STAPLED OLEFIN CO-AGONISTS OF THE GLUCAGON AND GLP-1 RECEPTORS

BACKGROUND OF THE INVENTION

The present invention is related to stapled co-agonist peptides of the glucagon and GLP-1 receptors, and the use of these stapled GLP-1 receptor/GCG receptor co-agonists for treatment of metabolic disorders.

Pre-proglucagon is a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide- 1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are involved 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 through 108 of pre-proglucagon. GLP-1 (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 maintain blood glucose levels.

GLP-1 has different biological activities compared to glucagon. Its actions include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake. GLP-1 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 receptor 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 musculoskeletal diseases.

Glucagon is a peptide hormone structurally related to GLP-1 that is well recognized for its acute ability to increase blood glucose through stimulation of glycogenolysis and gluconeogenesis (Jiang & Zhang, Am. J. Physio.l Endocrinol. Metab. 284: E671-E678 (2003)). Of lesser appreciation are the chronic effects of glucagon pharmacology characterized by increases in thermogenesis, satiety, lipolysis, fatty acid oxidation, and ketogenesis (Habegger et al., Nat. Rev. Endocrinol. 6: 689-697 (2010)). Repeated administration of glucagon was first reported decades ago to yield improvements in rodent metabolism, accompanied with lower body weight (Salter, Am. J. Clin. Nutr. 8: 535-539 (1960)). Nonetheless, the inherent risk of hyperglycemia, especially in insulinresistant states such T2DM, has complicated the translation of these observations to human study.

The hormone oxyntomodulin (OXM, glucagon-37) is a posttranslational product of preproglucagon processing in the intestine and central nervous system (CNS) and is secreted from L-cells in the gut in response to food intake. Discovered in 1983, OXM has been implicated in the regulation of food intake and energy expenditure (Jarrouse et al., Endocrinol. 115: 102-105 (1984); Schjoldager et al., Eur. J. Clin. Invest., 18: 499-503 (1988)). Central or peripheral administration of OXM in rats causes a decrease in short term food intake with minimal effects on gastric emptying (Dakin et al. Endocrinology, 142: 4244-4250 (2001), Dakin et al. Endocrinology, 145: 2687-2695 (2004)). Repeated intracerebroventricular administration of OXM in rats results in elevated core temperatures and reduced weight gain compared to pair-fed animals, suggesting effects on both caloric intake and energy expenditure (Dakin et al. Am. J.Physiol. Endocrinol. Metab., 283: E1173-E1177 (2002)).

In related studies, peripheral administration of OXM dose-dependently inhibited both fast-induced and dark phase food intake, but unlike GLP-1, had no effect on gastric emptying. OXM also reduced levels of fasting ghrelin and increased c-fos immunoreactivity, in the arcuate nucleus (ARC). Repeated seven-day IP administration of OXM caused a reduction in the rate of body weight gain and adiposity in rats (See Dakin et al. Endocrinology, 145: 2687-2695 (2004)).

Studies of OXM action in mice have demonstrated that although OXM can activate both the glucagon (GCG) and the GLP-1 receptors, the anorectic actions of OXM require only the GLP-1 receptor, as icv OXM inhibits food intake in glucagon receptor knockout mice. However, the anorectic effects of OXM are completely absent in GLP-1 receptor knockout mice. Furthermore, exendin-4, but not OXM, regulates energy expenditure in mice. Hence, OXM appears to be a weak agonist at the GLP-1 receptor, when used in pharmacological concentrations (See Baggio et al., Gastroenterol. 127: 546-58 (2004)). OXM was also found to ameliorate glucose intolerance in mice fed a high fat diet (Dakin et al., Am. J. Physiol. Endocrinol. Metab. 294: E142-E147 (2008) and increase the intrinsic heart rate in mice independent of the GLP-1 receptor (Sowden et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: R962-R970 (2007). OXM has also been shown to differentially affect GLP-1 receptor beta-arrestin recruitment and signaling through Galpha (Jorgensen et al., J. Pharma. Exp. Therapeut. 322: 148-154 (2007)) and to differentially affect hypothalamic neuronal activation following peripheral injection of OXM (Choudhri et al., Biochem. Biophys. Res. Commun. 350: 298-306 (2006)).

In humans, a single 90 minute intravenous infusion of OXM in normal weight healthy subjects reduced hunger scores and food intake at a buffet meal by about 19%. Cumulative twelve-hour caloric intake was reduced by about 11% with no reports of nausea or changes in food palatability (Cohen et al., J. Clin. Endocrinol. Metab., 88: 4696-4701 (2003); Lykkegaard et al., ADA Scientific Sessions, Abstract #1506-P (2003)). More recently, pre-prandial injections of OXM over a four-week period in obese healthy volunteers (BMI about 33) led to a significant reduction of caloric intake on the first day of treatment (about 25%) that was maintained over the course of the study (35% reduction after four weeks) (Wynne et al., Diabetes 54: 2390-2395 (2005)). Robust weight loss was observed at the end of the study in treated subjects (1.9%, placebo-corrected). Plasma levels of OXM were similar to that observed in the infusion study (peak concentration about 950 pM). The absence of any tachyphylaxis and a low incidence of mild and transient nausea (about 3%) despite the relatively high doses necessitated by the poor in vivo stability of OXM (plasma t½ < 12 minutes) renders this hormone one of the few obesity targets with both human validation and an attractive tolerability profile.

OXM has a very short half-life and is rapidly inactivated by the cell surface dipeptidyl peptidase IV (DPP-IV) (Zhu et al., J. Biol. Chem. 278: 22418-22423 (2002). However, DPP-IV inhibitors are weight-neutral in the clinic, suggesting that supraphysiological levels of OXM (900-1000 pM) may be required to achieve weight loss in humans. OXM peptide analogs for inducing weight loss in humans have been the object of Published International Application Nos. WO03/022304, WO2004/062685, WO2006/134340, and WO2010/096052.

Recently, two independent and simultaneous papers reported the use of relatively balanced GLP-1 receptor/GCG receptor co-agonists as being of enhanced efficacy and safety relative to pure GLP1R agonists in the treatment of rodent obesity, with simultaneous improvement in glycemic control (Day et al., Nat. Chem. Biol. 5: 749-757 (2009); Pocai eta al., Diabetes 58: 2258-2266 (2009)). Of related significance is work with oxyntomodulin (OXM), an endogenous precursor to glucagon, which is secreted postprandially by L-cells of the jejuno-ileum together with GLP-1 (Holst, Regul. Pept. 93: 45-51 (2000); Drucker, Nat. Clin. Pract. Endocrinol. Metab. 1: 22-31 (2005).

Glucagon peptide analogs and derivatives modified to have various degrees of activity at the GLP-1 receptor and GCG receptor have been disclosed in Published International Application Nos. WO2008/1010017, WO2009/155258, WO2011/075393, WO2012/177444, WO2012/177443; WO2016/065090; and WO2019/060660. Some of the disclosed glucagon peptide analogs were reported therein to have activity at both the GLP-1 receptor and GCG receptor; however, there remains a need for co-agonist peptides that have relatively balanced activity or potency at the GLP-1 receptor and GCG receptor.

BRIEF SUMMARY OF THE INVENTION

The present invention provides stapled co-agonist peptides of the glucagon (GCG) receptor and the glucagon-like protein 1 (GLP-1) receptor, which have an α-helical conformation due to intramolecular ring formation between two peptide amino acids. The alpha helical structure of the stapled peptides of the present invention results in an increase in physical stability due to decreased proteolytic degradation, and in a lower propensity to self-associate to form β-sheets via hydrogen bonding resulting in a decrease in fibril formation and interpeptide aggregation. Reduced fibril formation and peptide aggregation may result in an increase in peptide efficacy and allows for the use of lower doses of peptide for the same physiological or pharmacological effect. Additionally, the reduction in fibril formation and peptide aggregation leads to a reduction in undesirable amounts of precipitate formation in sterile peptide solutions and peptide formulations. The precipitate formation can lead to cloudy and gel-like suspensions which may adversely affect the dosing accuracy and may cause immunogenic reactions on injection.

The peptides of the present invention are useful for the treatment of metabolic diseases or disorders, such as but not limited to, diabetes (e.g., type 1 diabetes, Type 2 diabetes, or gestational diabetes), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and/or obesity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides stapled peptide co-agonists of the glucagon (GCG) receptor and the glucagon-like protein 1 (GLP-1) receptor that have the amino acid sequences provided below.

In one embodiment, the GCG receptor/GLP-1 receptor co-agonist peptides of the present invention comprise the amino acid sequence of native human glucagon (SEQ ID NO: 1) wherein

1) L-Serine at X² is replaced with α-aminoisobutyric acid (Aib), or D-Serine;
2) Tyrosine at X¹⁰ is replaced with Lysine, Lysine conjugated to a fatty acid, or Lysine conjugated to a fatty diacid;
3) L-Serine at X¹⁶ is replaced with α-aminoisobutyric acid (Aib), or Glutamic acid; and
4) X³⁰ is absent, or Lysine conjugated by a gamma-glutamic acid (yE) spacer;
- and may include up to eight additional amino acid substitutions;
- wherein the eight additional amino acid substitutions are selected from:
  - 1) Lysine at X¹² is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
  - 2) Arginine at X¹⁷ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
  - 3) Alanine at X¹⁹ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid;
  - 4) Glutamine at X²⁰ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
  - 5) Aspartic acid at X²¹ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
  - 6) Glutamine at X²⁴ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
  - 7) Methionine at X²⁷ is optionally replaced with Leucine, or (S)-2-amino-2-methylnon-8-enoic acid; and
  - 8) Asparagine at X²⁸ is optionally replaced with Aspartic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
- provided that the peptide contains at least two amino acids selected from (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, and (S)-2-aminohept-6-enoic acid, which cyclize to form a double bond containing ring; or a pharmaceutically acceptable salt thereof.

In a class of this embodiment, the two amino acids acids which cyclize to form a double bond containing ring are selected from: 1) one (R)-2-amino-2-methyloct-7-enoic acid and one (S)-2-amino-2-methylnon-8-enoic acid; 2) two (S)-2-amino-2-methylhept-6-enoic acids; and 3) two (S)-2-aminohept-6-enoic acids.

In another class of this embodiment, the two amino acids acids which cyclize to form a double bond containing ring are one (R)-2-amino-2-methyloct-7-enoic acid and one (S)-2-amino-2-methylnon-8-enoic acid.

In another class of this embodiment, the two amino acids acids which cyclize to form a double bond containing ring are two (S)-2-amino-2-methylhept-6-enoic acids.

In another class of this embodiment, the two amino acids acids which cyclize to form a double bond containing ring are two (S)-2-aminohept-6-enoic acids.

In another class of this embodiment, (R)-2-amino-2-methyloct-7-enoic acid and (S)-2-amino-2-methylnon-8-enoic acid cyclize to form a double bond containing ring; or two (S)-2-amino-2-methylhept-6-enoic acids cyclize to form a double bond containing ring; or two (S)-2-aminohept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, (R)-2-amino-2-methyloct-7-enoic acid and (S)-2-amino-2-methylnon-8-enoic acid cyclize to form a double bond containing ring.

In another class of this embodiment, two (S)-2-amino-2-methylhept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, two (S)-2-aminohept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, the alkene of one (R)-2-amino-2-methyloct-7-enoic acid and the alkene of one (S)-2-amino-2-methylnon-8-enoic acid cyclize to form a double bond containing ring; or the alkenes of two (S)-2-amino-2-methylhept-6-enoic acids cyclize to form a double bond containing ring; or the alkenes of two (S)-2-aminohept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, the alkene of one (R)-2-amino-2-methyloct-7-enoic acid and the alkene of one (S)-2-amino-2-methylnon-8-enoic acid cyclize to form a double bond containing ring.

In another class of this embodiment, the alkenes of two (S)-2-amino-2-methylhept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, the alkenes of two (S)-2-aminohept-6-enoic acids cyclize to form a double bond containing ring.

In another class of this embodiment, the GCG receptor/GLP-1 receptor co-agonist peptides of the present invention comprise the amino acid sequence of native human glucagon (SEQ ID NO: 1) wherein Tyrosine at X¹⁰ is replaced with is Lysine, or Lysine conjugated to a fatty acid; or a pharmaceutically acceptable salt thereof.

In another class of this embodiment, the GCG receptor/GLP-1 receptor co-agonist peptides of the present invention comprise the amino acid sequence of native human glucagon (SEQ ID NO: 1) with up to six additional amino acid substitutions are selected from:

1) Lysine at X¹² is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
2) Arginine at X¹⁷ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
3) Alanine at X¹⁹ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid;
4) Glutamine at X²⁰ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
5) Aspartic acid at X²¹ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
6) Glutamine at X²⁴ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
7) Methionine at X²⁷ is optionally replaced with Leucine, or (S)-2-amino-2-methylnon-8-enoic acid; and
8) Asparagine at X²⁸ is optionally replaced with Aspartic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid; or a pharmaceutically acceptable salt thereof.

In another class of this embodiment, the GCG receptor/GLP-1 receptor co-agonist peptides of the present invention comprise the amino acid sequence of native human glucagon (SEQ ID NO: 1) with up to four additional amino acid substitutions are selected from:

1) Lysine at X¹² is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
2) Arginine at X¹⁷ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
3) Alanine at X¹⁹ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid;
4) Glutamine at X²⁰ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
5) Aspartic acid at X²¹ is optionally replaced with (R)-2-amino-2-methyloct-7-enoic acid;
6) Glutamine at X²⁴ is optionally replaced with (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
7) Methionine at X²⁷ is optionally replaced with Leucine, or (S)-2-amino-2-methylnon-8-enoic acid; and
8) Asparagine at X²⁸ is optionally replaced with Aspartic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid; or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a peptide comprising the amino acid sequence

HX²QGTFTSDX¹⁰SX¹²YLDX¹⁶X¹⁷AX¹⁹X²⁰X²¹FVX²⁴WLX²⁷X²⁸TX³⁰-NH₂ (SEQ ID NO: 20) wherein
X² is α-aminoisobutyric acid (Aib), or D-Serine;
X¹⁰ is Lysine, Lysine conjugated to a fatty acid, or Lysine conjugated to a fatty diacid;
X¹² is (R)-2-amino-2-methyloct-7-enoic acid, or Lysine;
X¹⁶ is α-aminoisobutyric acid (Aib), or Glutamic acid;
X¹⁷ is (R)-2-amino-2-methyloct-7-enoic acid, or Arginine;
X¹⁹ is (S)-2-amino-2-methylnon-8-enoic acid, or Alanine;
X²⁰ is Glutamine, (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
X²¹ is Aspartic acid or (R)-2-amino-2-methyloct-7-enoic acid;
X²⁴ is Glutamine, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
X²⁷ is Leucine, (S)-2-amino-2-methylnon-8-enoic acid, or Methionine;
X²⁸ is Aspartic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, or (S)-2-aminohept-6-enoic acid;
X³⁰ is absent, or Lysine conjugated by a gamma-glutamic acid (yE) spacer;

provided that the peptide contains at least two amino acids selected from (R)-2-amino-2-methyloct-7-enoic acid, (S)-2-amino-2-methylnon-8-enoic acid, (S)-2-amino-2-methylhept-6-enoic acid, and (S)-2-aminohept-6-enoic acid, which cyclize to form a double bond containing ring; and wherein the peptide optionally includes a protecting group that, if present, is joined to the C-terminal carboxy group of the peptide; or a pharmaceutically acceptable salt thereof.