The present invention relates to compounds with an extended duration of action at the glucagon receptor as compared to native glucagon. Specifically provided are glucagon receptor agonists with modifications to the structure of native glucagon introduced to selectively agonize the glucagon receptor over an extended period of time. The glucagon receptor agonists may be useful either in combination with oilier therapeutic agents for treating disorders such as type 2 diabetes mellitus (T2DM) and/or obesity, or as monotherapies for treating a variety of disorders, such as obesity, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, hyperinsulinemia and/or nighttime hypoglycemia, as well as fatty liver syndrome in dairy cows.

Over the past several decades, the prevalence of diabetes has continued to rise. T2DM is the most common form of diabetes accounting for approximately 90% of all diabetes. T2DM is characterized by high blood glucose levels caused by insulin resistance. The current standard of care for T2DM includes diet and exercise, and treatment with oral medications, and injectable glucose lowering drugs, including mcretin-based therapies, such as glucago ~like-peptkle~] (GLP-'l) receptor agonists and dipeptidyi peptidase IV (DPP-IV) inhibitors. When treatment with oral medications and incretin-based therapies are insufficient, treatment with insulin is considered. Patients whose disease has progressed to the point that insulin therapy is required are generally started on a single daily injection of a long-acting, basal insulin, although mealtime injections of rapid-acting insulins may be included, as necessary, in some cases.

Despite the availability of these therapies, blood glucose levels in many patients with T2DM still remain inadequately controlled. Uncontrolled diabetes leads to several conditions that impact morbidity and mortality of patients. One of the main risk factors for T2DM is obesity. The majority of T2DM patients (-90%) are overweight or obese, it is documented that a decrease in body adiposity will lead to improvement in obesity-associated co-morbidities including hyperglycemia and cardiovascular events. Therefore, therapies effective in glucose control and weight reduction are needed for better disease management

In addition, the prevalence and awareness of nonalcoholic fatty liver disease (NAFLD) - which refers to a cluster of liver disorders associated with the accumulation of fat in the liver, and nonalcoholic steatohepatitis ( ASH) - which is a severe form of NALFD characterized by histological findings such as inflammation, hepatocyte injur and fibrosis, have also continued to rise. NASH is the most common liver disease in western countries, and affects between 3-5% of adults in the United States. Treatment typically includes prescribed changes in diet and exercise, and may involve bariatric surgery, pioglitazones, statins, omega 3 and vitamin E therapy (in the case of non-diabetic N ASH patients) to reduce liver fat, but there are no therapeutic agents approved to address the inflammation and/or fibrosis associated with NASH, Therefore additional therapies are needed.

Several peptides which are available and/or in development as therapeutic agents, including glucagon, are derived from pre-proglucagon, which is a polypeptide that is processed in tissue to form several structurally related peptides. Glucagon is a 29-amino acid peptide that corresponds with amino acids 53 to 81 of pre-proglucagon, having the following amino acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 1), Glucagon helps maintain the level of glucose in the blood by binding to and activating glucagon receptors on hepatocytes, causing the liver to release glucose - stored in the form of glycogen - through a process called glycogenolysis. As glycogen stores become depleted, glucagon stimulates the liver to synthesize additional glucose by gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycemia. Administration of glucagon is an established therapy for treating acute hypoglycemia, and emergency glucagon administration can restore normal glucose levels within minutes of administration. Certain glucagon analogs have been described as exhibiting improved solubility and stability. See, e.g., WO2015094875; WO2015094876; WO2015094878.

Other peptides derived from pre-proglucagon include GLP-1, glucagon-like-peptide-2 (GLP-2), and oxyntomodulin (OXM). GLP-1 is a 36 amino acid peptide, the major biologically active fragment of which (GLP-17..36) is produced as a 30-amino acid, ('.'-terminal amidated peptide that corresponds with amino acids 98 to 127 of pre-proglucagon, having the following amino acid sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 2). Whereas glucagon

stimulates the release of glucose to prevent hypoglycemia, GLP- 1 (SEQ ID NO: 2) stimulates insulin synthesis and secretion and has been shown to prevent hyperglycemia in diabetics. A variety of GLP-1 analogs are currently available, including exenatide, liraglutide, albiglutide and dulaglutide.

OXM is a 37 amino acid peptide composed of the complete 29 amino acid sequence of glucagon with an octapeptide carboxy terminal extension (amino acids 82 to 89 of pre-proglucagon and termed "intervening peptide 1" or IP-1), having the following amino acid sequence: HSQGTFTSDYS YLDSRRAQDFVQWLIVINTKRNRNNIA (SEQ ID NO: 3). OXM activates both the glucagon and GLP-1 receptors, with a slightly higher potency for the glucagon receptor over the GLP-1 receptor. Analogs of OXM having dual glucagon receptor and GLP-1 receptor activity have been described. See, e.g., WO201 1087672; WO2011087671.

Although not derived from pre-proglucagon, glucose-dependent insuiinotropic polypeptide (GIP) is another peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose. GIP is a 42 amino acid peptide having the following amino acid sequence: YAEGTFISDYSIAMDKIHQQDFVN WLLAQKGKKNDWKHMTQ (SEQ ID NO: 4). Certain analogs of GIP have been described as exhibiting both GIP and GLP-1 receptor activity. See, e.g., WO2015067715; WO201111 9657; WO2013164483.

In addition, similar to the dual activity of OXM described above, certain glucagon analogs have been described as having co-agonist activity both at the glucagon receptor and one or more of the GLP-1 or GIP receptors. For example, WO201 1075393 and WO 2012177444, purport to describe glucagon analogs having activity at both the glucagon and GLP-1 receptors. Similarly, WG2Q13192130 purports to describe glucagon analogs also having activity at the GIP receptor. Further, WO2015067716 purports to describe analogs having triple agonist activity at each of the glucagon, GLP-1 and GIP receptors.

Despite the variety of peptides and proteins proposed as T2DM and/or obesity therapies, therapies that are currently available and/or in development have limitations. In particular, while the dual or triple agonists described above may be stated to provide the glucose-lowering properties of a GLP-1 receptor and/or GIP receptor agonist along with the metabolic benefits of a glucagon receptor agonist, the activity levels of such peptides at each of the various receptors they agonize are fixed, making it difficult to achieve an ideal receptor activation balance to obtain, in vivo, high efficacy with minimal side effects. And therapies which do not involve glucagon receptor agonism lack the potential metabolic benefits of such a mechanism of action.

While concomitant administration of glucagon may be theoretically capable of attenuating such limitations, currently available glucagon products are impractical for use in such applications, in particular, the plasma half-life of glucagon is less than an hour, making it impractical for chronic use, particularly in embodiments involving

combinations with other therapies that are available and/or in development, as many such therapies are dosed as infrequently as once a day, and some are proposed for dosing as infrequently as once-weekly. In addition, wild type glucagon also has some activity at the GLP-1 receptor, which may complicate efforts to draw an appropriate balance of glucagon versus GLP-1 receptor activity in combination therapies wherein the other compound has its down GLP-1 receptor activity. Moreover, the solubility and chemical and physical stability characteristics of currently available glucagon products are also inappropriate for chronic use in such applications, and would not allow for co-formulation with other therapeutic agents.

Thus, there is a need for glucagon receptor agonists which have extended duration of action allowing for dosing as infrequently as once a day, thrice-weekly, twice-weekly or once a week. There is also a need for glucagon receptor agonists which have potent activity at the glucagon receptor, and high selectivity for activity at the glucagon receptor versus the GLP-1 receptor. There is also a need for glucagon receptor agonists with suitable characteristics to be co-formulated with other therapeutic agents. There is also a need for glucagon receptor agonists with solubility and stability characteristics allowing for long term storage and use.

The glucagon receptor agonists of the present invention seek to meet these needs.

Accordingly, the present invention provides glucagon receptor agonists with an extended duration of action, allowing for dosing as infrequently as once a day, thrice-weekly, twice- weekly or once a week. The present invention provides glucagon receptor agonists with optimal and selective activity at the glucagon receptor as compared, for example, to the GLP-1 and/or GIP receptors. The present invention provides glucagon receptor agonists which have physical and chemical characteristics suitable for chronic use and co-formulation with other treatments. The present invention pro ides glucagon receptor

agonists which, when used in combination with other diabetes treatments, result in enhanced glucose control, metabolic benefits, such as body weight lowering, and/or lipid benefits, such as PCSK9 lowering, when used in combination with other diabetes treatments. In particular, combinations of glucagon receptor agonists of the present invention with GLP-IR agonists or GIP-GLP-l co-agonists have beneficial synergistic effects on measures such as weight loss and body composition. The present invention also seeks to provide effective treatments for other disorders when used as a monotherapy and/or in combination with other therapies, including obesity, NAFLD, NASH, dyslipidemia, metabolic disorder, hyperinsulinemia and/or nighttime hypoglycemia.

Accordingly, an embodiment of the present invention provides a glucagon receptor agonist comprising Formula I:

YXiQGTFX2SDYSKYLDX3K AX4EFVX5WLLEX6X7 [Formula I] wherein

Xi is Aib;

X2 is T or L;

X3 is Aib;

X4 is K which is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]- acetyl)2-(yG]u)a-CO-(CH2)b-C02H, wherein a is 1 or 2 and b is 14 to 24; X5 is E or A;

X<5 is T or E;

X7 is either absent, or is a peptide selected from the group consisting of GPSSGAPPPS and GPSSG;

and the C-terminal amino acid is optionally amidated (SEQ ID NO: 5); or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention provides a glucagon receptor agonist comprising a formula consisting of Formula I:

YXiQGTFX2SDYSKYLDX3 AX4EFVX5WLLEX6X7 [Formula I] wherein

Xi is Aib;

X2 is T or L;

X3 is Aib;

X4 is K which is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]- acetyl)2-(YGlu)a-CO-(CH2)b-C02H, wherein a is 1 or 2 and b is 14 to 24;

X5 is E or A;

Xe is T or E;

X? is either absent, or is a peptide selected from the group consisting of GPSSGAPPPS and GPSSG;

and the C-terminal amino acid is optionally amidated (SEQ ID NO: 5); or a p armaceutically acceptable salt thereof.

Another embodiment of the present invention provides a glucagon receptor agonist consisting of Formula I:

YXiQGTFX2SDYSKYLDX3KKAX4EFVX5WLLEX6X7 [Formula I] wherein

Xi is Aib;

X2 is T or L;

X3 is Aib;

X4 is K which is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]- acetyl)2~(yG]u)a~CO~(Ci-l2)b~C02H, wherein a is 1 or 2 and b s 1.4 to 24; X5 is E or A;

X<5 is T or E;

X7 is either absent, or is a peptide selected from the group consisting of GPSSGAPPPS and GPSSG;

and the C-terminal amino acid is optionally amidated (SEQ ID NO: 5); or a pharmaceutically acceptable salt thereof.

In certam embodiments, the glucagon receptor agonist has the structure of Formula I wherein X2 is T. In certam embodiments, the glucagon receptor agonist has th structure of Formula I wherein X2 is L.

In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherem ¾ is E. In certam embodiments, the glucagon receptor agonist has the structure of any of the abov e embodiments wherein X5 is A.

In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherem ¾ is T. In certai embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherein ¾ is E.

In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherem X7 is GPSSGAPPPS. In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherein X7 is GPSSG. In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherem X7 is absent.

In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherein b is 16 to 20. In certain embodiments, the glucagon receptor agomst has the structure of any of the above embodiments wherein b is 16 to 18. In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherein b is 16. In certain embodiments, the glucagon receptor agonist has the structure of any of the above embodiments wherein b is 18.

In certain embodiments, the glucagon receptor agonist has the structure of

Formula I wherem: X2 is T; a is 2; b is 16; X5 is E; X6 is T; and X? is GPSSGAPPPS; and wherein the C-terminai amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 6).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherem: X2 is T; a is 2; b is 18; X5 is E; X6 is T; X7 is GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 7).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherein: X2 is L; a is 2; b is 16; X5 is E; X6 is T; and X? is GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 8).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherein: X2 is T; a is 2; b is 16; X5 is E; X6 is T; and X7 is GPSSG; and wherein the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 9).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherem: X2 is T; a is 2; b is 18; X5 is E; X6 is T; and X7 is GPSSG; and wherein the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 10).

In certain embodiments, the glucagon receptor agonist has the structure of Fonnula I wherein: X2 is T; a is 1 ; b is 16; X5 is A; X¾ is E; and X7 is absent (SEQ ID NO: 11).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherein: X2 is T; a is 1; b is 18; X5 is A; X6 is E; and X7 is absent (SEQ ID NO: 12).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherein: a is 2; X5 is E; Xf> is T; X7 is a peptide selected from the group consisting of GPSSGAPPPS and GPSSG; and the C-terminal amino acid is amidated (SEQ ID NO: 16).

In certain embodiments, the glucagon receptor agonist has the structure of Formula I wherein: X2 is T; a is 1; X5 is A; Xg is E; and X7 is absent (SEQ ID NO: 17).

In certain embodiments, the activity of the glucagon receptor agonist at the glucagon receptor is at least 100-fold higher than the activity of the glucagon receptor agonist at the GLP-1 receptor.

Another embodiment of the present invention provides a method of treating a disease selected from the group consisting of T2DM, obesity, fatty liver disease, NASH, dysiipidemia, metabolic syndrome, hyperinsulinemia and nighttime hypoglycemia, comprising administering to a patient in need thereof, an effective amount of a glucagon receptor agonist of the present invention.

Another embodiment of the present invention provides a method of treating a disease selected from the group consisting of T2DM, obesity, fatty liver disease, N ASH, dysiipidemia, metabolic syndrome, hyperinsulinemia and nighttime hypoglycemia, comprising administering to a patient in need thereof, an effective amount of a glucagon receptor agonist of the present invention in combination with an effective amount of one or more additional therapeutic agents. In certain embodiments the disease is T2DM. In certain embodiments the disease is obesity. In certain embodiments the disease is fatty liver disease. In certain embodiments the one or more additional therapeutic agents are selected from the group consisting of GLP-1 R agonists, GIP-GLP-1 co-agonists, insulin receptor agonists, oxyntomodulins, metformin, thiazolidinediones, sulfonylureas,

dipeptidyl peptidase-4 ("DPP-4") inhibitors, and sodium glucose co-transporter 2

("SGLT2") inhibitors. In certain embodiments the additional therapeutic agent is a GLP-1R agonist. In certain embodiments the GLP-1 R agonist is dulagiutide. In certain embodiments the additional therapeutic agent is a GIP-GLP-1 co-agonist. In certain embodiments the GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 15. In certain embodiments the additional therapeutic agent is an insulin receptor agonist.

In certain embodiments, the glucagon receptor agonist has synergistic effects on weight loss and body composition when used in combination with an additional therapeutic agent, such as a GLP-1 R agonist or a GIP-GLP-1 co-agonist.

Another embodiment of the present invention provides use of a glucagon receptor agonist of the present invention in therapy. Another embodiment of the present invention provides use of a glucagon receptor agonist of the present in vention in treating a disease selected from the group consisting of T2DM, obesity, fatty liver disease, NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia and nighttime hypoglycemia. In certain embodiments the disease is T2DM. In certain embodiments the disease is obesity. In certain embodiments the disease is fatty liver disease.

Another embodiment of the present invention provides a glucagon receptor agonist of the present in vention for use in simul taneous, separate, or sequential use in combination with one or more additional therapeutic agents for use in therapy. In certain embodiments the one or more additional therapeutic agents are selected from the group consisting of GLP-1 R agonists, GIP-GLP-1 co-agonists, insulin receptor agonists, oxyntomodulins, metformin, thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and sodium glucose co-transporter 2 ("SGLT2") inhibitors. In certain embodiments the additional therapeutic agent is a GLP-1 R agonist. In certain embodiments the GLP-1 R agonist is dulagiutide. In certain embodiments the additional therapeutic agent is a GIP-GLP-1 co-agonist. In certain embodiments the GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 15.

Another embodiment of the present invention provides use of a glucagon receptor agonist of the present invention in the manufacture of a medicament for the treatment of T2DM, obesity, fatty liver disease, NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia and nighttime hypoglycemia.

Another embodiment of the present invention provides a pharmaceutical composition compri sing a glucagon receptor agonist of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In certain embodiments the additional therapeutic agent is selected from the group consisting of GLP-IR agonists, GIP-GLP-l co-agonists, insulin receptor agonists, oxyntomoduiins, metformin, thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and SGLT2 inhibitors. In certain embodiments the additional therapeutic agent is a GLP-1 R agonist. In certain embodiments the GLP-1 R agonist is dulaglutide. In certain embodiments the additional therapeutic agent is a GIP-GLP-l co-agonist. In certain embodiments the GIP-GLP-l co-agonist has the structure of SEQ ID NO: 15. In certain embodiments the additional therapeutic agent is an insulin receptor agonist.

Another embodiment of the present invention provides a method of inducing non-therapeutic weight-loss comprising administration of an effective amount of a glucagon receptor agonist of the present invention.

Another embodiment of the present invention provides a method of treating fatty liver syndrome in a bovine, comprising administering to a bovine in need thereof, an effective amount of a glucagon receptor agonist of the present invention.

Another embodiment of the present invention provides a glucagon receptor agonist of the present invention for use in the treatment of fatty liver syndrome in a bovine.

When used herein the term "glucagon receptor agonists" means compounds comprising the amino acid sequence of native human glucagon (SEQ ID NO: 1), a glucagon analog, a glucagon derivative or a glucagon fusion protein, which bind to and activate the glucagon receptor, and maintain selective activity at the glucagon receptor relative to the GLP-1 receptor, resulting in an increase in serum glucose levels when administered as a monotherapy. Such binding characteristics and pharmacodynamic effects may be measured using known in vitro and in vivo methods, such as those described in the studies below. A glucagon analog is a molecule having a modification including one or more amino acid substitutions, deletions, inversions, or additions when compared with the amino acid sequence of native human glucagon (SEQ ID NO: 1), A glucagon derivative is a molecule having the amino acid sequence of native human

glucagon (SEQ ID NO: l) or of a glucagon analog, but additionally having at least one chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group. A glucagon fusion protein is a heterologous protein comprising glucagon, a glucagon analog or a glucagon derivative and a second polypeptide, such as an immunoglobulin Fc region.

The activities of the glucagon receptor agonists of the present invention are also selective for the glucagon receptor relative to GLP-1 receptor. When used herein, the terms "selective...relative to," "selectivity" and "selective against" refer to a compound that displays 50-, 100-, 200-, 250-, 500- or 1000-fold higher potency for the glucagon receptor over the GLP-1 receptor. Such selectivity may be measured using known in vitro methods, such as those described in the studies below.

The glucagon receptor agonists of the present invention have extended time action profiles allowing for dosing as infrequently as once daily, thrice-weekly, twice-weekly or once- weekly. The time action profile of a glucagon receptor agonist may be measured using known pharmacokinetic test methods.

The extended time action profi les of the glucagon receptor agonists of the present invention are achieved through the use of a fatty acid moiety conjugated to the epsilon-amino group of the side chain of the lysine amino acid at position 20. The fatty acid is conjugated to the epsilon-amino group of a lysine side-chain through a linker, which comprises [2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)a, wherein a is 1 or 2. The fatty acid and the gamma-glutamic acid in the linker act as albumin binders, and provide the potential to generate long-acting compounds. The fatty acid conjugated to the epsilon-amino group of the side chain of the lysine amino acid at position 20 by way of the linker comprises -CO-(CH2)b-C02H wherein b is 14 to 24. Thus, the complete linker-fatty acid structure comprises ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)a-CO-(CH2)b-C02H wherein a is 1 or 2 and b is 14 to 24. As shown in the chemical structures of Examples 1 -7 below, the first unit of [2-(2-Arnino-ethoxy)-ethoxy]-acetyl is linked to the epsilon-amino group of the lysine side-chain. The second unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl is then attached to the amino-group of the first unit of [2-(2-Aminoethoxy)-ethoxy] -acetyl. Then, the first unit of yGlu is attached to the amino group of the second unit of [2-(2-Arnino-ethoxy)-ethoxy] -acetyl through the y-carboxyl group of the side-chain. When a = 2, the second unit of yGlu is attached to the a-amino group of the first unit of yGlu through the γ-carboxyl group of the side-chain. Finally, the fatty acid is attached to the a-amino group of the first (when a = 1) or second (when a=2) unit of yGlu.

The glucagon receptor agonists of the invention are preferably formulated as pharmaceutical compositions administered by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and processes for preparing same are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy (D.B, Troy, Editor, 21st Edition, Lippincoti, Williams & Wilkins, 2006). The preferred route of administration is subcutaneous.

The glucagon receptor agonists of the present invention may react with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. (See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts:

Properties, Selection and Use, 2nd Revised Edition (Wiley -VCH, 2011)).

Pharmaceutically acceptable salts of the present invention include trifluoroacetate, hydrochloride, and acetate salts.

One particular benefit provided by the selectivity of the glucagon receptor agonists of the present invention for the glucagon receptor over the GLP-1 receptor is the ability to provide flexible treatment options when glucagon receptor agonists of the present invention are administered in combination with additional therapeutic agents, such that the ratio of activity at the glucagon receptor to activity at the other receptor(s) (e.g., GLP-1, GIP and/or insulin receptor). Thus, in certain preferred embodiments, the present invention provides a method of treatment of T2DM in a patient comprising administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a pharmaceutically acceptable salt thereof, in combination with an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is selected from the group consisting of a GLP-1 R agonist, a GIP-GLP-1 co-agonist or an insulin receptor agonist.

When used herein, the term "additional therapeutic agent(s)," means other compound(s) known to have beneficial therapeutic effects, such as treatments for T2DM and/or obesity, which are currently available and/or in development, including for example GLP-1 R agonists, GIP-GLP-1 co-agonists, insulin receptor agonists,

oxyntomodulins, metformin, thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and SGLT2 inhibitors.

When used herein, the term "in combination with" means administration of the glucagon receptor agonist of the present invention either simultaneously, sequentially or in a single combined formulation with the one or more additional therapeutic agents,

When used herein, the term "GLP-1 R agonist" refers to a compound comprising the amino acid sequence of native human GLP-1 (SEQ ID NO: 2), a GLP-1 analog, GLP-1 derivative or a GLP-1. fusion protein, which maintains activity at the GLP-1 receptor. GLP-1 receptor activity may be measured by methods known in the art, including using in vivo experiments and in vitro assays that measure GLP-1 receptor binding activity or receptor activation. A GLP-1 analog is a molecule having a modification including one or more amino acid substitixtions, deletions, inversions, or additions when compared with the amino acid sequence of native human GLP-1 (SEQ ID NO: 2). A GLP-1 derivative is a molecule having the amino acid sequence of native human GLP-1 (SEQ ID NO:2) or of a GLP-1 analog, but additionally having at least one chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxyiic acid group. A GLP-1 fusion protein is a heterologous protein comprising GLP-1 , a GLP-1 analog or a GLP-1 derivative and a second polypeptide, such as an immunoglobulin Fc region. GLP-1 R agonists currently available and/or in development include exenatide, liraglutide, iixisenatide, albiglutide, duiaglutide and semagiutide. In certain preferred embodiments wherein a glucagon receptor agonist of the present invention is administered in combination with a GLP-1 R agonist, the GLP-1 R agonist is duiaglutide. See, e.g., US 7,452,966.

When used herein, the term "GIP-GLP-1 co-agonist" refers to a compound which has activity at both the GIP and GLP-1 receptors. GIF receptor and GLP-1 receptor activity may be measured by methods known in the art, including using in vivo experiments and in vitro assays that measure GIP receptor and/or GLP-1 receptor binding activity or receptor activation. Although no GIP-GLP-1 co-agonists are currently available as T2DM treatments, multiple GIP analogs have been described as exhibiting both GIP and GLP-1 receptor activity, see, e.g., WO2013164483; WO 201 1119657.

In certain preferred embodiments wherein a glucagon receptor agonist of the present invention is administered in combination with a GIP-GLP-1 co-agonist, the GIP-GLP-1 co-agonist has the following structure:

YX1EGTFTSDYSIX2LDKIAQX3AX4VQWLIAGGPSSGAPPPS;

wherein

X] is Aib;

X;2 is Aib;

X3 is K which is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-amino- ethoxy)-ethoxy]-acetyl)2-(yGlu)a-CO

(CH2)b-C02H wherein a is 1 or 2 and b is 10 to 20;

X4 is Phe or 1-naphthylalanine (l-Nal);

and the C-terminal amino acid is optionally amidated (SEQ ID NO: 13), or a pharmaceutically acceptable salt thereof,

In certain preferred embodiments a is 2, b is 18; X4 is l-Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide. (SEQ ID NO: 14). I certain preferred embodiments a is 1 , b is 18; X4 is Phe; and the C-terminal amino acid is amidated as a C-terminal primary amide. (SEQ ID NO: 15). Such GIP-GLP-1 co-agonists may be prepared using techniques such as those which may be used to prepare glucagon receptor agonists of the present invention, as described below in the Peptide Synthesis examples.

When used herein, the term "insulin receptor agonist" refers to human insulin, or analogs or derivatives thereof, or any other protein which is capable of binding to and activating the insulin receptor. Insulin receptor activity may be measured by methods known in the art, including using in vivo experiments and in vitro assays that measure insulin receptor binding activity or receptor acti vation. An insulin analog is a molecule having a modification including one or more amino acid substitutions, deletions, inversions, or additions when compared with native human insulin, the structure of which is well known. An insulin derivative is a molecule having the amino acid sequence of native human insulin, or an analog thereof, but additionally having at least one chemical modification of one or more of its amino acid side groups, -carbon atoms, terminal amino group, or terminal carboxylic acid group. An insulin fusion protein is a heterologous protein comprising insulin, an insulin analog or an insulin derivative portion and a second polypeptide. Although any insulin receptor agonist may be considered for use in embodiments wherein a glucagon receptor agonist of the present invention is administered in combination with an insulin receptor agonist, preferred insulin receptor agonists are those having a basal, or extended, duration of action. Currently available insulin receptor agonists with basal activity include insulin glargine, insulin detemir, and insulin degludec, each of which is indicated for once-daily administration.

In certain embodiments, the present invention provides a method for treatment of T2DM in a patient comprising administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a

pharmaceutically acceptable salt thereof.

In certain embodiments, the present invention provides a method for treatment of obesity in a patient compri sing administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a

pharmaceutically acceptable salt thereof.

In certain embodiments, the present invention provides a method for treatment of fatty liver disease in a patient comprising administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the present invention provides a method for treatment of N ASH in a patient comprising administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a

pharmaceutically acceptable salt thereof.

In other embodiments, the present invention also provides a method of treatment of T2DM in a patient comprising administering to a patient in need of such treatment an effective amount of a glucagon receptor agonist of the present invention, or a

pharmaceutically acceptable salt thereof, in combination with one or more additional therapeutic agents, such as GLP-1R agonists, GIP-GLP-1 co-agonists, insulin receptor agonists, oxyntomodulins, metformin, thiazolidinediones, sulfonylureas, DPP -4 inhibitors, and SGLT2 inhibitors.

When used herein, the term "patient in need thereof refers to a mammal, preferably a human or a bovine, with a disease or condition requiring treatment, including for example, T2DM, obesity, fatty liver disease, NASH and/or metabolic syndrome.

When used herein, the term "effective amount" refers to the amount or dose of glucagon receptor agonist of the present invention, or a pharmaceutically acceptable salt thereof which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment. An effective amount can be readily determined by a person of skill in the art through the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular glucagon receptor agonist administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

Certain glucagon receptor agonists of the present invention are generally effective over a wide dosage range. For example, dosages for once-weekly administration may fall within the range of about 0.01 to about 30 mg per person per week. Glucagon receptor agonists of the present invention may be dosed daily, thrice-weekly, twice- weekly or once-weekly. Once- weekly administration is preferred.

As used herein, the term "treating" or "to treat" includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

The amino acid sequences of the present invention contain the standard single letter or three letter codes for the twenty naturally occurring amino acids. Additionally, "Aib" is alpha amino isobutyric acid. The present invention also encompasses novel intermediates and processes useful for the synthesis of glucagon receptor agonists of the present invention, or a pharmaceutically acceptable salt thereof. The intermediates and glucagon receptor agonists of the present invention may be prepared by a variety of procedures known in the art. In particular, the process using chemical synthesis is illustrated in the Examples below. The specific synthetic steps for each of the routes described may be combined in different ways to prepare glucagon receptor agonists of the present invention. The reagents and starting materials are readily available to one of ordinary skill in the art.

The invention is further illustrated by the following examples, which are not to b« construed as limiting.

Example 1 is a glucagon receptor agonist represented by the following description:

YXiQGTFX2SDYSKYLDX3 AX4EFVX5WLLEX6X7

wherein Xi is Aib; X2 is T; X3 is Aib; X4 is K which is chemically modified through conjugation to the epsilon-arnino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)a-CO-(CH2)b-C02H wherein a is 2 and b is 16; X5 is E; X6 is T; X7 is GPSSGAPPPS; and the C-terrninal amino acid is amidated as a C-terminal primary amide (SEQ ID NO; 6).

Below is a depiction of the structure of Example 1 using the standard single letter amino acid codes with the exception of residues Aib2, Aib 16 and K20, where the structures of these amino acid residues have been expanded:

The peptide of Example 1 is generated by solid-phase peptide synthesis using a Fmoc/t-Bu strategy carried out on a Symphony automated peptide synthesizer (PTI

Protein Technologies Inc.) starting from RAPP AM-Rink Amide resin and with couplings using 6 equivalents of amino acid activated with diisopropylcarbodiimide (DIG) and hydroxybenzotriazole (HOBt) (1 : 1 : 1 molar ratio) in dimethylformamide (DMF) for 90 min at 25°C.

Extended couplings for Pro31 (4h), Τφ25 (4h), Glu24 (4h), Va123 (lOh), G1u21 (4h), Aib 16 (4h), Asp 15 (4h), Thr7 (4h), Thr5 (4h), Gly4 (4h), Gln3 (4h) and Aib2 (24h) are necessary to improve the quality of the crude peptide. A Fmoc-Lys(Alloc)-OH building block is used for Lys20 coupling (orthogonal protecting group) to allow for site specific attachment of the fatty acid moiety later on in the synthetic process. The N-terminal residue is incorporated as Boc-Tyr(tBu)-OH using DiC-HOBt protocols as described above (24h coupling).

After finishing the elongation of the peptide-resin described above, the Alloc protecting group present in Lys20 is removed using catalytic amounts of Pd(PPh3)4 in the presence of PhSi¾ as a scavenger. Additional coupling/deprotection cycles using a

Fmoc/t-Bu strategy to extend the Lys20 side-chain involve F1110C-NH-PEG2-CH2COOH (ChemPep Catalog#280102), Fmoc-Glu(OH)-OtBu (ChemPep Catalog#l 00703) and HOOC-(CH2)i6-COOtBu. In all couplings, 3 equivalents of the building block are used with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

Concomitant cleavage from the resin and side chain protecting group removal are carried out in a solution containing trifluoroacetic acid (TFA): triisopropylsilane : 1,2-ethanedithiol: water : thioanisole 90:4:2:2:2 (v/v) for 2 h at 25°C followed by precipitation with cold ether. Crude peptide is purified to > 99% purity (15-20% purified yield) by reversed-phase HPLC chromatography with water / acetonitrile (containing 0.05% v/v TFA) gradient on a CI 8 column, where suitable fractions are pooled and lyophilized, resulting in a TFA salt.

In a synthesis performed essentially as described above, the purity of Example 1 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H 3 =1713.6; Calculated M+3H73 =1714.3; observed: M+4H74 =1285.7; Calculated M+4H 4 =1285.9).

Example 2

Example 2 is a glucagon receptor agonist represented by the following description:

YX1QGTFX2SDYS YLDX3KKAX4EFVX5WLLEX6X7

wherein: Xj is Aib, X2 is T; X3 is Aib; X4 is which is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)- ethoxy]-acetyl)2-(TGlu)a-CO-(CH2)b-C02H; a is 2; b is 18; X5 is E; X6 is T; X7 is

GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 7).

Below is a depiction of the structure of Example 2 using the standard single letter amino acid codes with the exception of residues Aib2, Aib 16 and K20, where the structures of these amino acid residues have been expanded:

The peptide according to Example 2 is synthesized similarly as described above in Example 1. HOOC-(CH2)i8-COOtBu is incorporated using 3 equivalents of the building block with PyBOP (3 equiv) and D1EA (6 equiv) in DMF for 4h at 25°C.

In a synthesis performed essentially as described above for Example 1, the purity of Example 2 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H+/3 -1723.2; Calculated M+3H+/3 -1723.6; observed: M+4H+/4 =1292.9; Calculated M+4H+/4 =1293.0).

Example 3

Example 3 is a glucagon receptor agonist represented by the following description:

YXiQGTF¾SDYSKYLDX3K AX4EFVX5WLLEX6X7

wherein: X: is Aib; X2 is L; X3 is Aib; X4 is which is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)a-CO-(CH2)b-C02H; a is 2; b is 16; X5 is E; X6 is T; and X7 is GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a C terminal primary amide (SEQ ID NO: 8).

Below is a depiction of the structure of Example 3 using the standard single letter amino acid codes with the exception of residues Aib2, Aibl6 and K20, where the structures of these amino acid residues have been ex anded:

The peptide according to Example 3 is synthesized similarly as described above for Example 1.

In a synthesis performed essentially as described above for Example 1, the purity of Example 3 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H+/3 =1717,4; Calculated M+3H73 =1718,3; observed: M+4H+/4 =1288.3; Calculated M+4H+/4 =1289.0),

Example 4 is a glucagon receptor agonist represented by the following description:

WE CLAIM

A glucagon receptor agonist compound comprising the formula:

YXiQGTFX2SDYSKYLDX3 AX4EFVX5WLLEX6X7

wherein

X[ is Aib;

X? is T;

X3 is Aib;

X4 is K which is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Arnino-ethoxy)-ethoxy]- acetyl)2-(yGlu)a-CO-(CH2)b-C02H, wherein a is 2 and b is 16;

X5 is E;

X{, is T;

X7 is GPSSGAPPPS;

and the C-terminal amino acid is amidated as a C-tenninal primary amide

or a pharmaceutically acceptable salt thereof.

A glucagon receptor agonist compound consisting of the formula:

YXiQGTFX2SDYSKYLDX3KKAX4EFVX5WLLEX6X7

wherein

Xi is Aib;

X2 is T;

X3 is Aib;

X4 is K which is chemically modified through conjugation to the epsilon- amino group of the side-chain with ([2-(2-Amino-ethoxy)-ethoxy]- acetyl)2-(yG]u)a-CO-(CH2)b-C02H, wherein a is 2 and b is 16;

X5 is E;

X¾ is T;

X7 is GPSSGAPPPS;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 6);

or a pharmaceutically acceptable salt thereof.

3. A method of treating a disease selected from the group consisting of T2DM, obesity, fatty liver disease, NASH, dyslipidemia, metabolic syndrome,

hyperinsulinemia and nighttime hypoglycemia, comprising administering to a patient in need thereof, an effective amount of the glucagon receptor agonist of any one of Claims 1-2.

4. The method of Claim 3, further comprising administering an effective amount of one or more additional therapeutic agents.

5. The method of Claim 4 wherein the additional therapeutic agent is a GLP-I R

agonist.

6. The method of claim 5 wherein the GLP-IR agonist is dulaglutide.

7. The method of Claim 4 wherein the additional therapeutic agent is a GIP-GLP-1 co- agonist.

8. The method of claim 7 wherein the GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 15.

9. The glucagon receptor agonist of any one of Claims 1 -2, for use in therapy.

10. The glucagon receptor agonist of any one of Claims 1 -2, for use in the treatment of T2DM, obesity, fatty liver disease, NAFLD, NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia or nighttime hypoglycemia.

1 1. The glucagon receptor agonist of any one of Claims 1-2 for use in simultaneous, separate, or sequential use in combination with one or more additional therapeutic agents selected from the group consisting of GLP-IR agonists, GIP-GLP-1 co- agonists and insulin receptor agonists for use in the treatment of T2DM, obesity, NAFLD, NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia or nighttime hypoglycemia.

12. The glucagon receptor agonist of any one of Claims 1-2 for use in simultaneous, separate, or sequential use in combination with dulaglutide for use in the treatment of T2DM, obesity, NAFLD, NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia or nighttime hypoglycemia.

13. The glucagon receptor agonist of any one of Claims 1-2 for use in simultaneous, separate, or sequential use in combination with a GIP-GLP-1 co-agonist having the structure of SEQ ID NO: 15 for use in the treatment of T2DM, obesity, NAFLD,

NASH, dyslipidemia, metabolic syndrome, hyperinsulinemia or nighttime hypoglycemia.

14. A phannaceutical composition comprising the glucagon receptor agonist of any one of Claims 1 -2 and a pharmaceutically acceptable carrier, diluent, or excipient. 15. The pharmaceutical composition of claim 14, further comprising an additional therapeutic agent.

16. The pharmaceutical composition of claim 15 wherein the additional therapeutic agent is a GLP- R agonist.

17. The pharmaceutical composition of claim 16 wherein the GLP- 1 R agonist is

dulaglutide.

18. The pharmaceutical composition of claim 15 wherein the additional therapeutic agent is a GIP-GLP-1 co-agonist.

19. The pharmaceutical composition of claim 18 wherein the GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 15.

20. The glucagon receptor agonist of any one of claims 1 -2, wherem the activity of the glucagon receptor agonist at the glucagon receptor is at least 100-fold higher than the potency of the glucagon receptor agonist at the GLP-1 receptor.

A method of inducing non-therapeutic weight-loss comprising administration of an effective amount of a glucagon receptor agonist of any one of claims 1 -2.