha amine and the side chain amine of the amino acid of the spacer to be alkylated, such that the glucagon analog is dialkylated.

Embodiments of the invention e such dialkylated molecules.

] When alkylation occurs through a hydroxyl group of a spacer, the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula II. In a specific exemplary embodiment, the amino acid is Ser.

When alkylation occurs through a thiol group of spacer, the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula III. In a specific exemplary embodiment, the amino acid is Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. In n embodiments, the hydrophilic bifunctional spacer comprises two or more reactive groups, e. g., an amine, a yl, a thiol, and a carboxyl group or any combinations thereof. In certain embodiments, the hydrophilic bifunctional spacer is comprises a hydroxyl group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer ses a thiol group and a carboxylate. In a specific embodiment, the spacer comprises an amino poly(alkyloxy)carboxylate. In this regard, the spacer can comprise, for example, NH2(CH2CHZO)n(CH2)mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12, such as, e.g., 8—amino—3,6- dioxaoctanoic acid, which is commercially available from es International, Inc.

(Louisville, KY).

In some embodiments, the spacer is a hydrophobic bifunctional spacer. In certain embodiments, the hydrophobic bifunctional spacer comprises two or more reactive groups, e. g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof. In certain embodiments, the hydrophobic bifunctional spacer comprises a hydroxyl group and a carboxylate. In other embodiments, the hydropholic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydropholic tional spacer ses a thiol group and a ylate. Suitable hydrophobic bifunctional spacers comprising a carboxylate and a hydroxyl group or a thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and 8- mercaptooctanoic acid.

The spacer (e. g., amino acid, dipeptide, tripeptide, hydrophilic bifunctional , or hydrophobic bifunctional spacer) in specific embodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e. g., 6, 7, 8, 9, or 10 atoms)) in length. In more specific embodiments, the spacer is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C12 to C18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, such that the total length of the spacer and alkyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms. In some embodiments, the length of the spacer and alkyl is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In ance with certain foregoing embodiments, the bifunctional spacer can be a synthetic or non-naturally occurring or ded amino acid comprising an amino acid backbone that is 3 to 10 atoms in length (e.g., o hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid). Alternatively, the spacer can be a ide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e. g., 6 to atoms) in . The dipeptide or tripeptide spacer can be composed of naturally- ing or coded and/or non-coded or non—naturally occurring amino acids, including, for example, any of the amino acids taught . In some embodiments, the spacer comprises an overall negative charge, e.g., comprises one or two negative- charged amino acids. In some embodiments, the dipeptide spacer is selected from the group consisting of: Ala-Ala, B-Ala- B-Ala, Leu-Leu, Pro-Pro, y—aminobutyric acid- y-aminobutyric acid, and y-Glu- y-Glu.

] Suitable methods of peptide alkylation via amines, hydroxyls, and thiols are known in the art. For example, a Williamson ether synthesis can be used to form an ether linkage n a yl group of the glucagon analog and the alkyl group. Also, a nucleophilic substitution reaction of the peptide with an alkyl halide can result in any of an ether, thioether, or amino linkage.

The alkyl group of the alkylated glucagon analog can be of any size, e. g., any length carbon chain, and can be linear or branched. In some embodiments, the alkyl group is a C4 to C30 alkyl. For example, the alkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl. In some embodiments, the alkyl group is a C8 to C20 alkyl, e.g., a C14 alkyl or a C16 alkyl.

In some specific embodiments, the alkyl group ses a steroid moiety of a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, holic acid, holic acid, glycocholic acid, and cholesterol acid.

In some embodiments of the disclosure, the on analog comprises an alkylated amino acid by ng a nucleophilic, long chain alkane with the glucagon analog, wherein the glucagon analog comprises a leaving group suitable for nucleophilic substitution. In specific aspects, the nucleophilic group of the long chain alkane comprises an amine, hydroxyl, or thiol group (e.g.,octadecylamine, tetradecanol, and canethiol). The leaving group of the on analog can be part of a side chain of an amino acid or can be part of the peptide backbone. Suitable leaving groups include, for example, N—hydroxysuccinimide, halogens, and sulfonate .

In certain embodiments, the glucagon analog is modified to comprise an alkyl group by ng the nucleophilic, long chain alkane with a spacer which is attached to the glucagon analog, wherein the spacer comprises the leaving group. In specific aspects, the long chain alkane comprises an amine, hydroxyl, or thiol group. In certain embodiments, the spacer comprising the leaving group can be any spacer discussed herein, e. g., amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers and hydrophobic bifunctional spacers further comprising a suitable leaving group.

With regard to these aspects of the disclosure, in which a long chain alkane is alkylated by the on analog or the , the long chain alkane may be of any size and can comprise any length of carbon chain. The long chain alkane can be linear or branched. In certain aspects, the long chain alkane is a C4 to C30 alkane. For example, the long chain alkane can be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane, C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane. In some ments, the long chain alkane comprises a C8 to C20 , e. g., a C14 alkane, C16 alkane, or a C18 alkane.

Also, in some embodiments, alkylation can occur n the glucagon analog and a cholesterol moiety. For example, the hydroxyl group of terol can displace a leaving group on the long chain alkane to form a cholesterol-glucagon analog product.

The alkylated glucagon s bed herein can be further modified to comprise a hydrophilic moiety. In some specific embodiments the hydrophilic moiety can comprise a polyethylene glycol (PEG) chain. The incorporation of a hydrophilic moiety can be accomplished through any suitable means, such as any of the methods described herein. In this regard, the alkylated glucagon analog can se a modified SEQ ID NO: 1 comprising one or more of the amino acid modifications described herein, in which at least one of the amino acids at position 10, 20, 24, and 29 comprise an alkyl group and at least one of the amino acids at position 16, 17, 21, 24, and 29, a position within a C-terminal extension or the inal amino acid are modified to a Cys, Lys, Om, homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalently bonded to a hydrophilic moiety (e. g., PEG). In some embodiments, the alkyl group is attached to position 10, optionally via a spacer comprising Cys, Lys, Om, homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cys residue at position 24.

Alternatively, the alkylated glucagon analog can comprise a spacer, wherein the spacer is both alkylated and modified to comprise the hydrophilic moiety.

Nonlimiting es of le spacers include a spacer comprising one or more amino acids selected from the group consisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

Stabilization of the alpha helix and alpha helix promoting amino acids Without being bound to any particular theory, the glucagon analogs described herein comprise a helical structure, e.g., an alpha helix. In some embodiments, the glucagon analog comprises amino acids which stabilize the alpha helical structure.

Accordingly, in some aspects, the glucagon analog comprises one or more alpha helix promoting amino acids. As used , the term “alpha helix promoting amino acid” refers to an amino acid which provides increased stability to an alpha helix of the on analog of which it is a part. Alpha helix promoting amino acids are known in the art. See, for example, Lyu et al., Proc Natl Acad Sci USA. 88: 5317-5320 (1991); Branden & Tooze, Introduction to Protein Structure, Garland Publishing, New York, NY, 1991; Fasman, Prediction ofProtein Structure and the Principles ofProtein Conformation, ed. Fasman, Plenum, NY, 1989). Suitable alpha helix promoting amino acids for purposes herein include, but are not limited to: e, norvaline, norleucine, alpha aminobutyric acid, alpha-aminoisobutyric acid, leucine, isoleucine, , and the like. In some embodiments, the alpha helix promoting amino acid is any amino acid which is part of an alpha helix found in a naturally-occurring n, e. g., Leu, Phe, Ala, Met, Gly, Ile, Ser, Asn, Glu, Asp, Lys, Arg.

In some embodiments, the alpha helix promoting amino acid es more stability to the alpha helix as ed to glycine or alanine. In some ments, the alpha helix promoting amino acid is an alpha, alpha di—substituted amino acid.

Alpha helix: Position oi alpha helix promoting amino acids In some embodiments, the glucagon analog comprises an amino acid sequence which is similar to native glucagon (SEQ ID NO: 1) and the glucagon analog comprises at least one alpha helix promoting amino acid. In some embodiments, the alpha helix promoting amino acid is located at any of positions 12 to 29 (according to the ing of native glucagon (SEQ ID NO: 1). In some embodiments, the glucagon analog comprises a modified amino acid sequence of SEQ ID NO: 1 and comprises at least one alpha helix promoting amino acid, e. g., at one or more of positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. In some embodiments, the glucagon analog comprises an alpha helix promoting amino acid at one, two, three, or all of positions 16, 17,20, and 21.

Alpha helix: Alphal Alpha Di-Substituted Amino Acids In some ments, the alpha helix promoting amino acid is an a1pha,a1pha di-substituted amino acid. In specific embodiments, the alpha, alpha di-substituted amino acid comprises R1 and R2, each of which is bonded to the alpha carbon, wherein each of R1 and R2 is independently selected from the group consisting of C1-C4 alkyl, optionally substituted with a hydroxyl, amide, thiol, halo, or R1 and R2 together with the alpha carbon to which they are attached form a ring (e.g., a C3—C8 ring). In some embodiments, each of R1 and R2 is selected from the group ting of: methyl, ethyl, propyl, and n-butyl, or R1 and R2 together form a cyclooctane or cycloheptane (e. g., laminocyclooctane-l-carboxylic acid). In some embodiments, R1 and R2 are the same. In some embodiments, R1 is different from R2. In certain aspects, each of R1 and R2 is a C1- C4 alkyl. In some aspects, each of R1 and R2 is a C1 or C2 alkyl. In some embodiments, each of R1 and R2 is , such that the alpha, alpha disubstituted amino acid is alpha- aminoisobutyric acid (AIB).

In some aspects, the glucagon s described herein comprises one or more alpha, alpha di-substituted amino acids and the glucagon analogs specifically lack a covalent intramolecular bridge (e. g., a lactam), since the alpha, alpha disubstituted amino acid is capable of izing the alpha helix in the absence of a covalent bridge. In some aspects, the glucagon analog ses one or more alpha, alpha di-substituted amino acids at the inus (around positions 12—29). In some embodiments, one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24, 28, or 29 of the glucagon analog is substituted with an (x, (ii-disubstituted amino acid, e.g., amino iso-butyric acid (AIB), an amino acid disubstituted with the same or a different group selected from methyl, ethyl, propyl, and n-butyl, or with a ctane or cycloheptane (e.g., 1-aminocyclooctane-lcarboxylic acid). For example, substitution of position 16 with AIB enhances GLP-l activity, in the absence of an intramolecular bridge, e.g., a non-covalent intramolecular bridge (e.g., a salt ) or a covalent intramolecular bridge (e. g., a lactam). In some embodiments, one, two, three or more of positions 16, 20, 21 or 24 are tuted with AIB. In specific embodiments, one or both of the amino acids corresponding to positions 2, 16, of native human glucagon (SEQ ID NO: 1) are substituted with an alpha, alpha disubstituted amino acid such as AIB.

In accordance with some embodiments, the glucagon analog g an intramolecular bridge comprises one or more substitutions within amino acid positions 12-29 with an (x, (it-disubstituted amino acid and an acyl or alkyl group covalently attached to the side chain of the amino acid at position 10 of the glucagon analog. In specific embodiments, the acyl or alkyl group is not naturally occurring on an amino acid.

In n aspects, the acyl or alkyl group is non-native to the amino acid at position 10.

Such acylated or alkylated glucagon peptides lacking an intramolecular bridge exhibit ed activity at the GLP-1 and glucagon receptors as compared to the non-acylated rpart peptides. Further ement in activity at the GLP-1 and glucagon receptors can be achieved by the acylated glucagon peptides lacking an intramolecular bridge by incorporating a spacer between the acyl or alkyl group and the side chain of the amino acid at position 10 of the analog. Acylation and alkylation, with or without incorporating spacers, are further described herein.

Alpha helix: Intramolecular Bridges In some embodiments, the alpha helix promoting amino acid is an amino acid which is linked to another amino acid of the glucagon analog via an olecular bridge. In such embodiments, each of these two amino acids linked via an intramolecular bridge is considered an alpha helix promoting amino acid. In some embodiments, the glucagon analog comprises one or two intramolecular s. In some specific embodiments, the glucagon analog comprises one intramolecular bridge in ation with at least one other alpha helix promoting amino acid, e.g., an alpha, alpha- disubstituted amino acid.

In some embodiments, the intramolecular bridge is a bridge which connects two parts of the glucagon analog via noncovalent bonds, including, for example, van der Waals interactions, hydrogen bonds, ionic bonds, hydrophobic interactions, dipole-dipole interactions, and the like. In this regard, the glucagon analog in certain aspects comprises a non-covalent intramolecular bridge. In some ments, the non-covalent intramolecular bridge is a salt .

] In some embodiments, the intramolecular bridge is a bridge which connects two parts of the analog via covalent bonds. In this regard, the glucagon analog in certain aspects comprises a covalent intramolecular bridge.

In some embodiments, the intramolecular bridge (e.g., non-covalent intramolecular bridge, covalent intramolecular bridge) is formed between two amino acids that are 3 amino acids apart, e.g., amino acids at positions i and i+4, n i is any integer between 12 and 25 (e.g., 12, 13, 14, 15, 16, 17, 18, 19,20, 21,22, 23, 24, and 25).

More particularly, the side chains of the amino acid pairs 12 and 16, 16 and 20 , 20 and 24 or 24 and 28 (amino acid pairs in which i = 12, 16, 20, or 24) are linked to one another and thus stabilize the glucagon alpha helix. Alternatively, i can be 17. In some specific embodiments, the on analog comprises an intramolecular bridge between amino acids 17 and 21. In some specific embodiments, the on analog comprises an intramolecular bridge n the amino acids at positions 16 and 20 or 12 and 16 and a second intramolecular bridge between the amino acids at positions 17 and 21. Glucagon analogs comprising one or more olecular bridges are contemplated herein. In specific embodiments, wherein the amino acids at positions i and i+4 are joined by an intramolecular bridge, the size of the linker is about 8 atoms, or about 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between two amino acids that are two amino acids apart, e.g., amino acids at positions j and j+3, wherein j is any integer between 12 and 26 (e.g., 12, 13,14,15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In some specific embodiments, j is 17. In specific ments, wherein amino acids at positions j and j+3 are joined by an intramolecular bridge, the size of the linker is about 6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed between two amino acids that are 6 amino acids apart, e. g., amino acids at positions k and k+7, wherein k is any integer between 12 and 22 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20,21, and 22). In some ic embodiments, k is l2, 13, or 17. In an exemplary embodiment, kis 17.

Alpha helix: Amino acids involved in intramolecular bridges Examples of amino acid gs that are capable of bonding (covalently or non-covalently) to form a six-atom linking bridge include Orn and Asp, Glu and an amino acid of Formula I, wherein n is 2, and homoglutamic acid and an amino acid of Formula I, wherein n is 1, wherein Formula I is: H2N—C—COOH (CH2)n wherein n = 1 to 4 [Formula I] Examples of amino acid gs that are e of bonding to form a seven- atom linking bridge include Orn-Glu (lactam ring); Lys—Asp (lactam); or Homoser- Homoglu (lactone). Examples of amino acid pairings that may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp (lactam); Orn-Homoglu (lactam); 4-aminoPhe- Asp (lactam); or Tyr-Asp (lactone). Examples of amino acid pairings that may form a tom linker include Homolys-Glu m); Lys-Homoglu m); 4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains on these amino acids may additionally be substituted with additional chemical groups, so long as the three- ional structure of the helix is not disrupted. One of ordinary skill in the art can envision alternative gs or alternative amino acid analogs, including chemically modified derivatives, that would create a izing structure of similar size and desired effect. For example, a homocysteine—homocysteine ide bridge is 6 atoms in length and may be further modified to e the desired effect.

Even without covalent e, the amino acid pairings bed above (or r pairings that one of ordinary skill in the art can envision) may also provide added stability to the alpha-helix through non—covalent bonds, for example, through formation of salt bridges or hydrogen-bonding interactions. Accordingly, salt bridges may be formed between: Orn and Glu; Lys and Asp; Homo-serine and Homo-glutamate; Lys and Glu; Asp and Arg; Homo-Lys and Asp; Om and Homo-Glutamate; 4-aminoPhe and Asp; Tyr and Asp; Homo-Lys and Glu; Lys and Homo-Glu; 4-aminoPhe and Glu; or Tyr and Glu. In some embodiments, the analog comprises a salt bridge between any of the following pairs of amino acids: Om and Glu; Lys and Asp; Lys and Glu; Asp and Arg; Homo-Lys and Asp; Orn and Homo-Glutamate; Homo—Lys and Glu; and Lys and Homo- Glu. Salt bridges may be formed between other pairs of oppositely d side chains.

See, e. g., Kallenbach et al., Role of the Peptide Bond in n Structure and Folding, in The Amide Linkage: Structural Significance in Chemistry, Biochemistry, and Materials e, John Wiley & Sons, Inc. (2000).

In some embodiments, the non—covalent intramolecular bridge is a hydrophobic bridge. In accordance with one embodiment, the alpha helix of the analog is stabilized through the incorporation of hydrophobic amino acids at positions j and j+3 or i and i+4. For instance, i can be Tyr and i+4 can be either Val or Leu; i can be Phe and i+4 can be Met; or i can be Phe and i+4 can be Ile. It should be understood that, for purposes herein, the above amino acid pairings can be reversed, such that the indicated amino acid at position i could alternatively be located at i+4, while the i+4 amino acid can be located at the i position. It should also be understood that suitable amino acid pairings can be formed for j and j+3.

Alpha helix: Covalent Intramolecular Bridge In some embodiments, the covalent intramolecular bridge is a lactam ring or lactam bridge. The size of the lactam ring can vary depending on the length of the amino acid side , and in one embodiment the lactam is formed by g the side chains of an omithine to a aspartic acid side chain. Lactam bridges and methods of making the same are known in the art. See, for example, Houston, Jr., et al., J Peptide Sci 1: 274-282 (2004), and Example 1 herein. In some embodiments, the analog comprises a modified sequence of SEQ ID NO: 1 and a lactam bridge between i and i+4, wherein i is as defined herein above. In some embodiments, the glucagon analog comprises two lactam bridges: one between the amino acids at positions 16 and 20 and another between the amino acids at positions 17 and 21. In some embodiments, the glucagon analog comprises one lactam bridge and one salt bridge. Further exemplary embodiments, are described herein in the section entitled “EXAMPLES.” Further ary embodiments include the following pairings, optionally with a lactam bridge: Glu at position 12 with Lys at position 16; native Lys at position 12 with Glu at position 16; Glu at on 16 with Lys at position ; Lys at position 16 with Glu at position 20; Glu at position 20 with Lys at position 24; Lys at position 20 with Glu at position 24; Glu at position 24 with Lys at position 28; Lys at position 24 with Glu at position 28.

In some embodiments, the nt intramolecular bridge is a lactone.

Suitable methods of making a lactone bridge are known in the art. See, for example, Sheehan et al., JAm Chem Soc 95: 875-879 .

In some aspects, olefin metathesis is used to cross-link one or two turns of the alpha helix of the analog using an all-hydrocarbon cross—linking system. The glucagon analog in this instance comprises (it-methylated amino acids bearing olefinic side chains of varying length and configured with either R or S stereochemistry at thej and j+3 or i and i+4 positions. In some embodiments, the olefinic side comprises (CH2)n, wherein n is any integer between 1 to 6. In some embodiments, n is 3 for a cross-link length of 8 atoms. In some embodiments, n is 2 for a cross-link length of 6 atoms. An ary glucagon analog comprising an olefinic link is bed herein as SEQ ID NO: 17.

Suitable methods of forming such intramolecular s are described in the art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000) and Walensky et al., Science 305: 1466-1470 . In alternative embodiments, the analog ses 0- allyl Ser es located on adjacent l turns, which are bridged together via ruthenium-catalyzed ring closing metathesis. Such ures of cross-linking are bed in, for example, Blackwell et al., Angew, Chem, Int. Ed. 37: 3281-3284 (1998).

In specific aspects, use of the unnatural thio-dialanine amino acid, lanthionine, which has been widely adopted as a peptidomimetic of cystine, is used to cross-link one turn of the alpha helix. Suitable methods of lanthionine—based ation are known in the art. See, for instance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004); Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J. Med. Chem. 35: 4185- 4194 (1992); Osapay et al., J. Med. Chem. 40: 2241-2251 (1997); Fukase et a1., Bull.

Chem. Soc. Jpn. 65: 2227-2240 (1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao, Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem. Soc. Chem. Commun. 1599—1600 (1993).

In some embodiments, a, w—diaminoalkane tethers, e.g., 1,4-diaminopropane and 1,5-diaminopentane) n two Glu residues at positions i and i+7 are used to stabilize the alpha helix of the analog. Such tethers lead to the ion of a bridge 9- atoms or more in length, depending on the length of the diaminoalkane tether. Suitable methods of ing peptides cross-linked with such tethers are bed in the art.

See, for example, Phelan et a1., J. Am. Chem. Soc. 119: 455-460 (1997).

In yet other embodiments, a disulfide bridge is used to cross-link one or two turns of the alpha helix of the analog. atively, a modified disulfide bridge in which one or both sulfur atoms are replaced by a ene group resulting in an isosteric macrocyclization is used to stabilize the alpha helix of the analog. Suitable methods of modifying peptides with disulfide bridges or sulfur—based cyclization are described in, for example, Jackson et al., J. Am. Chem. Soc. 113: 9391—9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).

In yet other embodiments, the alpha helix of the analog is stabilized via the binding of metal atom by two His residues or a His and Cys pair positioned atj and j+3, or i and i+4. The metal atom can be, for example, Ru(III), Cu(II), Zn(II), or Cd(II). Such methods of metal binding-based alpha helix stabilization are known in the art. See, for example, Andrews and Tabor, edron 55: 11711-11743 (1999); Ghadiri et al., J.

Am. Chem. Soc. 112: 1630-1632 ; and Ghadiri et a1., J. Am. Chem. Soc. 119: 9063- 9064 (1997).

The alpha helix of the analog can alternatively be stabilized h other means of peptide cyclizing, which means are reviewed in Davies, J. Peptide. Sci. 9: 471- 501 (2003). The alpha helix can be ized via the formation of an amide bridge, thioether bridge, thioester bridge, urea , ate bridge, sulfonamide bridge, and the like. For example, a thioester bridge can be formed between the inus and the side chain of a Cys residue. Alternatively, a thioester can be formed Via side chains of amino acids having a thiol (Cys) and a carboxylic acid (e.g., Asp, Glu). In another method, a cross-linking agent, such as a dicarboxylic acid, e.g.,suberic acid (octanedioic acid), etc. can introduce a link between two functional groups of an amino acid side chain, such as a free amino, hydroxyl, thiol group, and combinations thereof.

DPP-IV Resistant Peptides In some embodiments, the on analog comprises at position 1 or 2, or at both positions 1 and 2, an amino acid which achieves resistance of the glucagon analog to dipeptidyl peptidase IV (DPP IV) cleavage. In some embodiments, the glucagon analog comprises at position 1 an aminio acid selected from the group consisting of: idine, desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine, N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, or alpha, dimethyl imidiazole acetic acid (DMIA). In some embodiments, the glucagon analog comprises at position 2 an amino acid selected from the group consisting of: D-serine, D-alanine, , glycine, N-methyl serine, N—methyl alanine, or alpha, aminoisobutyric acid. In some embodiments, the glucagon analog comprises at position 2 an amino acid which achieves resistance of the glucagon analog to DPP IV and the amino acid which achieves resistance of the glucagon analog to DPP IV is not D—serine.

In some aspects, the glucagon analog comprising an amino acid which achieves ance of the glucagon analog to DPP IV further comprises an amino acid modification which stabilizes the alpha helix found in the C-terminal portion of glucagon, e. g.,through a covalent bond between amino acids at positions “i” and “i+4”, e. g., 12 and l6, l6 and 20, or 20 and 24. In some ments, this covalent bond is a lactam bridge between a glutamic acid at position 16 and a lysine at position 20. In some embodiments, this nt bond is an intramolecular bridge other than a lactam bridge. For example, suitable covalent bonding s include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, the use of (x, oa-diaminoalkane tethers, the formation of metal-atom s, and other means of e cyclization.

Modification of on 1 In some specific embodiments, the glucagon analog ses (a) an amino acid substitution of His at position 1 with a large, ic amino acid and (b) an olecular bridge that stabilizes that alpha-helix in the C-terminal portion of the molecule (e. g.,around positions 12—29). In specific embodiments, the amino acid at position 1 is replaced with Tyr, Phe, Trp, amino-Phe, nitro-Phe, chloro-Phe, Phe, 4- pyridyl-Ala, methyl-Tyr, or 3-amino Tyr. The intramolecular bridge, in some embodiments, is any of those described herein. In some aspects, the intramolecular bridge is between the side chains of two amino acids that are separated by three intervening amino acids, i.e., between the side chains of amino acids i and i+4. In some embodiments, the intramolecular bridge is a lactam bridge. In some embodiments, the glucagon analog comprises a large, aromatic amino acid at position 1 and a lactam bridge between the amino acids at positions 16 and 20 of the . Such a glucagon analog in some aspects further comprises one or more (e.g., two, three, four, five or more) of the other modifications bed herein. For example, the glucagon analog can comprise an amide in place of the C-terminal carboxylate. Also, in some embodiments, such glucagon analogs further comprise one or more of a large aliphatic amino acid at position 17, an imidazole containing amino acid at position 18, and a positive-charged amino acid at position 19. In some ments, the glucagon analogs comprising a modification at position 1 and an intramolecular bridge r comprises the amino acid sequence Ile- His-Gln at positions 17-19. Such modifications can be made without destroying activity of the on analog at the GLP-1 receptor and the glucagon receptor. In some embodiments, the glucagon analog additionally comprises an acylated or alkylated amino acid residue.

Modification of Position 3 In some embodiments, the third amino acid of SEQ ID NO: 1 (Gln3) is substituted with an acidic, basic, or hydrophobic amino acid residue and such modification causes the glucagon receptor activity to be d. In some ments, the , basic, or hydrophobic amino acid is glutamic acid, ornithine, norleucine. In some aspects, modification with one of these residues has led the glucagon analog to exhibit a ntially reduced or destroyed glucagon receptor activity. The glucagon analogs that are tuted with, for example, glutamic acid, ornithine, or norleucine in some s have about 10% or less of the ty of native glucagon at the glucagon receptor, e. g.,about 1-10%, or about 01-10%, or greater than about 0.1% but less than about 10%, while exhibiting at least 20% of the activity of GLP—1 at the GLP-1 receptor.

In some embodiments, the glucagon analogs t about 0.5%, about 1% or about 7% of the activity of native glucagon, while exhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.

In some embodiments, the glutamine at position 3 of SEQ ID NO: 1 of the on analog is substituted with a glutamine analog without a substantial loss of activity at the glucagon receptor, and in some cases, with an enhancement of glucagon receptor activity. In some embodiments, the glutamine analog is a naturally occurring or a non-naturally occurring or ded amino acid comprising a side chain of Structure I, II or III: -§—R1—CH2—XJl—R2 Structure I -§—R1—CH2JI—Y Structure II 1-H —CH2—S-CH2—R4, Structure III wherein R1 is C0_3 alkyl or C03 heteroalkyl; R2 is NHR4 or C13 alkyl; R3 is C13 alkyl; R4 is H or C1_3 alkyl; X is NH, O, or S; and Y is NHR4, SR3, or 0R3. In some embodiments, X is NH or Y is NHR4. In some embodiments, R1 is C04 alkyl or C1 heteroalkyl. In some embodiments, R2 is NHR4 or C1 alkyl. In some embodiments, R4 is H or C1 alkyl. In exemplary ments, an amino acid comprising a side chain of Structure I is provided where, R1 is CHz-S, X is NH, and R2 is CH3 (acetamidomethyl- ne, C(Acm)); R1 is CH2, X is NH, and R2 is CH3 (acetyldiaminobutanoic acid, Dab(Ac)); R1 is C0 alkyl, X is NH, R2 is NHR4, and R4 is H moyldiaminopropanoic acid, Dap(urea)); or R1 is CHz-CHZ, X is NH, and R2 is CH3 (acetylomithine, Om(Ac)).

In ary embodiments, an amino acid comprising a side chain of Structure II is provide where, R1 is CH2, Y is NHR4, and R4 is CH3 (methylglutamine, Q(Me)); In exemplary embodiments, an amino acid comprising a side chain of Structure III is provided where, R1 is CH2 and R4 is H (methionine-sulfoxide, M(O)); In specific embodiments, the amino acid at position 3 is substituted with Dab(Ac) For example, glucagon ts can comprise a modified amino acid sequence of SEQ ID NO: 595, SEQ ID NO: 601 SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, and SEQ ID NO: 606 of the sequence listing of International Patent Application No.

PCT/U82009/047438, filed on June 16, 2009, which is incorporated by reference in its entirety, wherein these amino acid sequences are modified as further described herein, e. g., modified to comprise at least three alpha helix promoting amino acids, modified to comprise (i) an acylated or alkylated amino acid at position 10, (ii) an alpha helix promoting amino acid at position 16, (iii) an aliphatic amino acid at position 17 and/or 18, and (iv) at least one charged amino acid located C-terminal to position 27, and, optionally, further modifications; modified to comprise at least three amino acids of the amino acids 18-24 of Exendin-4 (SEQ ID NO: 8) at the corresponding positions of the glucaogon analog.

Modification 0t Position 7 In some embodiments, the glucagon analog comprises a modified SEQ ID NO: 1 with an amino acid modification at position 7. In some aspects, the amino acid at position 7 of SEQ ID NO: 1 (Thr) is substituted with a large, aliphatic amino acid, e. g., Ile, Leu, Ala, and the like. Such modifications are believed to drastically reduce activity at the GLP-1 receptor of the glucagon . ation of Position 15 In some embodiments, the glucagon analogs comprise a modified SEQ ID NO: 1 with an amino acid modification at position 15 which improves stability. In some aspects, the amino acid at position 15 of SEQ ID NO: 1 is deleted or substituted with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid. Such modifications reduce degradation or cleavage of the analog over time, especially in acidic or alkaline buffers, e. g., buffers at a pH within the range of 5.5 to 8. In some embodiments, the on analogs sing this modification retains at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the original analog after 24 hours at 25°C.

Modification of Position 16 In accordance with one embodiment, analogs of glucagon are provided that have enhanced potency and optionally improved solubility and stability. In one embodiment, enhanced glucagon and GLP-1 y is provided by an amino acid modification at position 16 of native glucagon (SEQ ID NO: 1). By way of iting example, such enhanced potency can be provided by substituting the lly occurring serine at position 16 with glutamic acid or with another negative—charged amino acid having a side chain with a length of 4 atoms, or alternatively with any one of glutamine, homoglutamic acid, or steic acid, or a charged amino acid having a side chain ning at least one heteroatom, (e.g.,N, O, S, P) and with a side chain length of about 4 (or 3-5) atoms. In some embodiments, the glucagon analog comprises a modified SEQ ID NO: 1 comprising a substitution of the Ser at position 16 with an amino acid selected from the group consisting of glutamic acid, glutamine, utamic acid, steic acid, threonine or glycine. In some aspects, the serine residue at position 16 is tuted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid and homocysteic acid. In some ic aspects, the serine residue at position 16 is substituted with glutamic acid or a conservative substitution thereof (e. g. an Exendin-4 amino acid).

In alternative embodiments, the glucagon analog comprises a modified sequence of SEQ ID NO: 1 modified by a tution of Ser at position 16 with Thr or AIB or another alpha helix promoting amino acid as described above. In some embodiments, the alpha helix promoting amino acid forms a non—covalent intramolecular bridge with an amino acid at j+3 or i+4.

Modification at positions 17-18 In some embodiments, the glucagon analog comprises a modified SEQ ID NO: 1 in which the dibasic Arg-Arg site at positions 17 and 18 is eliminated. Without being bound to any particular , it is believed that elimination of the dibasic site in some embodiments es the in vivo efficacy of the glucagon . In some aspects, the glucagon analog is modified in this regard by substituting one or both of the amino acids at positions 17 and 18 of SEQ ID NO: 1 with an amino acid which is not basic, e. g., with an tic amino acid. In some embodiments, one of the amino acids at position 17 or 18 is deleted or an amino acid is inserted in between positions 17 and 18. In some embodiments, the Arg at position 17 is tuted with another amino acid as described , e. g., Gln, an amino acid comprising a hydrophilic moiety, an alpha helix promoting amino acid. In some embodiments, the alpha helix promoting amino acid forms a non-covalent intramolecular bridge with an amino acid at j+3 or i+4. In some embodiments, the Arg at position 18 is substituted with r amino acid as described herein. In exemplary aspects, the amino acid at position 18 is an alpha, alpha, disubstituted amino acid, e. g., AIB. In some aspects, the amino acid at position 18 is a small aliphatic amino acid, e. g., Ala. In some specific aspects, the amino acid at position 18 is a small aliphatic amino acid, e.g., Ala, and the Arg at position 17 remains unmodified.

Modification of Position 20 Enhanced activity at the GLP-1 or is also provided by an amino acid modification at on 20. In some embodiments, the ine at position 20 is replaced with an alpha helix promoting amino acid, e.g. AIB, as described above. In some embodiments, the alpha helix promoting amino acid forms a non-covalent intramolecular bridge with an amino acid at j—3 or i—4. In some specific embodiments the amino acid is a hydrophilic amino acid having a side chain that is either charged or has an ability to hydrogen-bond, and is at least about 5 (or about 4-6) atoms in length, for example, lysine, citrulline, arginine, or ornithine, and optionally forms a salt bridge with another alpha helix promiting amino acid at position 16, e.g. a negative charged amino acid. Such modifications in some particular aspects reduce degradation that occurs through deamidation of Gln and in some embodiments, increase the activity of the glucagon analog at the GLP-1 receptor. In some aspects, the amino acid at position 20 is Glu or Lys or AIB.

Modification at Positions 21x 23x 24, and 28 In some embodiments, position 21 and/or position 24 is modified by substitution with an alpha helix promoting amino acid. In some embodiments, the alpha helix promoting amino acid forms a non-covalent intramolecular bridge with an amino acid at j-3 or i-4. In some aspects, the alpha helix promoting amino acid is AIB.

In ary ments, the amino acid at position 23 is a He.

In exemplary aspects, the amino acid at position 28 is an alpha, alpha, disubstituted amino acid, e. g., AIB.

Charged C-terminus In some embodiments, the glucagon analog is modified by amino acid tutions and/or additions that introduce a charged amino acid into the C-terminal portion of the analog. In some ments, such modifications e stability and solubility. As used herein the term "charged amino acid" or “charged residue” refers to an amino acid that comprises a side chain that is negative-charged (i.e., de-protonated) or ve-charged (i.e., protonated) in aqueous solution at physiological pH. In some aspects, these amino acid substitutions and/or ons that introduce a charged amino acid modifications are at a position C—terminal to position 27 of SEQ ID NO: 1. In some embodiments, one, two or three (and in some instances, more than three) charged amino acids are introduced within the C-terminal portion (e.g., position(s) C-terminal to position 27). In accordance with some embodiments, the native amino ) at positions 28 and/or 29 are substituted with a charged amino acids, and/or in a further embodiment one to three d amino acids are also added to the C-terminus of the . In exemplary embodiments, one, two or all of the charged amino acids are negative-charged.

The negative-charged amino acid in some embodiments is aspartic acid, glutamic acid, cysteic acid, homocysteic acid, or utamic acid. In some aspects, these modifications increase solubility, rovide at least 2—fold, 5-fold, 10-fold, 15-fold, 25- fold, 30-fold or greater solubility relative to native on at a given pH between about .5 and 8, e.g., pH 7, when measured after 24 hours at 25°C. inal tion In accordance with some embodiments, the glucagon analogs disclosed herein are modified by truncation of the C-terminus by one or two amino acid residues. Such modified glucagon peptides, as shown herein, retain similar activity and y at the glucagon receptor and GLP-1 receptor. In this regard, the glucagon peptides can comprise amino acids 1-27 or 1-28 of the native glucagon analog (SEQ ID NO: 1), optionally with any of the additional modifications described .

Charge-neutral C-terminus In some embodiments, the glucagon analog comprises a modified SEQ ID NO: 1 in which the the carboxylic acid of the C-terminal amino acid is replaced with a charge- neutral group, such as an amide or ester. Without being bound to any particular theory, such modifications in certain aspects increases ty of the glucagon analog at the GLP-1 receptor. Accordingly, in some embodiments, the glucagon analog is an amidated peptide, such that the C-terminal residue comprises an amide in place of the alpha carboxylate of an amino acid. As used herein a general reference to a peptide or analog is intended to encompass peptides that have a modified amino terminus, carboxy terminus, orboth amino and y termini. For example, an amino acid chain composing an amide group in place of the terminal carboxylic acid is intended to be encompassed by an amino acid sequence designating the rd amino acids.

Other modifications In some embodiments, the glucagon analogs additionally or alternatively comprise the following amino acid cations: (i) Substitution of Ser at position 2 with Ala; (ii) Substitution of Tyr at position 10 with Val or Phe, or Trp; (iii) tution of Lys at position 12 with Arg; (iv) Substitution of Arg at position 17 with Gln or a small aliphatic amino acid, e.g., Ala, or a large aliphatic amino acid, e.g., Ile; (V) Substitution of Arg at position 18 with a small aliphatic amino acid, e.g., Ala; or an imidazole- containing amino acid, e. g., His; (vi) Substitution of Ala at position 19 with a ve- charged amino acid, e. g., Gln; (vii) Substitution of Val at position 23 with He, and (viii) Substitution of Thr at position 29 with Gly or Gln.

In some embodiments, the ity of the glucagon analog is increased by modification of the methionine at position 27, for example, by substitution with e or norleucine. Such modifications can reduce oxidative degradation. Stability can also be increased by modification of the Gln at position 20 or 24 or 28, e.g.,by substitution with Ala, Ser, Thr, or AIB. Such modifications can reduce degradation that occurs through deamidation of Gln. Stability can be increased by modification of Asp at position 21, e. g.,by substitution with another acidic residue, e.g., Glu. Such modifications can reduce ation that occurs through dehydration of Asp to form a cyclic succinimide intermediate followed by isomerization to partate.

In some embodiments, the glucagon analogs described herein are glycosylated, ed, ylated, phosphorylated, esterified, N-acylated, cyclized via, e. g., a disulfide bridge, or converted into a salt (e.g., an acid addition salt, a basic addition salt), and/or optionally dimerized, multimerized, or polymerized, or conjugated.

Any of the modifications described herein, including, for example, the modifications which se or decrease glucagon receptor activity and which increase GLP-1 receptor activity, can be applied individually or in combination. Combinations of the cations that increase GLP-1 receptor activity may provide higher GLP-l activity than any of such modifications taken alone.

EXEMPLARYEMBODIMENTS The t disclosures provide peptides comprising a structure r to that of native human glucaon and exhibiting enhanced agonist ty at the GLP-1 receptor, compared to native human glucagon. Glucagon normally has about 1% of the activity of native-GLP-l at the GLP-1 receptor, while GLP-l normally has less than about 0.01% of the activity of native glucagon at the glucagon receptor. Accordingly, the peptides of the present disclosures exhibit greater than 1% of the activity of native-GLP-l at the GLP-1 or. In exemplary embodiments, the peptides of the present disclosures exhibit r than or about 5%, greater than or about 10%, r than or about 15%, greater than or about 20%, greater than or about 25%, greater than or about 30%, greater than or about 35%, greater than or about 40%, greater than or about 45%, greater than or about 50%, greater than or about 55%, greater than or about 60%, r than or about 65%, greater than or about 70%, greater than or about75%, greater than or about 80%, greater than or about 85%, greater than or about 90%, or greater than or about 95% of the activity of native-GLP-l at the GLP-1 receptor. In exemplary aspects, the peptides of the present disclosures exhibit ty at the GLP-1 receptor which is greater than that of native GLP-l. Accordingly, in exemplary aspects, the peptides of the t disclosures exhibit greater than or about 100% of the activity of -GLP-l at the GLP-1 receptor. In exemplary aspects, the peptides of the present disclosures exhibit greater than or about 150%, greater than or about 200%, greater than or about 250%, greater than or about 300%, greater than or about 350%, greater than or about 400%, greater than or about 450%, greater than or about 500%, greater than or about 550%, greater than or about 600%, greater than or about 650%, greater than or about 700%, greater than or about 750%, greater than or about 800%, r than or about 850%, greater than or about 900%, greater than or about 950%, or greater than or about 1000% of the activity of native-GLP-l at the GLP-1 receptor.

In ary embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 12.

In exemplary embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 13.

In ary embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 14.

In exemplary embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 15.

In exemplary embodiments, the e comprises the amino acid ce of SEQ ID NO: 16 and exhibits at least 100-fold selectivity for the human GLP-1 receptor versus the GIP receptor.

In exemplary embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 17.

The t disclosures further provides variant peptides sing an amino acid sequence which is highly similar to the amino acid sequence of one of the presently disclosed peptides. In exemplary embodiments, the variant peptide of the present disclosures comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to amino acids 1-29 of the amino acid sequence of the peptide of any of SEQ ID NOs: 12-17, wherein the variant peptide retains the activity of the parent peptide at the GLP-1 receptor, glucagon receptor, and GIP receptor (e.g., exhibits at least lOO-fold selectivity for the human GLP-1 receptor versus the GIP receptor), and optionally a GLP- 1 potency of at least 1%; or n the EC50 of the peptide at the GIP receptor is less than 100-fold different from its EC50 at the GLP—1 receptor). In exemplary embodiments, the t peptide of the present disclosures comprises exhibits at least 100-fold r selectivity for the human GLP-1 receptor versus the GIP receptor.

In exemplary embodiments, the variant peptide of the present disclosures ses an amino acid sequence based on an amino acid sequence of a peptide of the t disclosures but differs at one or more amino acid positions, including, but not limited to position 1, position 2, position 3, position 7", position 10, position 12, position , position 16, position 17, position 18, on 20, position 21, position 23, position 24, position 27, position 28, position 29. In exemplary aspects, the variant peptide may comprise a conservative substitution relative to the parent peptide, may comprise any of the amino acid modifications described herein, or may comprise an amino acid cation that returns to the amino acid present at that position in the native glucagon sequence (SEQ ID NO: 1). In exemplary aspects, the variant peptide of the present disclosures ses an amino acid sequence based on an amino acid sequence of a e of the present sures but differs in one or more of the following ways: a) the variant peptide comprises an acylated amino acid or an alkylated amino acid; b) an acylated amino acid or an alkylated amino acid is ed with the corresponding amino acid of native glucagon (SEQ ID NO: 1) at that position or a conservative substitution of the native amino acid, and optionally a new acylated or alkylated amino acid is introduced at a different position; c) the variant peptide comprises an amino acid ntly attached to a hydrophilic moiety; d) an amino acid ntly attached to a hydrophilic moiety is replaced with the corresponding amino acid of native on (SEQ ID NO: 1) at that position, and optionally a new amino acid covalently attached to a hydrophilic moiety is introduced at a different position; e) the C-terminal amino acid of the variant peptide ses a C- terminal amide in place of a C-terminal alpha carboxylate; f) an amino acid at any of positions 1 through 29 is replaced with the corresponding amino acid of native glucagon (SEQ ID NO: 1) at that position; g) or any combinations thereof.

With regard to any of the ing variant peptides, in ary embodiments, the variant peptide comprises a hydrophilic moiety covalently attached to an amino acid at position l6, 17, 21, 24, 29, a position within a C-terminal extension, or at the C-terminus. In exemplary aspects, the variant peptide comprises a Cys, Lys, Orn, homocysteine, and Ac-Phe covalently attached to a hilic moiety , optionally, wherein the Cys, Lys, Orn, homocysteine, or Ac—Phe is located at position l6, 17, 21, 24, 29, a position within a C-terminal extension, or at the C—terminus of the t peptide.

In exemplary aspects, the hydrophilic moiety is a polyethylene .

In ary aspects, the variant peptide comprises an ed or alkylated amino acid, optionally, at on 10. In exemplary aspects, the variant peptide comprises an acylated or alkylated amino acid which comprises a C8 to C20 alkyl chain, a C12 to C18 alkyl chain, or a C14 or C16 alkyl chain. In exemplary aspects, the vrariant peptide comprises an acylated or alkylated amino acid which an ed or alkylated amino acid of Formula I, Formula II, or Formula III, optionally, wherein the amino acid of Formula I is Lys.

In exemplary aspects, the variant peptide of the present disclosures comprises an ed or alkylated amino acid, wherein the acyl group or alkyl group is covalently attached to the amino acid via a spacer, optionally, wherein the spacer is an amino acid or a dipeptide. In exemplary embodiments, the spacer comprises one or two acidic residues.

In any of the foregoing exemplary embodiments, the peptide or variant peptide of any of the present sures exhibits an (EC50 at the glucagon or)/(EC50 at the GLP-1 receptor) is about 20 or less (e.g., 20, 19, 18, 17,. 16, 15, l4, l3, 12, ll, 10, 9, 8,7, 6, 5, 4, 3, 2, l, 0.5, 0.25, 0.10, 0. 05, 0.025, 0.01, 0.001).

] In any of the ing exemplary embodiments, the peptide or variant peptide of any of the present disclosures exhibits an (EC50 at the glucagon receptor)/(EC50 at the GLP-1 receptor) is more than 20 (e.g., 21, 25, 30, 40, 50 , 60, 70, 80, 90, 100, 250, 500, 750, 1000, or more).

In any of the foregoing exemplary embodiments, the peptide or variant peptide of any of the present disclosures ts an EC50 at the GLP—1 receptor which is two- to ten-fold (e. g., 3-, 4-, 5-, 6-, 7-, 8-, ) greater than the EC50 at the on or.

EXCLUSIONS In exemplary embodiments, any one of the following peptides is excluded from the glucagon analogs described herein, gh any of the following peptides comprising one or more further modifications thereto as described herein ting the desired GLP-l or co-agonist ty, pharmaceutical compositions, kits, and treatment methods using such compounds may be included in the invention: The peptide of SEQ ID NO: 1 with an [Arg12] substitution and with a C—terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys20] substitutions and with a C—terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys24] substitutions and with a C-terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys29] substitutions and with a C-terminal amide; The peptide of SEQ ID NO: 1 with a [Glu9] substitution; The peptide of SEQ ID NO: 1 missing Hisl, with [Glu9, Glul6, Lys29] tutions and C-terminal amide; The peptide of SEQ ID NO: 1 with [Glu9, Glul6, Lys29] tutions and with a C-terminal amide; The peptide of SEQ ID NO: 1 with [Lys13, Glu17] substitutions linked via lactam bridge and with a C-terminal amide; The e of SEQ ID NO: 1 with [Lysl7, Glu21] substitutions linked via lactam bridge and with a C—terminal amide; The peptide of SEQ ID NO: 1 missing Hisl, with [Glu20, Lys24] substitutions linked via lactam bridge. In some embodiments, the glucagon analog is not any of the peptides disclosed in any of International Patent Application No. PCTIUS2009/034448, filed on February 19, 2009, and published on August 26, 2010, as ; International Patent Application No. , filed on December 18, 2009, and published on August 26, 2010, as ; International Patent Application No. , filed on June 16, 2009, and published on er 23, 2009 as ; International Patent Application No. PCTIUS2008/053857, filed on February 13, 2008, and published on August 21, 2008, as W0 2008/101017; International Patent Application No. , filed on December 9, 2010; International Patent Application No. , filed on June 16, 2009, and published on January 28, 2010, as WO2010/011439; International Patent Application No. , filed on June 16, 2010, and published on December 23, 2010, as WO2010/148089; International Patent Application No. 2011/022608, filed on January 26, 2011; and US. Provisional Application No. ,285, filed on er 22, 2010; each of which are incorporated by reference in their entirety. In some embodiments, the glucagon analog does not include all or part of the sequence KRNRNNIA linked to the C—terminus after on 29, e. g. KRNR.

METHODS OF MAKING PEPTIDES The glucagon analogs of the disclosure can be obtained by methods known in the art. Suitable methods of de novo synthesizing peptides are described in, for e, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and US Patent No. 5,449,752.

Also, in the instances in which the analogs of the disclosure do not comprise any non-coded or non-natural amino acids, the glucagon analog can be recombinantly produced using a nucleic acid encoding the amino acid sequence of the analog using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and l et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

In some embodiments, the glucagon analogs of the sure are isolated. In some ments, the on analogs of the disclosure are purified. . It is recognized that “purity” is a relative term, and not to be necessarily construed as absolute purity or absolute enrichment or absolute selection. In some aspects, the purity is at least or about 50%, is at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% (e. g., at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or is approximately 100%.

In some embodiments, the peptides described herein are commercially synthesized by companies, such as Synpep n, CA), Peptide Technologies Corp. ersburg, MD), and le Peptide Systems (San Diego, CA). In this respect, the peptides can be synthetic, recombinant, isolated, andfor purified.

CONJUGATES The invention r provides conjugates comprising one or more of the glucagon analogs described herein conjugated to a heterologous moiety,wherein the conjugate exhibits enhanced activity at the GLP-1 receptor, as compared to native glucagon, and exhibits at least lOO-fold greater ivity for the human GLP-1 receptor versus the GIP receptor. As used herein, the term “heterologous moiety” is synonymous with the term “conjugate moiety” and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the glucagon analogs described herein. Exemplary conjugate moieties that can be linked to any of the analogs described herein include but are not limited to a heterologous peptide or polypeptide (including for example, a plasma protein), a targeting agent, an globulin or portion thereof (e. g.,van'able , CDR, or Fc region), a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents. In some ments a conjugate is provided comprising an analog of the present invention and a plasma protein, wherein the plasma protein is selected from the group consisting of n, transferin, ogen and globulins. In some ments the plasma protein moiety of the conjugate is albumin or erin. The conjugate in some embodiments comprises one or more of the glucagon analogs described herein and one or more of: a peptide (which is distinct from the glucagon and/or GLP-1 or active glucagon analogs described herein), a polypeptide, a nucleic acid le, an antibody or fragment thereof, a polymer, a quantum dot, a small le, a toxin, a diagnostic agent, a ydrate, an amino acid.

In some embodiments, the heterologous moiety is a peptide which is distinct from the glucagon and/or GLP-1 receptor active analogs described herein and the conjugate is a fusion peptide or a chimeric peptide. In some embodiments, the heterologous moiety is a peptide extension of 1-21 amino acids. In specific embodiments, the extension is ed to the C-terminus of the glucagon analog, e.g., to amino acid at position 29.

In some specific aspects, the extension is a single amino acid or dipeptide. In ic embodiments, the extension comprises an amino acid selected from the group consisting of: a charged amino acid (e.g., a ve-charged amino acid (e. g., Glu), a positive-charged amino acid), an amino acid comprising a hydrophilic moiety. In some aspects, the extension is Gly, Glu, Cys, Gly-Gly, Gly-Glu.

In some embodiments, the extension comprises an amino acid sequence of SEQ ID NO: 9 (GPSSGAPPPS), SEQ ID NO: 10 (GGPSSGAPPPS), SEQ ID NO: 8 (KRNRNNIA), or SEQ ID NO: 11 (KRNR). In specific s, the amino acid ce is ed through the C-terminal amino acid of the glucagon analog, e.g., amino acid at position 29. In some embodiments, the amino acid sequence of SEQ ID NOs: 13-16 is bound to amino acid 29 of the glucagon analog through a peptide bond. In some specific embodiments, the amino acid at position 29 of the glucagon analog is a Gly and the Gly is fused to one of the amino acid sequences of SEQ ID NOs: 8-11.

In some embodiments, the logous moiety is a polymer. In some embodiments, the polymer is selected from the group consisting of: polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl rylate), poly(ethyl methacrylate), poly(buty1methacry1ate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), sobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl , polyvinyl esters, polyvinyl halides, poly(vinyl e), and polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co—polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose e phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt, opylene, polyethylenes including poly(ethylene glycol), thylene oxide), and poly(ethylene terephthalate), and polystyrene.

] In some aspects, the polymer is a biodegradable r, including a synthetic biodegradable polymer (e,g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, ethanes, poly(butic acid), poly(valeric acid), and poly(lactidecocaprolactone )), and a natural biodegradable r (e.g., alginate and other ccharides including dextran and cellulose, collagen, chemical derivatives thereof itutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), as well as any copolymer or mixture thereof. In general, these materials degrade either by enzymatic ysis or exposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as a bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(buty1methacrylate), poly(isobuty1 methacrylate), poly(hexy1methacry1ate), poly(isodecyl methacrylate), poly(laury1 methacrylate), poly(pheny1 methacrylate), poly(methy1 acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or a hilic polymer. Hydrophilic polymers are further described herein under “Hydrophilic Moieties.” Suitable water-soluble rs are known in the art and e, for e, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; ), ypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl ellulose, hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylic acid mers, polymethacrylic acid, polymethylmethacrylate, maleic anhydride/methyl vinyl ether mers, poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylic acid, acidic y polymers, ypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymer, polymethylvinylether co—maleic anhydride, carboxymethylamide, potassium methacrylate divinylbenzene co-polymer, polyoxyethyleneglycols, hylene oxide, and derivatives, salts, and combinations thereof.

In specific embodiments, the polymer is a polyalkylene , including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, ose, fructose), a disaccharide (e. g., sucrose, lactose, maltose), an oligosaccharide (e. g., raffinose, stachyose), a polysaccharide (a starch, amylase, ectin, cellulose, chitin, e, laminarin, xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, in some embodiments, is a fatty acid, eicosanoid, glandin, leukotriene, oxane, N-acyl ethanolamine), glycerolipid (e. g., mono—, di-, bstituted glycerols), glycerophospholipid (e. g., atidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), olipid (e. g., sphingosine, ceramide), sterol lipid (e.g., steroid, terol), prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fat—soluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.

In some embodiments, the heterologous moiety is attached via non-covalent or covalent bonding to the analog of the present disclosure. In certain aspects, the heterologous moiety is attached to the analog of the present disclosure Via a linker.

Linkage can be accomplished by covalent chemical bonds, physical forces such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.

A variety of non-covalent ng systems may be used, ing biotin-avidin, ligand/receptor, enzyme/substrate, c ucleic acid binding n, lipid/lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.

The glucagon analog in some embodiments is linked to conjugate moieties via direct covalent e by reacting targeted amino acid residues of the analog with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these ed amino acids. Reactive groups on the analog or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group. Derivatizing agents include, for example, idobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Alternatively, the conjugate moieties can be linked to the analog indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the ant loaded carrier.

Cysteinyl residues are most commonly reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid, chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are tized by reaction with bromotrifluoroacetone, alpha-bromo—B—(S—imidozoyl)propionic acid, chloroacetyl ate, lmaleimides, 3-nitro—2—pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri—4—nitrophenol, or chloronitrobenzooxa-l,3- diazole.

] Histidyl residues are derivatized by on with diethylpyrocarbonate at pH .5-7.0 because this agent is relatively specific for the histidyl side chain. Para- henacyl e also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. l and amino-terminal es are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino- containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. tization of ne residues requires that the on be performed in alkaline conditions because of the high pKa of the guanidine functional group.

Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, with ular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic ium nds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O—acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N—R‘), where R and R' are different alkyl groups, such as 1-cyclohexyl(2-morpholinylethyl) carbodiimide or l-ethyl(4-azonia—4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or yl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, ns: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine, and/or amidation or esterification of the C—terminal carboxylic acid group.

Another type of covalent cation involves chemically or tically coupling glycosides to the analog. Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of tyrosine, or tryptophan, or (f) the amide group of glutamine.

These methods are described in W087/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

In some embodiments, the glucagon analog is conjugated to a logous moiety via covalent linkage n a side chain of an amino acid of the glucagon analog and the heterologous . In some embodiments, the on analog is conjugated to a heterologou moiety via the side chain of an amino acid at position l6, 17, 21, 24, or 29, a on Within a C-terminal ion, or the C—terminal amino acid, or a combination of these positions. In some aspects, the amino acid covalently linked to a heterologous moiety (e. g., the amino acid sing a heterologous moiety) is a Cys, Lys, Orn, homo- Cys, or Ac-Phe, and the side chain of the amino acid is ntly bonded to a heterologous moiety.

In some embodiments, the conjugate comprises a linker that joins the glucagon analog to the heterologous moiety. In some aspects, the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In some ments, the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some ments, the length of the linker is long enough to reduce the potential for steric hindrance. If the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers may be any length. Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length. Such fusion proteins may alternatively be produced by recombinant genetic engineering methods known to one of ordinary skill in the art.

Con'u ates: Fc usions As noted above, in some embodiments, the analogs are conjugated, e.g., fused to an immunoglobulin or portion thereof (e.g.,variable region, CDR, or Fc region).

Known types of immunoglobulins (Ig) include IgG, IgA, IgE, IgD or IgM. The Fc region is a inal region of an Ig heavy chain, which is responsible for binding to Fc receptors that carry out activities such as recycling (which results in ged half-life), dy ent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fc region stretches from Cys226 to the C-terminus of the heavy chain. The "hinge region" generally extends from Glu216 to Pro230 of human IgG1 (hinge regions of other IgG isotypes may be aligned with the IgGl sequence by aligning the cysteines involved in cysteine bonding). The Fc region of an IgG includes two constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341. The CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447. References made to amino acid ing of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et a1. 1991, Sequences of Proteins of Immunological Interest, US. Department of Public Health, Bethesda, Md.

In a related ments, the Fc region may comprise one or more native or modified constant regions from an immunoglobulin heavy chain, other than CH1, for e, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequence that include the FcRn binding site. FcRn, a salvage receptor, is responsible for recycling immunoglobulins and returning them to circulation in blood. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography ister et al. 1994, Nature 9). The major contact area of the PC with the FcRn is near the junction of the CH2 and CH3 domains. Fc—FcRn contacts are all within a single Ig heavy chain. The major contact sites include amino acid residues 248, 7, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 7, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not e FcyR binding site(s). FcyR are responsible for ADCC and CDC. Examples of positions within the Fc region that make a direct contact with FcyR are amino acids 9 (lower hinge region), amino acids 265-269 (B/C 100p), amino acids 297—299 (C'/E 100p), and amino acids 327-332 (F/G) loop (Sondermann et al., Nature 406: 267-273, 2000). The lower hinge region of IgE has also been implicated in the Fch binding , et al., Biochemistry 36, 15568- 15578, 1997). Residues involved in IgA receptor binding are described in Lewis et al., (J Immunol. 175:6694-701, 2005). Amino acid residues involved in IgE receptor binding are described in Sayers et al. (J Biol Chem. 279(34):35320—5, 2004).

Amino acid modifications may be made to the Fc region of an immunoglobulin. Such variant Fc regions comprise at least one amino acid modification in the CH3 domain of the Fc region (residues 342-447) and/or at least one amino acid cation in the CH2 domain of the Fc region (residues 231-341). Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et a1. 2001, J. Biol. Chem. 27626591). Other mutations may reduce binding of the Fc region to FcyRI, A, FcyRIIB, and/or FcyRIIIA without significantly reducing affinity for FcRn. For e, substitution of the Asn at position 297 of the Fc region with Ala or another amino acid removes a highly conserved N-glycosylation site and may result in reduced immunogenicity with concomitant prolonged ife of the Fc , as well as d binding to FcyRs (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 2; Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modifications at positions 233—236 of IgG1 have been made that reduce binding to FcyRs (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 2912613). Some exemplary amino acid substitutions are described in US Patents 7,355,008 and 7,381,408, each incorporated by reference herein in its entirety.

Coniugates: Hydrophilic moieties The glucagon s described herein can be further modified to improve its solubility and stability in s solutions at physiological pH, while retaining the high biological activity relative to native glucagon. Hydrophilic moieties such as PEG groups can be ed to the analogs under any suitable conditions used to react a protein with an activated polymer molecule. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e. g., an aldehyde, amino, ester, thiol, a—haloacetyl, maleimido or hydrazino group) to a ve group on the target compound (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group). Activating groups which can be used to link the water e polymer to one or more ns include without limitation sulfone, maleimide, dryl, thiol, triflate, tresylate, azidirine, oxirane, 5-pyridyl, and alpha-halogenated acyl group (e. g., alpha-iodo acetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid).

If attached to the analog by reductive alkylation, the polymer selected should have a single ve aldehyde so that the degree of polymerization is controlled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev. 54: 4??-485 (2002); Roberts et al., Adv.

Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 .

In specific aspects, an amino acid residue of the analog having a thiol is modified with a hydrophilic moiety such as PEG. In some ments, the thiol is modified with maleimide-activated PEG in a Michael addition reaction to result in a PEGylated analog comprising the thioether linkage shown below: Peqflde O SngwONO)ECH H3 0 o In some embodiments, the thiol is modified with a haloacetyl-activated PEG in a nucleophilic substitution reaction to result in a PEGylated analog comprising the thioether linkage shown below: Pepfide LSWHWOVOIEHS Suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyols (e.g., POG), yethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde, copolymers of ne /propylene glycol, monomethoxy-polyethylene glycol, mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol, carboxymethylcellulose, etals, polyvinyl alcohol (PVA), nyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ne/maleic anhydride copolymer, poly (beta-amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxide copolymers, colonic acids or other ccharide polymers, Ficoll or dextran and mixtures thereof. Dextrans are polysaccharide polymers of glucose ts, predominantly linked by Oil-6 es.

Dextran is available in many molecular weight ranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branched rs are contemplated. Resulting preparations of conjugates may be essentially monodisperse or polydisperse, and may have about 0.5, 0.7, l, 1.2, 1.5 or 2 polymer moieties per analog.

In some embodiments, the on analog is ated to a hydrophilic moiety via covalent linkage between a side chain of an amino acid of the glucagon analog and the hydrophilic moiety. In some embodiments, the glucagon analog is conjugated to a hilic moiety via the side chain of an amino acid at position 16, 17, 21, 24, or 29, a position within a C-terminal extension, or the inal amino acid, or a combination of these positions. In some aspects, the amino acid covalently linked to a hydrophilic moiety (e. g., the amino acid comprising a hydrophilic moiety) is a Cys, Lys, Orn, homo- Cys, or Ac-Phe, and the side chain of the amino acid is covalently bonded to a hydrophilic moiety (e. g., PEG).

Coniugates: rPEG In some embodiments, the conjugate of the present disclosure comprises the analog having glucagon and/or GLP-l agonist activity fused to an accessory analog which is capable of forming an extended conformation similar to chemical PEG (e. g., a recombinant PEG (rPEG) molecule), such as those described in International Patent Application Publication No. WO2009/023270 and U.S. Patent Application Publication No. US20080286808. The rPEG molecule in some s is a polypeptide comprising one or more of glycine, serine, glutamic acid, aspartic acid, alanine, or proline. In some aspects, the rPEG is a homopolymer, e. g., poly-glycine, poly-serine, poly-glutamic acid, poly-aspartic acid, poly-alanine, or poly—proline. In other embodiments, the rPEG comprises two types of amino acids ed, e.g., poly(Gly—Ser), poly(Gly-Glu), poly(Gly-Ala), poly(Gly-Asp), poly(Gly-Pro), poly(Ser—Glu), etc. In some aspects, the rPEG comprises three different types of amino acids, e.g., poly(Gly—Ser—Glu). In ic aspects, the rPEG increases the half—life of the Glucagon and/or GLP—l agonist analog. In some aspects, the rPEG comprises a net positive or net ve charge. The rPEG in some aspects lacks secondary structure. In some embodiments, the rPEG is greater than or equal to 10 amino acids in length and in some embodiments is about 40 to about 50 amino acids in length. The ory e in some aspects is fused to the N— or C- terminus of the analog of the present disclosure through a peptide bond or a proteinase ge site, or is ed into the loops of the analog of the present disclosure. The rPEG in some aspects comprises an affinity tag or is linked to a PEG that is greater than 5 kDa. In some embodiments, the rPEG s the analog of the present disclosure with an increased hydrodynamic radius, serum half-life, protease resistance, or solubility and in some aspects confers the analog with decreased immunogenicity.

Coniugates: Multimers ] The invention further provides multimers or dimers of the analogs disclosed herein, including homo- or hetero- ers or homo— or hetero— dimers. Two or more of the analogs can be linked together using standard linking agents and procedures known to those skilled in the art. For example, dimers can be formed between two peptides through the use of bifunctional thiol crosslinkers and bi-functional amine crosslinkers, particularly for the analogs that have been tuted with cysteine, lysine ornithine, homocysteine or acetyl phenylalanine es. The dimer can be a homodimer or alternatively can be a heterodimer. In certain embodiments, the linker connecting the two (or more) analogs is PEG, e. g., a 5 kDa PEG, 20 kDa PEG. In some embodiments, the linker is a disulfide bond. For example, each monomer of the dimer may comprise a Cys residue (e. g., a terminal or internally positioned Cys) and the sulfur atom of each Cys residue participates in the formation of the disulfide bond. In some aspects, the monomers are connected via terminal amino acids (e.g., N-terminal or C-terminal), via al amino acids, or via a terminal amino acid of at least one monomer and an internal amino acid of at least one other monomer. In ic aspects, the rs are not connected via an N-terminal amino acid. In some s, the monomers of the multimer are attached together in a “tail-to-tail” orientation in which the inal amino acids of each monomer are attached together.

CEUTICAL COMPOSITIONS USES AND KITS Salts In some embodiments, the glucagon analog is in the form of a salt, e. g., a pharmaceutically acceptable salt. As used herein the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the analog, or separately prepared by reacting a free base function with a le acid. Many of the compounds sed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid on salts may be prepared from inorganic and organic acids. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, sodium bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, lfate, heptanoate, hexanoate, fumarate, hydrochloride, romide, odide, 2-hydroxyethansulfonate (isothionate), lactate, maleate, methane sulfonate, nate, 2-naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, ylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, anate, phosphate, glutamate, bicarbonate, p—toluenesulfonate, and undecanoate.

Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, ic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- e-sulfonic acid, salicylic acid, and the like. Examples of acids which can be ed to form pharmaceutically able acid addition salts e, for e, an inorganic acid, e. g., hydrochloric acid, romic acid, sulphuric acid, and phosphoric acid, and an organic acid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Basic addition salts also can be prepared in situ during the final isolation and purification of the source of salicylic acid, or by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, methylammonium, diethylammonium, and ethylammonium, amongst others. Other representative organic amines useful for the formation of base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.

Further, basic nitrogen-containing groups can be quaternized with the analog of the present disclosure as lower alkyl halides such as methyl, ethyl, , and butyl chlorides, bromides, and iodides; long chain s such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

Formulations ] In accordance with some embodiments, a pharmaceutical composition is provided n the ition ses a glucagon analog of the present disclosure, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The pharmaceutical composition can se any pharmaceutically acceptable ingredient, including, for example, acidifying , ves, adsorbents, aerosol propellants, air cement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing , dyes, emollients, emulsifying agents, emulsion izers, fillers, film forming , flavor enhancers, flavoring , flow enhancers, gelling agents, granulating agents, humectants, lubricants, hesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening , therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity- sing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one or a combination of the following components: acacia, acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum te, amylopectin, a-amylose, ascorbic acid, ascorbyl ate, aspartame, bacteriostatic water for injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl te, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, araben sodium, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, dibasic anhydrous m phosphate, dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calcium propionate, calcium silicate, m sorbate, calcium stearate, calcium sulfate, calcium sulfate hemihydrate, canola oil, er, carbon dioxide, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, B-carotene, carrageenan, castor oil, hydrogenated castor oil, ic fying wax, cellulose acetate, cellulose e phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethyl cellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil, cresol, m- cresol, o-cresol, p-cresol, croscarmellose , vidone, cyclamic acid, cyclodextrins, dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate, dibutyl te, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-B-cyclodextrin, cyclodextrin-type compounds such as Captisol®, dimethyl ether, dimethyl phthalate, dipotassium te, disodium edentate, disodium hydrogen phosphate, te calcium, docusate potassium, docusate sodium, dodecyl gallate, dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid, eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium, ethylparaben sodium, ethyl vanillin, se, fructose liquid, fructose milled, fructose pyrogen-free, powdered fructose, fumaric acid, n, glucose, liquid glucose, glyceride es of saturated vegetable fatty acids, glycerin, glyceryl behenate, glyceryl eate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitostearate, glycine, glycols, urol, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, high fructose syrup, human serum albumin, hydrocarbons (HC), dilute hydrochloric acid, hydrogenated vegetable oil, type II, yethyl cellulose, oxyethyl-B-cyclodextrin, hydroxypropyl cellulose, low-substituted hydroxypropyl ose, oxypropyl-B- cyclodextrin, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, imidurea, indigo carmine, ion exchangers, iron oxides, pyl alcohol, isopropyl myristate, pyl ate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols, anhydrous lanolin, lecithin, ium aluminum silicate, magnesium carbonate, normal magnesium carbonate, magnesium carbonate anhydrous, magnesium carbonate hydroxide, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium te, magnesium trisilicate, magnesium trisilicate anhydrous, malic acid, malt, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methyl methacrylate, methyl oleate, methylparaben, methylparaben potassium, methylparaben sodium, microcrystalline cellulose and ymethylcellulose , mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, ic acid, paraffin, peanut oil, petrolatum, atum and lanolin alcohols, pharmaceutical glaze, phenol, liquified , phenoxyethanol, phenoxypropanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin, polacrilin potassium, mer, polydextrose, hylene , polyethylene oxide, polyacrylates, polyethylene-polyoxypropylene-block polymers, polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene tes, polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, postassium e, potassium citrate anhydrous, potassium hydrogen phosphate, potassium metabisulfite, monobasic potassium phosphate, potassium propionate, potassium sorbate, povidone, propanol, nic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl e, propylparaben, propylparaben potassium, propylparaben sodium, protamine sulfate, rapeseed oil, Ringer's solution, saccharin, saccharin ammonium, saccharin calcium, rin sodium, safflower oil, saponite, serum ns, sesame oil, dal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride, sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium lauryl sulfate, sodium metabisulfite, sodium ate, dibasic, sodium phosphate, monobasic, sodium phosphate, tribasic, anhydrous sodium propionate, sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl te, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty esters), sorbitol, sorbitol solution "50%, soybean oil, ceti wax, , corn starch, potato starch, pregelatinized starch, sterilizable maize starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugars, compressible sugar, confectioner’s sugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), oma oil, thimerosal, titanium dioxide, alpha tocopherol, tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol, delta-tocopherol, gamma—tocopherol, tragacanth, tin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl—B—cyclodextrin, trimethyltetradecylammonium bromide, tris buffer, trisodium edentate, vanillin, type I hydrogenated vegetable oil, water, soft water, hard water, carbon dioxide-free water, pyrogen-free water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for irrigation, waxes, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or any excipient in the ok of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington ’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by nce in its entirety, discloses various components used in formulating pharmaceutically acceptable itions and known techniques for the preparation thereof. Except insofar as any conventional agent is incompatible with the pharmaceutical compositions, its use in pharmaceutical compositions is plated. Supplementary active ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing ent(s) may be present in the ceutical ition at any concentration, such as, for example, at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/V, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at most B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% wiv, 0.1% w/v, 0.001% w/v, or 0.0001%. In other embodiments, the foregoing component(s) may be t in the pharmaceutical composition at any tration range, such as, for e from about A to about B.

In some embodiments, A is 0.0001% and B is 90%.

The pharmaceutical compositions may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical ition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 11, ing on the ation and route of administration. In certain embodiments, the pharmaceutical compositions may comprise buffering agents to achieve a physiological compatible pH. The buffering agents may include any compounds capabale of buffering at the desired pH such as, for example, phosphate buffers (e. g.,PBS), triethanolamine, Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, late, MES, and others. In certain embodiments, the strength of the buffer is at least 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 120 mM, at least 150 mM, or at least 200 mM. In some embodiments, the strength of the buffer is no more than 300 mM (e.g.,at most 200 mM, at most 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM, at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10 mM, at most 5 mM, at most 1 mM).

Routes of Administration ] The following discussion on routes of administration is merely provided to illustrate exemplary embodiments and should not be construed as limiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the analog of the present disclosure ved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, s, lozenges, and troches, each containing a ermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) le emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, e, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, m stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, ing agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the analog of the present disclosure in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the analog of the t disclosure in an inert base, such as n and in, or sucrose and , emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.

The s of the disclosure, alone or in combination with other suitable components, can be delivered via pulmonary administration and can be made into l formulations to be administered via inhalation. These aerosol formulations can be placed into rized acceptable lants, such as dichlorodifluoromethane, propane, en, and the like. They also may be ated as pharmaceuticals for nonpressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. In some embodiments, the analog is formulated into a powder blend or into microparticles or nanoparticles. Suitable pulmonary formulations are known in the art. See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei and Garren, Pharmaceutical ch, 7(6): 565-569 (1990); Kawashima et a1., J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993); International Patent Application ation Nos. WO 2007!133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous and non- s, isotonic sterile injection ons, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, uscular, intraspinal, or intravenous. The analog of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2— dimethyl-153—dioxolanemethanol, , poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a ceutically acceptable tant, such as a soap or a detergent, suspending agent, such as pectin, ers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations e oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) ic detergents such as, for example, dimethyl dialkyl ammonium s, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic ents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-B-aminopropionates, and 2-alkyl -imidazoline quaternary ammonium salts, and (6) mixtures thereof.

The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition ing only the on of the e liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile s, granules, and tablets of the kind known in the art.

Injectable formulations are in accordance with the invention. The ements for effective pharmaceutical carriers for injectable compositions are well- known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622- 630 (1986)).

Additionally, the analog of the present sures can be made into suppositories for rectal stration by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. ations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the analog of the disclosure can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

Dose The analogs of the disclosure are ed to be useful in methods of treating a disease or medical condition in which glucagon receptor agonism, GLP-1 receptor agonism, or Glucagon receptor/GLP-l receptor nism plays a role. For purposes of the disclosure, the amount or dose of the analog of the present sure administered should be sufficient to , e.g., a eutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the analog of the present disclosure should be sufficient to stimulate CAMP ion from cells as described herein or sufficient to decrease blood glucose levels, fat levels, food intake levels, or body weight of a mammal, in a period of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks or longer, e. g., 5 to 20 or more weeks, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular analog of the present disclosure and the condition of the animal (e. g., human), as well as the body weight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art. For purposes herein, an assay, which ses ing the extent to which blood glucose levels are lowered upon administration of a given dose of the analog of the present disclosure to a mammal among a set of mammals of which is each given a different dose of the analog, could be used to determine a starting dose to be administered to a mammal.

The extent to which blood glucose levels are lowered upon administration of a certain dose can be assayed by methods known in the art, including, for instance, the methods described herein as Example 4.

The dose of the analog of the present disclosure also will be determined by the existence, nature and extent of any e side effects that might accompany the administration of a particular analog of the present disclosure. Typically, the attending physician will decide the dosage of the analog of the present disclosure with which to treat each individual patient, taking into consideration a y of factors, such as age, body , general health, diet, sex, analog of the present disclosure to be administered, route of administration, and the severity of the condition being treated. By way of e and not intending to limit the invention, the dose of the analog of the present sure can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg body weight/day.

In some embodiments, the pharmaceutical composition comprises any of the analogs disclosed herein at a purity level suitable for administration to a patient. In some embodiments, the analog has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharmaceutically acceptable diluent, carrier or ent. The pharmaceutical composition in some aspects comprise the analog of the present disclosure at a concentration of at least A, wherein A is about about 0.001 mg/ml, about 0.01 mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mgi'ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml or higher. In some embodiments, the pharmaceutical composition comprises the analog at a concentration of at most B, wherein B is about 30 mg/ml, about mg/ml, about 24 mg/ml, about 23, mgx’ml, about 22 mgi’ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18 mgfml, about 17 mgi’ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml, about 13 mgfml, about 12 mgfml, about 11 mg/ml, about 10 mg/ml, about 9 mg/ml, about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about 4 mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1 mg/ml. In some embodiments, the itions may contain an analog at a concentration range of A to B mg/ml, for example, about 0.001 to about 30.0 mg/ml.

Targeted Farms One of ordinary skill in the art will y appreciate that the analogs of the disclosure can be modified in any number of ways, such that the therapeutic or prophylactic efficacy of the analog of the present disclosures is increased through the cation. For instance, the analog of the present disclosure can be conjugated either directly or indirectly h a linker to a targeting moiety. The practice of conjugating compounds, e.g., glucagon analogs described , to targeting es is known in the art. See, for instance, Wadhwa et a1., J Drug Targeting, 3, 111—127 (1995) and US.

Patent No. 5,087,616. The term "targeting moiety" as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the ing moiety directs the delivery of the analog of the present disclosures to a population of cells on which surface the receptor (the glucagon receptor, the GLP-1 receptor) is expressed. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, es, hormones, growth factors, cytokines, and any other natural or non-natural ligands, which bind to cell surface receptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cell receptor (TCR), B—cell receptor (BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.). As used herein a "linker" is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers may e for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid—labile moieties, base-labile es and enzyme-cleavable groups. The term "linker" in some embodiments refers to any agent or molecule that bridges the analog of the present disclosures to the targeting moiety. One of ordinary skill in the art recognizes that sites on the analog of the present disclosures, which are not necessary for the function of the analog of the present disclosures, are ideal sites for attaching a linker andfor a ing moiety, provided that the linker and/or targeting moiety, once attached to the analog of the present disclosures, do(es) not interfere with the function of the analog of the present disclosures, i.e., the ability to stimulate CAMP secretion from cells, to treat es or obesity.

Controlled Release Formulations Alternatively, the glucagon analogs described herein can be modified into a depot form, such that the manner in which the analog of the present disclosures is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of analog of the present disclosures can be, for example, an implantable composition sing the analog of the present disclosures and a porous or non-porous material, such as a polymer, n the analog of the present sures is encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the analog of the present disclosures are released from the implant at a predetermined rate.

] The pharmaceutical composition in certain aspects is modified to have any type of in vivo release profile. In some aspects, the ceutical composition is an immediate e, controlled release, sustained release, extended release, delayed e, or bi-phasic e formulation. Methods of formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publication Nos. 58, W02004/033036; WO2000/032218; and .

The instant compositions may further se, for e, micelles or liposomes, or some other encapsulated form, or may be stered in an extended release form to provide a prolonged storage and/or delivery effect. The disclosed pharmaceutical formulations may be administered according to any regime including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, every three weeks, monthly, or thly.

Combinations The glucagon analogs described herein may be administered alone or in combination with other eutic agents which aim to treat or prevent any of the diseases or medical conditions described herein. For example, the glucagon s described herein may be co-administered with (simultaneously or sequentially) an anti- diabetic or anti-obesity agent. Anti-diabetic agents known in the art or under investigation e insulin, leptin, Peptide YY (PYY), Pancreatic Peptide (PP), fibroblast growth factor 21 (FGFZl), Y2Y4 receptor agonists, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), iride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); ides such as min (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase—4 (DPP-4) inhibitors such as liptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) tors; glucokinase activators (GKA); glucagon receptor antagonists (GRA); or FBPase (fructose 1,6- bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under igation include appetite suppressants, including phenethylamine type stimulants, phentermine (optionally with amine or dexfenfluramine), diethylpropion (Tenuate®), phendimetrazine (Prelu- 2®, 1®), etamine (Didrex®), sibutramine (Meridia®, Reducti1®); rimonabant (Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide) or Contrave (bupropion and xone); or lipase inhibitors, similar to XENICAL (Orlistat) or Cetilistat (also known as ATL-962), or GT 389-255.

The es described herein in some embodiments are co-administered with an agent for ent of non-alcoholic fatty liver disease or NASH. Agents used to treat non-alcoholic fatty liver disease include oxycholic acid (a.k.a., Actigall, URSO, and Ursodiol), Metformin (Glucophage), rosiglitazone (Avandia), Clofibrate, rozil, Polymixin B, and Betaine.

The peptides described herein in some embodiments are inistered with an agent for treatment of a neurodegenerative disease, e.g., Parkinson’s Disease. Anti- Parkinson’s e agents are furthermore known in the art and include, but not limited to, levodopa, carbidopa, anticholinergics, bromocriptine, pramipexole, and ropinirole, amantadine, and line.

In view of the foregoing, the invention further provides pharmaceutical compositions and kits additionally comprising one of these other therapeutic agents. The additional therapeutic agent may be administered simultaneously or sequentially with the analog of the t disclosure. In some aspects, the analog is administered before the additional therapeutic agent, while in other aspects, the analog is administered after the additional therapeutic agent.

Uses It is contemplated that the glucagon analogs described herein and related pharmaceutical compositions are useful for treatment of a disease or medical condition, in which e. g., the lack of activity at the glucagon receptor, the GLP-1 receptor, or at both receptors, is a factor in the onset and/or progression of the disease or l condition.

Accordingly, the invention provides a method of treating or preventing a disease or l condition in a patient, wherein the disease or medical ion is a disease of l condition in which a lack of GLP—1 receptor tion and/or glucagon receptor activation is associated with the onset and/or progression of the disease of medical condition. The method comprises providing to the patient an analog in accordance with any of those described herein in an amount effective to treat or prevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolic syndrome. Metabolic Syndrome, also known as metabolic syndrome X, insulin resistance syndrome or Reaven‘s syndrome, is a disorder that affects over 50 million Americans.

Metabolic Syndrome is typically characterized by a clustering of at least three or more of the ing risk factors: ( 1) abdominal y (excessive fat tissue in and around the n), (2) atherogenic dyslipidemia (blood fat disorders including high cerides, low HDL cholesterol and high LDL cholesterol that enhance the accumulation of plaque in the artery walls), (3) elevated blood pressure, (4) insulin resistance or glucose rance, (5) prothrombotic state (e.g.,high fibrinogen or nogen activator inhibitor-1 in blood), and (6) pro-inflammatory state (e.g.,elevated C-reactive protein in blood). Other risk factors may include aging, hormonal nce and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronary heart disease and other disorders related to the accumulation of vascular plaque, such as stroke and peripheral vascular disease, referred to as atherosclerotic cardiovascular disease (ASCVD). Patients with Metabolic Syndrome may progress from an n resistant state in its early stages to full blown type II diabetes with r increasing risk of ASCVD. Without intending to be bound by any particular theory, the relationship between insulin resistance, Metabolic Syndrome and vascular disease may involve one or more concurrent pathogenic mechanisms including ed insulin-stimulated vasodilation, insulin ance—associated reduction in NO availability due to ed oxidative stress, and alities in adipocyte—derived hormones such as adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B—76B (2004)).

According to the 2001 National Cholesterol Education Program Adult Treatment Panel (ATP 111), any three of the following traits in the same individual meet the ia for Metabolic Syndrome: (a) abdominal obesity (a waist ference over 102 cm in men and over 88 cm in women); (b) serum triglycerides (150 mg/dl or above); (c) HDL cholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women); (d) blood pressure (130/85 or more); and (e) fasting blood glucose (110 mg/dl or above).

According to the World Health Organization (WHO), an individual having high insulin levels (an ed fasting blood glucose or an elevated post meal glucose alone) with at least two of the following criteria meets the criteria for Metabolic Syndrome: (a) abdominal obesity (waist to hip ratio of r than 0.9, a body mass index of at least 30 kg/m2, or a waist measurement over 3? inches); (b) cholesterol panel showing a triglyceride level of at least 150 mgi'dl or an HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 or more, or on treatment for high blood pressure). (Mathur, Ruchi, “Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May 11, 2009).

] For purposes herein, if an individual meets the criteria of either or both of the criteria set forth by the 2001 National Cholesterol Education m Adult Treatment Panel or the WHO, that individual is considered as afflicted with Metabolic Syndrome. t being bound to any particular theory, es described herein are useful for treating Metabolic Syndrome. Accordingly, the invention provides a method of preventing or treating Metabolic Syndrome, or reducing one, two, three or more risk factors thereof, in a subject, comprising providing to the subject an analog described herein in an amount effective to prevent or treat Metabolic Syndrome, or the risk factor thereof.

In some embodiments, the method treats a hyperglycemic medical condition.

In certain aspects, the hyperglycemic medical condition is diabetes, diabetes mellitus type I, diabetes mellitus type II, or gestational diabetes, either insulin-dependent or non- insulin-dependent. In some aspects, the method treats the hyperglycemic medical condition by reducing one or more complications of diabetes including nephropathy, retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In some aspects, the obesity is drug-induced obesity. In some aspects, the method treats y by preventing or reducing weight gain or sing weight loss in the patient. In some aspects, the method treats obesity by reducing appetite, decreasing food intake, ng the levels of fat in the t, or decreasing the rate of movement of food through the gastrointestinal .

Because obesity is associated with the onset or progression of other diseases, the s of treating obesity are further useful in methods of reducing complications associated with y including vascular e (coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, onset of diabetes type II, hyperlipidemia and musculoskeletal diseases. The invention accordingly provides methods of treating or preventing these obesity—associated complications.

In some embodiments, the disease or medical condition is Nonalcoholic fatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liver disease ranging from simple fatty liver (steatosis), to oholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). All of the stages of NAFLD have in common the accumulation of fat (fatty infiltration) in the liver cells (hepatocytes).

Simple fatty liver is the abnormal accumulation of a certain type of fat, ceride, in the liver cells with no inflammation or scarring. In NASH, the fat accumulation is associated with varying degrees of inflammation itis) and g (fibrosis) of the liver. The inflammatory cells can destroy the liver cells ocellular necrosis). In the terms "steatohepatitis" and "steatonecrosis", steato refers to fatty infiltration, hepatitis refers to ation in the liver, and necrosis refers to destroyed liver cells. NASH can ultimately lead to scarring of the liver (fibrosis) and then irreversible, advanced scarring (cirrhosis). Cirrhosis that is caused by NASH is the last and most severe stage in the NAFLD spectrum. (Mendler, Michel, “Fatty Liver: Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH),” ed. Schoenfield, Leslie J ., MedicineNet.com, August 29, 2005).

Alcoholic Liver Disease, or Alcohol—Induced Liver Disease, encompasses three pathologically distinct liver diseases related to or caused by the excessive consumption of alcohol: fatty liver (steatosis), chronic or acute hepatitis, and cirrhosis.

Alcoholic hepatitis can range from a mild hepatitis, with abnormal laboratory tests being the only indication of disease, to severe liver dysfunction with complications such as jaundice (yellow skin caused by bilirubin retention), c encephalopathy (neurological dysfunction caused by liver failure), ascites (fluid accumulation in the abdomen), bleeding esophageal varices (varicose veins in the esophagus), abnormal blood clotting and coma. Histologically, alcoholic hepatitis has a characteristic ance with ballooning degeneration of cytes, inflammation with neutrophils and sometimes Mallory bodies (abnormal aggregations of cellular intermediate filament proteins).

Cirrhosis is characterized anatomically by widespread nodules in the liver combined with fibrosis. (Worman, Howard J ., “Alcoholic Liver Disease”, Columbia University Medical Center website).

Without being bound to any particular theory, the analogs bed herein are useful for the treatment of Alcoholic Liver Disease, NAFLD, or any stage thereof, ing, for example, steatosis, hepatitis, hepatitis, c inflammation, NASH, sis, or complications thereof. Accordingly, the invention provides a method of preventing or treating lic Liver Disease, NAFLD, or any stage thereof, in a subject comprising providing to a subject an analog described herein in an amount effective to prevent or treat Alcoholic Liver Disease, NAFLD, or the stage f. Such treatment s include reduction in one, two, three or more of the following: liver fat content, incidence or progression of cirrhosis, incidence of cellular carcinoma, signs of ation, e.g.,abnormal hepatic enzyme levels (e.g., aspartate aminotransferase AST and/or alanine aminotransferase ALT, or LDH), elevated serum ferritin, elevated serum bilirubin, and/or signs of fibrosis, levated TGF—beta levels. In preferred embodiments, the peptides are used treat patients who have progressed beyond simple fatty liver osis) and exhibit signs of inflammation or hepatitis. Such methods may result, for example, in reduction of AST and/or ALT levels.

GLP-1 and exendin-4 have been shown to have some neuroprotective effect.

The invention also provides uses of the on analogs described herein in treating neurodegenerative diseases, including but not limited to Alzheimer's disease, Parkinson's e, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, other demyelination related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS- ated ia, or other dementias, a central nervous system cancer, traumatic brain injury, spinal cord injury, stroke or cerebral ia, cerebral vasculitis, epilepsy, gton‘s disease, Tourette‘s syndrome, in Barre syndrome, Wilson e, Pick‘s disease, neuroinflammatory disorders, encephalitis, encephalomyelitis or meningitis of viral, fungal or bacterial origin, or other central nervous system infections, prion diseases, cerebellar ataxias, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy, ssive supranuclear palsy, ia, muscle spasticity, tremor, retinitis pigmentosa, striatonigral degeneration, mitochondrial encephalo-myopathies, neuronal ceroid lipofuscinosis, hepatic encephalopathies, renal encephalopathies, metabolic encephalopathies, toxin- induced encephalopathies, and radiation-induced brain damage.

In some embodiments, the disease or medical condition is hypoglycemia. In some embodiments, the patient is a diabetic patient and the hypoglycemia is induced by the administration of insulin. In specific aspects, the method comprises providing the analog of the present disclosure in combination with insulin so that the analog buffers the ycemic effects of the bolus administration of insulin.

In some embodiments, the glucagon analogs are used in conjunction with parenteral administration of nutrients to non-diabetic patients in a hospital setting, e. g., to patients receiving parenteral ion or total parenteral nutrition. Nonlimiting examples include surgery patients, ts in comas, patients with digestive tract illness, or a nonfunctional gastrointestinal tract (e.g. due to surgical removal, blockage or impaired absorptive capacity, s disease, ulcerative s, gastrointestinal tract obstruction, gastrointestinal tract fistula, acute pancreatitis, ischemic bowel, major gastrointestinal surgery, certain congenital gastrointestinal tract anomalies, prolonged ea, or short bowel syndrome due to surgery, ts in shock, and patients oing healing processes often receive parenteral administration of carbohydrates along with various combinations of lipids, electrolytes, minerals, vitamins and amino acids. The glucagon analogs and the eral nutrition composition can be administered at the same time, at different times, before, or after each other, provided that the glucagon analog is exerting the desired biological effect at the time that the parenteral nutrition composition is being digested. For example, the parenteral ion may be administered, 1, 2 or 3 times per day, while the glucagon analog is administered once every other day, three times a week, two times a week, once a week, once every 2 weeks, once every 3 weeks, or once a month.

] As used herein, the terms "treat," and "prevent" as well as words stemming rom, do not necessarily imply 100% or complete ent or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill hi the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a disease or medical condition in a mammal. Furthermore, the ent or prevention provided by the method can include treatment or prevention of one or more conditions or symptoms of the e or l condition. For example, with regard to methods of treating obesity, the method in some embodiments, achieves a decrease in food intake by or fat levels in a patient. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

With regard to the above methods of treatment, the patient is any host. In some embodiments, the host is a mammal. As used herein, the term “mammal” refers to any vertebrate animal of the mammalia class, including, but not limited to, any of the monotreme, ial, and placental taxas. In some embodiments, the mammal is one of the mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. In certain embodiments, the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). In certain embodiments, the mammals are from the order actyla, ing Bovines (cows) and S wines (pigs) or of the order Perssodactyla, including Equines (horses). In some instances, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids s and apes). In particular embodiments, the mammal is a human.

The on analogs of the present disclosure can be provided in accordance with one embodiment as part of a kit. Accordingly, in some embodiments, a kit for administering a glucagon analog, e. g., a glucagon agonist peptide, to a patient in need thereof is provided wherein the kit comprises a glucagon analog as described herein.

In one embodiment the kit is ed with a device for administering the glucagon composition to a patient, e.g.,syringe needle, pen device, jet injector or other needle-free or. The kit may alternatively or in addition include one or more containers, e. g., vials, tubes, bottles, single or multi-chambered pre-filled syringes, cartridges, infusion pumps (external or implantable), jet injectors, pre-filled pen devices and the like, optionally containing the glucagon analog in a lyophilized form or in an aqueous solution. The kits in some embodiments comprise instructions for use. In accordance with one embodiment the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device. In another embodiment the kit comprises a e and a , and in one ment the e glucagon composition is prepackaged within the syringe.

The following examples are given merely to illustrate the present invention and not in any way to limit its scope.

EXAMPLES EXAMPLE 1 Synthesis ofpeptide nts agon Materials: All peptides described herein in the EXAMPLES were ed unless specified otherwise.

MBHA resin (4-methylbenzhydrylamine polystyrene resin was used during peptide synthesis. MBHA resin, 100—180 mesh, 1% DVB cross-linked polystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmoc protected amino acids were purchased from Midwest Biotech. The solid phase peptide syntheses using Boc-protected amino acids were performed on an Applied Biosystem 430A Peptide Synthesizer. Fmoc protected amino acid synthesis was med using the d tems Model 433 Peptide Synthesizer.

Peptide synthesis (Boc amino acids/ HF cleavage): Synthesis of these analogs was performed on the Applied Biosystem Model 430A Peptide Synthesizer. Synthetic peptides were constructed by sequential on of amino acids to a cartridge containing 2 mmol of Boc ted amino acid. Specifically, the synthesis was carried out using Boc DEPBT—activated single couplings. At the end of the coupling step, the peptidyl-resin was treated with TFA to remove the N-terminal Boc protecting group. It was washed repeatedly with DMF and this repetitive cycle was repeated for the desired number of coupling steps. After the assembly, the sidechain protection, Fmoc, was removed by 20% piperidine treatment and acylation was conducted using DIC. The peptidyl-resin at the end of the entire sis was dried by using DCM, and the peptide was cleaved from the resin with anhydrous HF.

For the lactamization, orthogonal protecting groups were selected for Glu and Lys (e.g.,Glu(Fm), Lys(Fmoc)). After removal of the ting groups and before HF ge, cyclization was performed as described previously (see, e. g., International Patent Application ation No. WO2008/101017).

HF treatment of the yf—resin The yl-resin was treated with anhydrous HF, and this typically yielded approximately 350 mg (~50% yield) of a crude deprotected-peptide. Specifically, the peptidyl-resin (30mg to 200mg) was placed in the hydrogen e (HF) reaction vessel for cleavage. 500 uL of p-cresol was added to the vessel as a carbonium ion scavenger.

The vessel was attached to the HF system and submerged in the methanol/dry ice mixture. The vessel was ted with a vacuum pump and 10 ml of HF was distilled to the reaction . This reaction mixture of the yl-resin and the HF was stirred for one hour at 00 C, after which a vacuum was established and the HF was quickly evacuated (10-15 min). The vessel was removed lly and filled with approximately ml of ether to precipitate the e and to extract the ol and small molecule organic protecting groups resulting from HF treatment. This mixture was filtered utilizing a teflon filter and repeated twice to remove all excess cresol. This filtrate was discarded. The precipitated peptide dissolves in approximately 20 ml of 10% acetic acid (aq). This filtrate, which contained the desired peptide, was collected and lyophilized.

An analytical HPLC analysis of the crude solubilized peptide was conducted under the following conditions [4.6 X 30 mm Xterra C8, 1.50 mL/min, 220 nm, A buffer 0.1% TFA/10% ACN, B buffer 0.1% TFAi’100% ACN, gradient 5—95%B over 15 minutes]. The extract was diluted twofold with water and loaded onto a 2.2 X 25 cm Vydac C4 preparative reverse phase column and eluted using an acetonitrile gradient on a Waters HPLC system (A buffer of 0.1% TFA/10% ACN, B buffer of 0.1 % TFA/10% CAN and a gradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLC analysis of the purified peptide demonstrated greater than 95% purity and electrospray ionization mass spectral analysis was used to confirm the identity of the e.

Peptide Acylation Acylated peptides were prepared as follows. es were synthesized on a solid support resin using either a CS Bio 4886 Peptide Synthesizer or d Biosystems 430A Peptide Synthesizer. In situ lization chemistry was used as described by Schnolzer et al., Int. J. Peptide Protein Res. 40: 3 (1992). For acylated peptides, the target amino acid residue to be acylated (e.g., position ten, relative to the amino acid position numbering of SEQ ID NO: 3) was tuted with an N a —FMOC lysine residue.

Treatment of the completed N—terminally BOC protected e with 20% piperidine in DMF for 30 minutes removed FMOC/formyl groups. Coupling to the free s—amino Lys e was achieved by coupling a ten-fold molar excess of either an FMOC-protected spacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex. CH3(CH2)14-COOH)and PyBOP or DEPBT coupling reagent in DMF/DIEA. Subsequent removal of the spacer amino acid’s FMOC group is followed by repetition of coupling with an acyl chain. Final treatment with 100% TFA resulted in removal of any side chain protecting groups and the N-terminal BOC group. Peptide resins were neutralized with 5% DlEA/DMF, dried, and then cleaved from the support using HFx'p—cresol, 95:5, at 0°C for one hour. Following ether extraction, a 5% HOAc on was used to solvate the crude e. A sample of the solution was then verified to contain the correct molecular weight peptide by ESI-MS.

Correct es were purified by RP-HPLC using a linear gradient of 10% CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm x 250 mm protein column was used for the cation. Acylated peptide analogs generally completed elution by a buffer ratio of 20:80. Portions were pooled together and checked for purity on an analytical RP-HPLC. Pure fractions were lyophilized yielding white, solid peptides.

If a peptide comprised a lactam bridge and target residues to be acylated, acylation is carried out as described above upon addition of that amino acid to the e backbone.

Peptide PEGylation For peptide PEGylation, 40 kDa methoxy poly(ethylene glycol) tamide (NOF) was reacted with a molar equivalent of peptide in 7M Urea, 50mM Cl buffer using the minimal amount of solvent needed to dissolve both peptide and PEG into a clear solution (generally less than 2 mL for a reaction using 2-3 mg peptide). Vigorous stirring at room temperature commenced for 4—6 hours and the reaction analyzed by analytical RP-HPLC. PEGylated products appeared distinctly from the starting al with decreased retention times. Purification was performed on a Vydac C4 column with conditions similar to those used for the initial e purification. Elution occurred around buffer ratios of 50:50. Fractions of pure PEGylated peptide were found and lyophilized. Yields were above 50%, varying per reaction.

Analysis using mass spectrometry ] The mass spectra were obtained using a Sciex API-III electrospray quadrapole mass spectrometer with a standard ESI ion source. Ionization conditions that were used are as follows: ESI in the positive-ion mode; ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing and curtain gas used was nitrogen flow rate of .9 L/min. Mass spectra were recorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwell time. The sample (about 1mg/mL) was dissolved in 50% aqueous acetonitrile with 1% acetic acid and introduced by an external e pump at the rate of 5 uL/min.

When the peptides were analyzed in PBS solution by ESI MS, they were first ed using a ZipTip solid phase extraction tip containing 0.6 uL C4 resin, according to instructions provided by the manufacturer (Millipore Corporation, Billerica, MA, see the Millipore website of the world wide web at millipore.com/catalogue.nsf/docs/C5737).

High Performance Liquid Chromatography (HPLC) analysis: Preliminary es were med with these crude peptides to get an approximation of their relative conversion rates in ate Buffered Saline (PBS) buffer (pH, 7.2) using high performance liquid chromatography (HPLC) and MALDI analysis. The crude peptide s were dissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of the resulting solution was stored in a 1.5 ml HPLC vial which was then sealed and incubated at 37 OC. ts of 100p] were drawn out at various time intervals, cooled to room temperature and analyzed by HPLC.

The HPLC es were performed using a Beckman System Gold Chromatography system using a UV detector at 214 nm. HPLC analyses were performed on a 150 mm x 4.6 mm C18 Vydac column. The flow rate was 1 ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent B ned 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to 70%B in 15 minutes). The data were collected and analyzed using Peak Simple Chromatography software.

] The l rates of hydrolysis were used to measure the rate constant for the dissociation of the respective prodrugs. The concentrations of the prodrug and the drug were estimated from their peak areas respectively. The first order dissociation rate constants of the prodrugs were determined by ng the logarithm of the concentration of the prodrug at various time intervals. The slope of this plot gives the rate constant ‘k’.

The half lives of the degradation of the various prodrugs were then calculated by using the formula tl/2 = .693/k.

EXAMPLE 2 Synthesis of the Peptide ofSeq ID No. I 7 The peptide seq ID no. 17 was synthesized by solid phase using Fmoc/t-Bu chemistry. For the preparation of the e, 0.5 g of 2—Chlorotrityl—resin (100-200 mesh, 1.18 mmol/g) (GL Biochem) were first derivatized by tion with 0.8 equivalents (eq.). ln(Trt)-OH, 4 eq. relative to amino acid diisopropylethylamine (DIPEA) in dry DCM oromethane) for 2 hours. The peptide sequence assembly was then performed on a peptide ynthesizer APEX396 (Advanced Biotech). All the amino acids were dissolved at a 0.5M concentration in DMF (N,N—dimethyl formamide). The acylation reactions were performed for 60 min with 6—fold excess of activated amino acid over the resin free amino groups. Double acylations were performed for Aib2, Aibl6, Asp15, Hisl. The amino acids were activated with equimolar amounts of HATU (2-(lH- 7-Azabenzotriazol-l-yl)-l,l ,3,3-tetramethyl uronium orophosphate), solution 0.5 M in DMF, and a 2-fold molar excess of DIEA (N,N—diisopropylethylamine), solution 2 M in NMP (l-methylpyrrolidinone).

The side chain protecting groups were: tert-butyl for Asp and Glu, Ser, Thr and Tyr; trityl for Asn, Gln and His; tert—butoxy-carbonyl for Lys, Trp; and, 2,2,4,6,7- pentamethyldihydrobenzofuransulfonyl for Arg; Boc-His(Trt)-OH was used in the synthesis.

The lysine at position 10, to be derivatized on the side chain, was incorporated as loc). At the end of the assembly the Alloc protecting group was removed and the synthesis was completed by condensation of the two y—carboxyglutamic acid residues (4 eq.) and the palmitic acid (4 eq.) using HBTU (4 eq.; (O-benzotriazol-l-yl-N,N,N',N'— tetramethyluronium hexafluorophosphate), and DIPEA (8 eq.) as activators.

At the end of the synthesis, the dry peptide-resins were individually treated with 25 mL of the cleavage mixture, 88% trifluoroacetic acid (TFA), 5% phenol, 2% triisopropylsilane and 5% water for 2 hours at room ature. The resin was filtered and the volume of the solution was reduced then added to cold -t-butyl ether in order to precipitate the peptide. After centrifugation, the peptide pellets were washed with fresh cold -t-butyl ether to remove the organic scavengers. The process was repeated twice. Final pellets were dried, ended in H20, 20% acetonitrile, and lyophilized.

The crude peptides were purified by reverse-phase HPLC using preparative Waters XBridge C18 (50 X 150 mm, 5 pm) and using as s (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile. Analytical UPLC was performed on a Waters Chromatograph, with a BEH130, C18 Acquity 1.7 pm , 2.1 x 100 mm, s), at 45 °C, using H20, 0.1% TFA (A) and CH3CN, 0.1% TFA (B) as solvents.

The purified peptide was characterized by electrospray mass spectrometry on a Waters SQ Detector.

EXAMPLE 3 The y of each peptide to induce cAMP was measured in a firefly luciferase-based reporter assay. The CAMP tion that is induced is directly proportional to the glucagon fragment binding to the glucagon or GLP-1 receptor.

HEK293 cells co-transfected with the glucagon or GLP—1 receptor, respectively, and rase gene linked to a CAMP sive element were employed for the bioassay.

The cells were serum-deprived by culturing 16 hours in Dulbecco-modified Minimum Essential Medium (Invitrogen, Carlsbad, CA) supplemented with 0.25% Bovine Growth Serum (HyClone, Logan, UT) and then incubated with serial dilutions of glucagon fragments for 5 hours at 37 0C, 5% CO2 in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences, San Jose, CA). At the end of the incubation, 100 ML of LucLite luminescence substrate t (Perkin Elmer, Wellesley, MA) were added to each well. The plate was shaken briefly, incubated 10 min in the dark and light output was measured on MicroBeta-1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). The effective 50% trations (EC50) were calculated by using Origin software (OriginLab, Northampton, MA).

EXAMPLE 4 The in vitro t potencies of the peptide of SEQ ID NO. 17 were determined using Chinese Hamster Ovary cells (CHO-Kl) stably expressing human GLP- 1R, GCGR or GIPR. The GLP-lR and GCGR stable cell line isogenic pools were generated by InvitrogenTM using their Jump-InTM targeted ation technology, whereas the stable CHO/GIPR lines were generated through classic cloning and transfection techniques (random integration) with limited dilution for selection of clones.

The es were tested in vitro for their relative abilities to stimulate CAMP tion in the various cell lines using the DiscoveRX HithunterTM CAMP XS+ assay kit (cat# 90- 0075L) as per kit instructions. The peptide 50% effective concentrations (EC50s) were calculated by non-linear regression analysis 4—parameter curve fitting with GraphPad Prism 4 software (GraphPad Software, San Diego, CA) by plotting luminescence values versus e doses.

EXAMPLE 5 es having the amino acid sequences as described in the sequence listing were made as ially described in Example 1 or 2 and subsequently tested for in vitro agonist activity at each of the glucagon receptor and GLP-1 receptor as essentially described in Example 3 or 4. In some instances, the in vitro agonist activity at the GIP receptor was also tested. The results are shown below in Table l. a hoe 08m 900 .

E0 ouch 0020 mmo‘mmfi 000 000; 000; 000m 08 03m hava. :25 350 00“: 0.000 $90 000.0 000.0 90.0 000.0 8208on 8:80on 08m sownuaw have 05 900 . :0 ouch 000.0 oowd 000.0 000.0 30.0 mafia mean 35050 35030 003 295 Z H 3 Z m4m<rw 563052430>mmo<<me<mq§m30QEQEQEBEEOQE: oEEm-m>MHQA§AOmm 228m205031530>mmo<<mm3§mGBaasmmmenfioom: 4303000250009dems0oégégimmflgofi: 28-omEfiBgmmgéfioag03H0-203630050500003: I a NH ow MH-Mo$U>nHm£m<MMMQAWMmWQmHmHUOQEE am .82 Q5 9:05 a 0mm @2039 3520:6500 0065.2 08 08 moEHQOQ 8% 8% 000m 00:0: 8550 003? oar—L 03 .v .m .om 22% 050 m Mezzqgoiooim 000m— 2F 29886— 29:36— As shown in Table 1, many of the peptides exhibit an EC50 at the GLP-1 receptor in the nanomolar or picomolar range.

EXAMPLE 6 Diet d obesity (DIO) mice are divided into groups of eight mice per group and the initial average body weight of each group is determined. Each group of mice is subcutaneously injected daily with a dose of a peptide or vehicle control for one week. The peptides tested in this study were the peptides of SEQ ID NOs: 12-16. The administered doses varied between 1 and 10 nmol/kg for each of the peptides tested.

Body weight, body composition, food , and blood glucose levels were determined periodically throughout the test period.

] To better determine the effect of these peptides on blood e levels, a second experiment with db/db mice are performed. In this experiment, db/db mice are divided into groups of eight mice per group and the initial average body weight of each group is determined. Each group of mice is subcutaneously injected with a single dose of a peptide of one of SEQ ID NOs: 12-16, wherein the dose is within the range of 3 and 30 nmole/kg. Body weight, body composition, food intake, and blood glucose levels were determined periodically throughout the test period.

EXAMPLE 7 The in vivo effects of certain peptides of the invention were confirmed in diet- induced obese (DIO) mice that were ined on a high fat diet for 16 weeks and had an initial body weight of ~ 47 g. Mice were stered a vehicle control or a dose of a peptide daily for nine days. The peptides tested in this study ed a peptide of SEQ ID NO: 12, a peptide of SEQ ID NO: 17, a peptide of SEQ ID NO: 18, and a peptide of SEQ ID NO: 19. Doses of each peptide were varied — each of the peptides of SEQ ID NO: 12, 17, and 19 was administered at a dose of either 3 nmol/kg or 9 nmol/kg, while the peptide of SEQ ID NO: 18 was administered at a dose of 1 nmol/kg, 3 nmol/kg, or 9 nmol/kg. Cumulative body weight change (in grams) was measured each day of the study and the results are shown in Figure 1. Data are expressed as mean 1 SEM.

Each of the peptides tested in this study trated a body-weight ng effect. Each group of mice that were stered a peptide at a 3 nmol/kg dose or a 9 nmol/kg dose demonstrated a decreased body weight (as compared to vehicle-treated mice) as early as Day 1 of the study. On Day 9 of the study, mice that were administered 3 nmol/kg of the peptide of SEQ ID NO: 12 ted an approximate 5.3% body weight reduction, while mice that were administered 9 nmol/kg dose of the peptide of SEQ ID NO: 12 exhibited an approximate 29.8% body weight loss. On Day 9 of the study, mice that were administered 3 nmol/kg of the peptide of SEQ 1]) NO: 17 exhibited an approximate 9.6% body weight reduction, while mice that were administered 9 nmol/kg of the peptide of SEQ ID NO: 17 demonstrated an approximate 35.5% weight loss. On Day 9 of the study, mice that were administered 3 g of the peptide of SEQ 1]) NO: 18 exhibited an approximate 12.3% body weight reduction, while mice that were administered 9 nmol/kg of the peptide of SEQ ID NO: 18 demonstrated an approximate 26.8% body weight loss. On Day 9 of this study, mice that were administered 3 nmol/kg of the peptide of SEQ ID NO: 19 demonstrated an approximate 8.1% body weight reduction, whereas mice that were administered 9 nmolfkg of the peptide of SEQ ID NO: 19 exhibited an approximate 31.5% body weight loss.

] In addition to cumulative body weight change, ambient glucose levels of the D10 mice were ed. As shown in Figure 2, mice that were administered a 3 nmol/kg or a 9 nmol/kg dose of the peptide of SEQ ID NO: 18 demonstrated a significant decrease in ambient glucose by Day 4 (-29% vs. vehicle; P < 0.05). Mice that were given 9 nmol/kg of the peptide of SEQ ID NO: 19 demonstrated an approximate 40% decrease in glucose levels on Day 4 red to vehicle control mice; P < 0.05), an imate 39% se in glucose levels on Day 7 (compared to e control mice; P < 0.05), and an approximate 26% decrease in glucose levels on Day 9 (compared to vehicle control mice; P < 0.05).

Mice that were given 9 nmolikg of the peptide of SEQ ID NO: 17 demonstrated an approximate 48% decrease in glucose levels on Days 4 and 7 (compared to e control mice; P < 0.05), an approximate 45% decrease in glucose levels on Day 9 (as compared to vehicle control mice; P < 0.05). Mice that were given 3 g of the peptide of SEQ ID NO: 17 demonstrated an approximate 28% reduction in glucose levels on Day 7 (as compared to vehicle control mice; P < 0.05) and an approximate 19% decrease in glucose levels on Day 9 (as compared to vehicle control mice; P < 0.05).

EXAMPLE 8 The in vivo effects of a peptide of the invention were confirmed in obese rhesus monkeys by administering daily for 21 days a vehicle control, a commercially ble product (Liraglutide), a peptide of SEQ ID NO: 20, or a peptide of SEQ ID NO: 17. Liraglutide was stered at a dose of 20 ug/kg s.c., while each of the peptides of SEQ ID NO: 17 and SEQ ID NO: 20 was stered at a dose of 3 [lg/kg s.c. A11 peptides have comparable pharmacokinetic (t1/2 ~10 h) and similar plasma protein binding in rhesus monkeys (> 98%). Body weight and food intake were measured.

As shown in Figure 3A, obese monkeys that were administered the peptide of SEQ ID NO: 17 exhibited or weight loss (—1.2 kg vs. baseline and vehicle, P < 0.05), compared to Liraglutide and to the peptide of SEQ ID NO: 20.

A significant decrease in food intake (expressed as % change vs. baseline in Figure 3B), was observed following treatment with liraglutide (-13.1 i 1.9%) or with the peptide of SEQ ID NO: 17 (-25.7% i 3.2%, P < 0.05 vs. Liraglutide). ing a one week wash-out, the animals treated with the peptide of SEQ ID NO: 17 regained much of the weight that was lost during the chronic treatment (Day 28). In Figures 3A and 3B, the data are expressed as mean i SEM. * P < 0.05 vs vehicle, "P < 0.05 peptide of SEQ ID NO: 17 vs. Liraglutide.

EXAMPLE 9 ] The in vivo effects of a peptide of the invention were tested in diabetic rhesus monkeys (n=7) that were aged 16-26 years and having an average body weight of ~22 kg.

Diabetic rhesus monkeys (n=7) that were aged 16—26 years with an e body weight of ~22 kg were administered a vehicle control, Liraglutide at a dose of 10 ug/kg s.c. or a peptide of SEQ ID NO: 17 at a dose of 1 ug/kg s.c. for 10 days.

Body weight was measured during the study and it was found that no body weight effect was observed in thse animals during the ent period.

At the end of each treatment, fasting blood glucose (FBG) was ed first followed by a Meal Tolerance Test (MTT) to assess glucose tolerance. As shown in Figure 4, diabetic monkeys treated with the peptide of SEQ ID NO: 17 demonstrated a icantly sed FBG (120 i 20 vs. 158 i 15 mgme, peptide of SEQ ID NO: 17 vs. vehicle (-25%), P<0.05; Figure 4). Also, the monkeys treated with the peptide of SEQ ID NO: 17 demonstrated an improved glucose tolerance during MTT as compared to vehicle control monkeys (Figure 4). Data are expressed in Figure 4 as mean i SEM. >“P < 0.05 versus vehicle.

All references, including ations, patent ations, and patents, cited herein are hereby incorporated by nce to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the” and similar referents in the context of describing the invention (especially in the context of the ing claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring dually to each separate value falling within the range and each endpoint, unless otherwise ted herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

All methods described herein can be med in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this ion are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the ing description. The inventors expect skilled ns to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as ted by able law. Moreover, any ation of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (11)

We claim:

1. A peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 17 or a pharmaceutically able salt thereof.

2. The peptide of Claim 1 consisting of the amino acid sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt thereof.

3. A conjugate comprising a peptide of claim 1 or 2 conjugated to a heterologous moiety, or a pharmaceutically acceptable salt thereof, wherein the conjugate exhibits enhanced ty at the GLP-1 receptor, as compared to native glucagon, and exhibits at least 100-fold greater selectivity for the human GLP-1 receptor versus the GIP receptor.

4. The conjugate of Claim 3, wherein the heterologous moiety comprises one or more of: a peptide, a polypeptide, a nucleic acid molecule, an antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagonostic agent, or a pharmaceutically acceptable salt thereof.

5. The conjugate of Claim 4, wherein the logous moiety is a peptide and the conjugate is a fusion peptide or a ic peptide, or a pharmaceutically acceptable salt thereof.

6. The conjugate of Claim 5, comprising an extension of 1-21 amino acids C- terminal to the amino acid at position 31 of the peptide, or a pharmaceutically able salt thereof.

7. The conjugate of Claim 6, wherein the extension is selected from the group consisting of: Gly, Glu, Cys, Gly-Gly, Gly-Glu, PPPS (SEQ ID NO: 9) or GGPSSGAPPPS (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof.

8. A dimer or multimer comprising a peptide of any one of the ing claims, or a ceutically acceptable salt f. (11193765_1):GGG

9. A pharmaceutical composition comprising the e of claim 1 or 2, or a pharmaceutically acceptable salt thereof; a conjugate of Claim 3 to 7, or a pharmaceutically acceptable salt thereof; a dimer or multimer of Claim 8, or a pharmaceutically acceptable salt thereof; or a combination f, and a pharmaceutically able carrier.

10. The pharmaceutical composition of claim 9, comprising an iabetic or anti-obesity agent.

11. Use of the peptide of claim 1 or 2, or a pharmaceutically able salt thereof; a conjugate of Claim 3 to 7, or a pharmaceutically acceptable salt thereof; a dimer or multimer of Claim 8, or a combination thereof in the manufacture of a medicament for the treatment of a disease or medical condition selected from the group consisting of: metabolic syndrome, diabetes, obesity, liver steatosis, and a neurodegenerative disease.