WO2021148664A1 - Oxyntomodulin mimetics - Google Patents

Abstract

Disclosed herein are oxyntomodulin mimetics, and their use as medicaments in the treatment of various diseases and disorders including, but not limited to, excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis, alcoholic and non-alcoholic fatty liver disease, diabetes (Type I and Type II), obesity, the regulation of blood glucose levels, the regulation of response to glucose tolerance tests, and the regulation of food intake.

Description

Oxyntomodulin Mimetics

Field of the Invention

The present invention relates to oxyntomodulin mimetics, and extends to their use as medicaments in the treatment of various diseases and disorders including, but not limited to, excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis, alcoholic and non- alcoholic fatty liver disease, diabetes (Type I and Type II), obesity, the regulation of blood glucose levels, the regulation of response to glucose tolerance tests, and the regulation of food intake.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome (Masuoka & Chalasani, 2013) and is increasingly becoming common in parallel with the increasing prevalence of obesity (Brunt et al., 2015; Rinella, 2015). NAFLD is characterized by excessive fat accumulation in the liver in the absence of excessive alcohol consumption or any other specific causes of hepatic steatosis (Masuoka & Chalasani, 2013)- a lipid accumulation above 5% of the liver weight is classified as NAFLD (Rinella, 2015). When the balance between lipid uptake and utilization is abrogated, lipids accumulate in the liver. NAFLD encompasses a variety of liver pathologies with different clinical manifestations, extending from simple lipid accumulation in the hepatocytes to nonalcoholic steatohepatitis (NASH) with intralobular inflammation, hepatocellular ballooning and fibrosis (Day et al., 1998; Farrell & Larter, 2006; Tilg & Moschen, 2010), and is projected to be the leading cause of liver transplants in the future (Wong et al., 2015). Importantly, accumulation of fatty acids and increased inflammation is associated with insulin resistance, and a significant proportion of those affected develop or already present with type 2 diabetes (Rinella, 2015). Presently, there are no approved pharmacological treatments for NASH.

Lifestyle changes focusing on healthy eating, weight loss and regular exercise are a cornerstone in NAFLD therapy in adults (Hannah & Harrison, 2016b; Orci et al., 2016; Vilar-Gomez et al., 2015) and children (Africa, Newton, & Schwimmer, 2016) , and bariatric surgery has been shown to reverse NASH and even substantial fibrosis (Hannah & Harrison, 2016a). Surgery however is only performed in a minority of the patients and is associated with peri- and postoperative hazards, hence there is clearly a need for pharmacological therapies to treat NASH (Musso, Cassader, & Gambino, 2016; Ratziu, 2016).

Worldwide, there are about 250 million diabetics and the number is projected to double in the next two decades. Over 90% of this population suffers from type 2 diabetes mellitus (T2DM). It is estimated that only 50-60% of persons affected with T2DM or in stages preceding overt T2DM are currently diagnosed.

T2DM is a heterogeneous disease characterized by abnormalities in carbohydrate and fat metabolism. The causes of T2DM are multi-factorial and include both genetic and environmental elements that affect b-cell function and insulin sensitivity in tissues such as muscle, liver, pancreas and adipose tissue. As a consequence impaired insulin secretion is observed and paralleled by a progressive decline in b-cell function and chronic insulin resistance. The inability of the endocrine pancreas to compensate for peripheral insulin resistance leads to hyperglycaemia and onset of clinical diabetes. Tissue resistance to insulin- mediated glucose uptake is now recognized as a major pathophysiologic determinant of T2DM.

A success criterion for an optimal T2DM intervention is the lowering of blood glucose levels, which can be both chronic lowering of blood glucose levels and increased ability to tolerate high glucose levels after food intake, described by lower peak glucose levels and faster clearance. Both of these situations exert less strain on b-cell insulin output and function.

Type I diabetes is characterised by a loss of the ability to produce insulin in response to food intake and hence an inability to regulate blood glucose to a normal physiological level.

Over the past 30 years, a dramatic increase in obesity has been registered. Alone in the US; over one-third of the adult population is either obese or overweight. Numerous disease types; diabetes, fatty liver disease, cardiovascular disease, and cancer have been shown to be associated with obesity. Due to the metabolic heterogeneity of obesity and the alarming rates of the prevalence of obesity, a healthcare burden has emerged. For example, in a meta-analysis reported by Kim et A1. (2016), the annual medical costs of obesity in the US was estimated to account for $149.4 billion in 2014 (D. D. Kim &

Basu, 2016). Furthermore, an increase of ~ 9 % in global estimates of obesity prevalence was found between 1975-2016 (http://apps.who.int/gho/data/ node.main.A896).

Obesity results in impaired life quality, thereby conveying an increased risk of co-morbidities and mortality. Therefore, prevention of obesity is highly sought after by the discovery of efficacious obesity treatments.

Most available obesity and type 2 diabetes treatments primarily treat symptoms. Furthermore, they often have limited therapeutic indices resulting in restricted dosing, cardiovascular complications, and hypoglycemia (Clemmensen et al., 2019).

Different approaches have been investigated in order to lessen obesity. In common, obesity treatments focus on creating a negative energy balance by increasing energy expenditure or decreasing energy intake.

Regulation of appetite has been found to create a negative energy balance effectively. However, drugs acting through neurotransmission (serotonergic receptors, cannabinoid receptors, etc.) have unfortunately been found to cause mood disorders (Cohen & Weinstein, 2018; Halford, Boyland, Lawton, Blundell, & Harrold, 2011). In the later years, an increased understanding of the process involved in obesity has led to progress in pharmacological approaches to combat obesity. Understanding of the physiological role of ghrelin, leptin, and gut-derived peptide hormones has expanded (Clemmensen et al., 2019).

Upon obesity, lifestyle modifications remain the first-line treatment. However, these recommendations are often not useful in long-term weight loss control. For many years, pharmacological therapeutics had relatively weak efficacies (5 - 10 % body weight loss) together with unwanted side- effects such as anxiety and depression (Hope, Tan, & Bloom, 2018; O'Neil et al., 2018).

Morbidly obese patients who have been unable to sustain weight loss by non-surgical means are often offered a surgical intervention known to be effective in terms of body weight loss and improving glucose homeostasis and insulin sensitivity. Despite the effectiveness of surgical procedures, there is a risk of experiencing post-surgical complications such as hypoglycemia after food intake (Hope et al., 2018). Bariatric surgery is a costly solution as it requires both specialist surgeons and facilities.

Furthermore, the weight loss associated with the surgeries is of varying degree. Therefore, this is not applicable for whole populations.

Incretins, however, play an essential role in postprandial insulin release and glucose homeostasis. In addition to weight loss, the incretin hormones have been shown to have a role in glucose control after bariatric surgery (Dimitriadis, Randeva, & Miras, 2017).

The therapeutic potential of incretins, particularly, proglucagon-derived peptides, have for many years been investigated. Several medications to aid type 2 diabetes treatment (Liraglutide [glucagon-like peptide 1 (GLP-1)] analog, Semaglutide (GLP-1 analog), etc.) have been developed during the past years.

In common, most of the drugs utilize the signaling pattern of products of the precursor polypeptide preproglucagon (Drucker, 2005). The processing of proglucagon results in different bioactive products with the ability to control blood glucose and energy expenditure.

One of the most widely studied peptide hormones GLP-1 (often used as a template in drug discovery), is secreted from the L-cells of the gut. GLP-1 acts directly on the b-cells to increase glucose-stimulated insulin secretion and also through the central nervous system to decrease food intake. Furthermore, GLP-1 slows gastric emptying.

Another well studied peptide hormone is glucagon, secreted from the pancreatic alpha cells. Glucagon acts as a counterregulatory hormone. Upon physiological challenges affecting euglycemia, levels of glucagon changes. Glucagon opposes insulin action by increasing hepatic glucose production. Furthermore, several studies have indicated a potential role of glucagon in the regulation of energy metabolism (Drucker, 2018).

The diabetogenic effects of glucagon have for long been considered a contraindication against its use for weight management in obese diabetic individuals. However, expanded understanding of the physiological role of glucagon has resulted in a revived interest in its use. Glucagon signaling results in an increased lipolysis, and thermogenic activation, which potentiates and increases energy expenditure and results in an overall negative energy balance (Miiller, Finan, Clemmensen, Dimarchi, & Tschop, 2019).

By combining the beneficial effects of glucagon with GLP-1 mediated effects, many metabolic comorbidities can be treated. The GLP-1 component is believed to preserve normoglycemia despite increased hepatic glucose production driven by glucagon. For that reason, many companies have initiated programs to develop GLP-l/glucagon therapeutics (Clemmensen et al., 2019).

The GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) belong to class B of the GPCR family. The ligands in this family can roughly be divided into three domains based on their primary amino acid sequence. An N-terminal receptor activating domain, a central a-helix for structure, and a C- terminal end for initial ligand binding to the extracellular domain of the target receptor (Culhane, Liu, Cai, & Yan, 2015).

Interestingly, tissue-specific post-translational modification of proglucagon results in several bioactive peptides of different length. In the gut, endocrine L cells secrete the 37-amino acid peptide hormone oxyntomodulin in proportion to nutrient ingestion. It consists of the 29-amino acids of glucagon and an 8-amino acid C-terminal tail. Oxyntomodulin elicits a biological response through interaction with both the GLP1R and GCGR, affecting gastric acid secretion, glucose homeostasis, and energy balance (Pocai, 2014).

The sequence of glucagon is highly conserved over a wide range of species, as shown below:

Human: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

(SEQ ID NO: 1)

Porcine:

(SEQ ID NO: 2)

African clawed frog:

(SEQ ID NO: 1)

Chicken :

(SEQ ID NO: 3) Gila Monster:

(SEQ ID NO: 1)

American Bullfrog:

(SEQ ID NO: 4)

King Cobra: — E- FTEH— NMK-R— -H— IN-

(SEQ ID NO: 5)

Alligator gar:

(SEQ ID NO: 6)

Chinchilla :

(SEQ ID NO: 7)

Ghost Shark:

Sea Lamprey:

Platypus :

(SEQ ID NO: 10)

As noted above, oxyntomdoulin consists of the sequence of glucagon followed by eight additional amino acids at the C- terminus of the the glucagon sequence. Unfortunately, databases such as UniProt do not directly recognize searches for "oxyntomodulin". However, a limited number of native oxyntomodulin sequences can nevertheless be found via literature searches for glucagon sequences. Interestingly, this does reveal a broad sequence variation at the 8-amino acid C-terminal tail.

C-terminal tails (amino acid positions 30-37):

Human:

Porcine :

African clawed frog: Chicken :

Gila Monster: American Bullfrog: King Cobra:

Alligator gar:

Ghost Shark:

Sea Lamprey:

Platypus :

The short circulatory half-life (~ 12 min) of oxyntomodulin makes it challenging for development into a therapeutic agent (Schjoldager, Baldissera, Mortensen, Christiansen, & Hospital, 1988). Degradation of oxyntomodulin is mediated by proteases such as Dipeptidyl peptidase IV and Neprilysin.

There are numerous approaches known in the art for attempting to improve the in-vivo half-life of peptide drugs. Such approaches include improving proteolytic stability (by, e.g., protecting the N- and C-termini, replacing amino acids with D-amino acids or unnatural amino acids, cyclising the peptide, etc.) and reducing renal clearance (by, e.g., conjugating the peptide to macromolecules, such as large polymers, albumin, immunoglobulins, etc.). One approach for trying to improve the pharmacokinetic and pharmacodynamic properties of peptide drugs is to acylate the peptide.

However, it is also known in the art that making such modifications to drug peptides can be deleterious in terms of, for example, reduced drug potency and unpredictable adverse side reactions, such as drug sensitisation. As such, it is not possible to predict whether such modifications necessarily would improve the therapeutic profile of a peptide drug, and developing peptide drugs that have an improved in-vivo half-life remains a challenging prospect.

Accordingly, there remains a need to develop novel GLP1R/GCGR dual agonists for use as therapeutic agents in treating the aforementioned conditions.

Summary of the Invention

Described herein are novel oxyntomodulin mimetics that are GLP1R/GCGR dual agonists that (as compared to native human oxyntomodulin) exhibit improved properties, in terms of agonizing one or both of GLP1R and/or GCGR, and/or as regards the peptide's duration of action and in-vivo half-life. The oxyntomodulin mimetics described herein elicit a prominent effect in terms of reduction in caloric intake, body weight lowering, improved glucose control, and a protracted duration of action.

Accordingly, in a first aspect the present invention provides an oxyntomodulin mimetic having the amino acid sequence: HX_2QGTFX₇X₈DYSKX₁₃LDX₁₆X₁₇X₁₈AX₂₀X₂₁FVQWLMNTX₃₀ (SEQ ID NO: 21) wherein

X₂ = a-aminoisobutyric acid (CAS Number 62-57-7), also referred to herein after as "AiB"

X₇ = T or S

X₈ = S or N, subject to the proviso that where X₇ is S then X₈ is not N Xi₃ = Y or H X₁₆ = a-aminoisobutyric acid (AiB) or K

Xi₇ = R or K X₁₈ = R or Y

X₂₀ = R, K or Q

X₂₁ = D or E

X₃₀ is an or amino acid sequence or amino acid selected from

KRNGX₃₄X₃₅GX₃₇ ( SEQ ID NO : 22) , KRNGX₃₄X₃₅G ( SEQ ID NO : 23) ,

KRNGX₃₄X_{35 (}SEQ ID NO: 24) , KRNGX_{34 (}SEQ ID NO: 25) , KRNG (SEQ

ID NO: 26), KRN, KR and K

X₃₄ , where present, is Q or E

X₃₅, where present, is Q or E

X₃₇ , where present, is Q or E.

In a second aspect, the present invention provides a pharmaceutical composition comprising an oxyntomodulin mimetic according to the first aspect.

In a third aspect, the present invention provides an oxyntomodulin mimetic according to the first aspect, for use as a medicament. In particular, the oxyntomodulin mimetic according may be for use in treating excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, diabetes (Type I and/or Type II), obesity, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake.

In a fourth aspect the present invention provides a method of treating excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, diabetes (Type I and/or Type II), obesity, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake, comprising administering to a patient in need of said treatment an effective amount of an oxyntomodulin mimetic according to the firt aspect.

Figures

Figure 1: Area under the curve of oxyntomodulin mimetics during a 24h stimulation study. Cells were stimulated with either OXM-064, OXM-073 or OXM-079 at 200 nM for 3 hours, 6 hours and 24 hours and b-arrestin levels were measured at both receptors. Dotted line designates the mean AUC levels for OXM-000.

Figure 2: Area under the curve of oxyntomodulin mimetics during a 24h stimulation study. Cells were stimulated with OXM-079, OXM-094, OXM-095, OXM-096, OXM-097, OXM-099 or OXM- 100 at 200 nM for 3 hours, 6 hours, and 24 hours and b- arrestin levels were measured at both receptors. Figure 3: Area under the curve of oxyntomodulin mimetics during a 24h stimulation study. Cells were stimulated with either OXM-064, OXM-102, or OXM-OOO at 200 nM for 3 hours, 6 hours and 24 hours, and b-arrestin levels were measured at both receptors.

Figure 4: Representative b-arrestin recruitment - dose response by OXM-079 and OXM-079 (A16.17) as a function of GLP1R and GCGR. Cells were stimulated with ligands at a starting concentration of 20 mM. The ligands were diluted by 8-fold at both receptors. Receptor activation is shown as % of Emax whereas data is shown as meaniSEM.

Figure 5: Acute effects of OXM-102 in female C57BL/6JOLAHSD mice. Acute changes in body weight upon a single subcutaneous administration of Vehicle (0.9 % NaCl), OXM-102 (A16.15) 10 nmol/kg, OXM-102 (A16.15) 30 nmol/kg or OXM-102 (A16.15) 100 nmol/kg. Body weight was measured every 24 hours during a 72 hour study. Data is shown as body weight change as percent of baseline.

Figure 6: Acute effects of oxyntomodulin mimetics of different compositions in female C57BL/6JOLAHSD mice. Acute changes in body weight upon a single subcutaneous administration of Vehicle (0.9 % NaCl), OXM-102 (A16.15), 50 nmol/kg, OXM-064 (A16.12) 50 nmol/kg, OXM-064 (A16.13) 50 nmol/kg, OXM-064 (A.13.15) 50 nmol/kg or OXM-064 (A17.15) 50 nmol/kg. Body weight was measured every 24 hours during a 96 hour study. Data is shown as body weight change as percentage from baseline.

Figure 7: Acute effects of OXM-102 with either C-18 or C-20 fatty diacid in female C57BL/6JOLAHSD mice. Acute changes in body weight upon a single subcutaneous administration of Vehicle (0.9 % NaCl), OXM-102 (A16.15) 100 nmol/kg or OXM-102 (A16.17) 100 nmol/kg. Body weight was measured every 24 hours during a 96 hour study. Data is shown as body weight change as percentage from baseline (Figure 7A), and food intake in grams per four animals per day (Figure 7B).

Figure 8: Acute effects of oxyntomodulin mimetics in female C57BL/6JOLAHSD mice. Acute changes in body weight upon a single subcutaneous administration of Vehicle (0.9 % NaCl), OXM-102 (A16.17) 100 nmol/kg and OXM-079 (A16.17)50 nmol/kg.

Body weight was measured every 24 hours during a 96 hour study. Data is shown as body weight change as percentage from baseline.

Figure 9: Acute effects of oxyntomodulin mimeticss in female C57BL/6JOLAHSD mice. Acute changes in body weight upon a single subcutaneous administration of Vehicle (0.9 % NaCl), OXM-102 (A16.17) 200 nmol/kg, OXM-079 (A16.14) 200 nmol/kg,

OXM-079 (A16.16) 200 nmol/kg and OXM-079 (A16.17) 200 nmol/kg. Body weight was measured every 24 hours during a 96 hour study. Data is shown as body weight change as percentage from baseline.

Figure 10: OXM-102 (A16.15) potently reduces appetite, body weight and improves glucose tolerance. (Figure 10A) Body weight expressed as percentage from baseline. (Figure 10B) Cumulative food intake. (Figure IOC) Plasma glucose during an Oral Glucose Tolerance Test. (Figure 10D) Total AUC of the Oral glucose tolerance test. (Figure 10E) Incremental AUC of the Oral glucose tolerance test.

Figure 11: Effects of OXM-102 (A16.15) independent of energy intake. (Figure 11A) Body weight expressed as percentage of Vehicle. (Figure 11B) Cumulative food intake. (Figure 11C) Plasma glucose during an Oral Glucose Tolerance test. (Figure 11D) total AUC of the Oral glucose tolerance test. (Figure H E) Incremental AUC of the Oral glucose tolerance test.

Figure 12: Thioflavin flourescence during the fibrillation process of OXM-102 with different acylations. Eighteen hours incubation of OXM-102 (A16.17), OXM-102 (A16.18) and OXM-102

(A16.15) at 200 mM with floursencese measurments every hour. The peptides were dissolved in a 5 mM sodium phosphate buffer. Dual amylin and calcitonin receptor agonist (KBP-372) serves as fibrilliation positive control. The relative fluorescence units (RFU) is shown as a function of time and data is presented as mean ±SEM.

Detailed Description

Oxyntomodulin mimetics

As noted above, the present invention relates to oxyntomodulin mimetics that have the following amino acid sequence :

HX2QGTFX₇X8DYSKX13LDX16X17X18AX20X21FVQWLMNTX30 (SEQ ID NO: 21) wherein X₂ , X₇ , X₇ , X₁₃ , Xie , X₁₇ , X₁₈ , X₂₀ , X₂₁ and X₃₀ are as previously defined.

In preferred embodiments X₃₀ is KRNGX₃₄X₃₅GX₃₇ ( SEQ ID NO: 22), KRNG ( SEQ ID NO: 26) or K (and wherein, as previously defined, X₃₄ , X₃₅ and X₃₇ are each indepently selected from Q or E). Most preferably, X₃₀ is KRNGX₃₄X₃₅GX₃₇ ( SEQ ID NO: 22). For example, X₃₀ may be, but is not limited to, KRNGQQGQ ( SEQ ID NO 27) or KRNGEEGE ( SEQ ID NO: 28).

In preferred embodiments X20 is R or K. Most preferably, X20 is R.

In preferred embodiments, X₇ is T and Xs is S.

In some embodiments, X13 is Y, Xis is R and X21 is D. In some other embodiments, X13 is H, X₁₈ is Y and X21 is E. In some embodiments, X₁₆ is AiB and X₁₇ is R.

In more preferred embodiments, however, one or other of X₁₆ and Xi₇ is K, wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group.

Most preferably, X₁₆ is K wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group. Preferably, X₁₇ is then R.

Alternatively, X₁₇ may be K wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group. Preferably, X₁₆ is then AiB.

Where one or other of X₁₆ and X₁₇ is K, wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group, it is preferred that said acyl group is selected from any one of the following: a C₁₈ or longer fatty acid with an optional linker, more preferably a C₁₈ to C₃₀ fatty acid with an optional linker, and most preferably a C₁₈ to C₂₂ fatty acid with an optional linker; or a C₁₈ or longer fatty diacid with an optional linker, more preferably a C₁₈ to C₃₀ fatty diacid with an optional linker, and most preferably a C₁₈ to C₂₂ fatty diacid with an optional linker.

Preferably said linker is present, and is a Gamma-Glutamic acid (yGlu) based linker. Most preferably, the linker comprises or consists of two yGlu residues linked together.

A linker consisting of two yGlu residues linked together (i.e. a two y-Glu repeat, yGlu-yGlu) is particularly preferred .

Particularly preferred acyl groups are listed and shown below (in the depicted chemical formula, the wavy bond designates the peptide backbone, and the dotted line constitutes the conjugation to the lysine moiety). yGlu-yGlu-C₁₈ diacid

Particularly preferred oxyntomodulin mimetics include those having an amino acid sequence in accordance with one the the following sequences:

wherein AiB is a-aminoisobutyric acid and wherein KAc is a lysine residue wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group.

The oxyntomodulin mimetics of the present invention may be acylated at their N-terminal or otherwise modified to reduce the positive charge of the first amino acid, and independently of that may be amidated at their C-terminal.

The oxyntomodulin mimetics of the invention may be produced using any suitable method known in the art for generating peptides, such as synthetic (chemical) and recombinant technologies. Preferably, the oxyntomodulin mimetics are produced using a synthetic method. Synthetic peptide synthesis is well known in the art, and includes (but is not limited to) solid phase peptide synthesis employing various protecting group strategies (e.g. using Fmoc, Boc, Bzl, tBu, etc .).

As noted supra, in some embodiments the N-terminal side of the oxyntomodulin mimetic is modified to reduce the positive charge of the first amino acid. For example, an acetyl, propionyl, or succinyl group may be substituted onto the first amino acid residue. Alternative ways of reducing positive charge include, but are not limited to, polyethylene glycol-based PEGylation, or the addition of another amino acid such as glutamic acid or aspartic acid at the N- terminus . Alternatively, other amino acids may be added to the N-terminus of the peptides discussed supra including, but not limited to, lysine, glycine, formyl glycine, leucine, alanine, acetyl alanine, and dialanyl.

The oxyntomodulin mimetics of the present invention may be in free acid form. Alternatively, and as noted supra, in some embodiments the C-terminal amino acid of the oxyntomodulin mimetic is instead amidated.

Synthetic chemical methods may be employed for amidating the C-terminal amino acid. One technique for manufacturing amidated versions of a peptide is to react precursors (having glycine in place of the C-terminal amino group of the desired amidated product) in the presence of peptidylglycine alpha- amidating monooxygenase in accordance with known techniques wherein the precursors are converted to amidated products in reactions described, for example, in US4708934 and EP0308067 and EP0382403.

Production of amidated peptides may also be accomplished using the process and amidating enzyme set forth by Consalvo, et al in US7445911; Miller et al, US2006/0292672; Ray et al, 2002, Protein Expression and Purification, 26:249-259; and Mehta, 2004, Biopharm. International, July, pp. 44-46.

The production of amidated peptides may proceed, for example, by producing glycine-extended precursor in E. coli as a soluble fusion protein with glutathione-S-transferase, or by direct expression of the precursor in accordance with the technique described in US6103495. Such a glycine extended precursor has a molecular structure that is identical to the desired amidated peptide except at the C-terminus (where the desired peptide terminates --X--NH₂, while the precursor terminates --X-gly, X being the C-terminal amino acid residue of the desired peptide). An alpha-amidating enzyme described in the publications above catalyzes conversion of precursors to desired product peptide. That enzyme is preferably recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells), as described in the Biotechnology and Biopharm. articles cited above.

Free acid forms of the oxyntomodulin mimetics of the present invention may be produced in like manner, except without including a C-terminal glycine on the "precursor", which precursor is instead the final peptide product and does not require the amidation step.

Pharmaceutical formulations

The present invention also relates to pharmaceutical compositions comprising an oxyntomodulin mimetic as described supra.

The oxyntomodulin mimetics of the invention may preferably be formulated for parenteral administration. For example, the oxyntomodulin mimetics may be formulated for injection, preferably for subcutaneous injection.

For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection), solutions of the oxyntomodulin mimetic in either sesame or peanut oil or in aqueous propylene glycol may be employed, for example. The aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parenteral application, examples of suitable preparations include solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Peptides may be formulated in sterile form in multiple or single dose formats such as being dispersed in a fluid carrier such as sterile physiological saline or 5% saline dextrose solutions commonly used with injectables.

The oxyntomodulin mimetics of the present invention may also be formulated for enteral administration. For example, the oxyntomodulin mimetics may be formulated in a pharmaceutical composition for oral administration.

Oral enteral formulations are for ingestion by swallowing for subsequent release in the intestine below the stomach, and hence delivery via the portal vein to the liver, as opposed to formulations to be held in the mouth to allow transfer to the bloodstream via the sublingual or buccal routes.

Suitable dosage forms for use in oral adminstration include tablets, mini-tablets, capsules, granules, pellets, powders, effervescent solids and chewable solid formulations. Such formulations may include gelatin which is preferably hydrolysed gelatin or low molecular weight gelatin. Such formulations may be obtainable by freeze drying a homogeneous aqueous solution comprising an oxyntomodulin mimetic and hydrolysed gelatin or low molecular weight gelatin and further processing the resulting solid material into said oral pharmaceutical formulation, and wherein the gelatin may have a mean molecular weight from 1000 to 15000 Daltons.

Such formulations may include a protective carrier compound such as 5-CNAC or others as disclosed herein.

Whilst oral formulations such as tablets and capsules are preferred, suitable compositions for oral administration may also take the form of syrups, elixirs or the like, whilst other enteral formulations such as suppositories or the like may also be used.

In preferred embodiments, the oxyntomodulin mimetics of the invention may be formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide. Alternatively, or in additionally, the oxyntomodulin mimetics may be formulated with a carrier for oral administration, and optionally wherein the carrier increases the oral bioavailability of the peptide. An exemplary carrier may comprise 5-CNAC, SNAD, or SNAC.

As noted above, oxyntomodulin mimetics of the present invention may be formulated for enteral, especially oral, administration by admixture with a suitable carrier compound. Suitable carrier compounds include those described in US Patent No. 5,773,647 and US Patent No. 5866536 and amongst these, 5-CNAC (N-(5-chlorosalicyloyl)-8-aminocaprylic acid, commonly as its disodium salt) is particularly effective. Other preferred carriers or delivery agents are SNAD (sodium salt of 10-(2-Hydroxybenzamido)decanoic acid) and SNAC (sodium salt of N-(8-[2-hydroxybenzoyl]amino)caprylic acid). In an embodiment, a pharmaceutical composition of the present invention comprises a delivery effective amount of carrier such as 5-CNAC, i.e. an amount sufficient to deliver the compound for the desired effect. Generally, the carrier such as 5-CNAC is present in an amount of 2.5% to 99.4% by weight, more preferably 25% to 50% by weight of the total composition .

As noted above, the oral dosage form of the oxyntomodulin mimetics of the present invention can take any known form, e.g. liquid or solid dosage forms. The liquid dosage forms include solution emulsions, suspensions, syrups and elixirs. In addition to the active compound (i.e. the oxyntomodulin mimetic), and any carrier, such as 5-CNAC, the liquid formulations may also include inert excipients commonly used in the art such as, solubilizing agents e.g. ethanol; oils such as cottonseed, castor and sesame oils; wetting agents; emulsifying agents; suspending agents; sweeteners; flavourings; and solvents such as water. The solid dosage forms include capsules, soft-gel capsules, tablets, caplets, powders, granules or other solid oral dosage forms, all of which can be prepared by methods well known in the art. The pharmaceutical compositions may additionally comprise additives in amounts customarily employed including, but not limited to, a pH adjuster, a preservative, a flavorant, a taste-masking agent, a fragrance, a humectant, a tonicifier, a colorant, a surfactant, a plasticizer, a lubricant such as magnesium stearate, a flow aid, a compression aid, a solubilizer, an excipient, a diluent such as microcrystalline cellulose, e.g. Avicel PH 102 supplied by EMC corporation, or any combination thereof. Other additives may include phosphate buffer salts, citric acid, glycols, and other dispersing agents. The composition may also include one or more enzyme inhibitors, such as actinonin or epiactinonin and derivatives thereof; aprotinin, Trasylol and Bowman-Birk inhibitor. Further, a transport inhibitor, i.e. a [rho]- glycoprotein such as Ketoprofin, may be present in the compositions of the present disclosure. The solid pharmaceutical compositions can be prepared by conventional methods e.g. by blending a mixture of the active compound, the carrier if present, and any other ingredients, kneading, and filling into capsules or, instead of filling into capsules, molding followed by further tableting or compression-molding to give tablets. In addition, a solid dispersion may be formed by known methods followed by further processing to form a tablet or capsule. Preferably, the ingredients in the pharmaceutical compositions are homogeneously or uniformly mixed throughout the solid dosage form.

Treatment of patients

The present invention also relates to the use of oxyntomodulin mimetics as described supra as medicaments. The oxyntomodulin mimetics can be administered to a patient to treat a number of diseases, disorders or conditions. As used herein, the term "patient" means any organism belonging to the kingdom Animalia. In preferred embodiments, the term "patient" refers to vertebrates, more preferably, mammals, and most preferrably humans.

Preferably, the oxyntomodulin mimetics are be used in treating one or more of the following: excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, diabetes (Type I and/or Type II), obesity, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake. For example, the peptides may be used for appetite suppression, for reducing an undesirably high fasting blood glucose level, for reducing an undesirably high peak serum glucose level, for reducing an undesirably high response to a glucose tolerance test, for mitigating insulin resistance, for reducing an undesirably high peak serum insulin level, for reducing an undesirably high HbAlc, for producing a decrease in liver triglycerides and/or for reducing fat accumulation in the liver of a subject.

There are a number of art-recognized measures of normal range for body weight in view of a number of factors such as gender, age and height. A patient in need of treatment or prevention regimens set forth herein include patients whose body weight exceeds recognized norms or who, due to heredity, environmental factors or other recognized risk factor, are at higher risk than the general population of becoming overweight or obese. In accordance with the present disclosure, it is contemplated that the oxyntomodulin mimetics may be used to treat diabetes where weight control is an aspect of the treatment.

Except where otherwise stated, the preferred dosage of the oxyntomodulin mimetics of the present invention is identical for both therapeutic and prophylactic purposes.

Except where otherwise noted or where apparent from context, dosages herein refer to weight of active compounds (i.e. oxyntomodulin mimetics) unaffected by or discounting pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are desirably included. As described supra, any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry for delivery of peptide active agents is appropriate for use herein, and the terms "excipient", "diluent", or "carrier" includes such non-active ingredients as are typically included, together with active ingredients in such dosage form in the industry.

In some embodiments, the method of treatment comprises parenteral administration to a patient in need of treatment of a pharmaceutically effective amount of the oxyntomodulin mimetic.

In other embodiments, the method of treatment comprises enteral administration to a patient in need of treatment of a pharmaceutically effective amount of the oxyntomodulin mimetic.

Said methods may include a preliminary step of determining whether the patient suffers from a particular condition, and/or a subsequent step of determining to what extent said treatment is effective in mitigating the condition in said patient. By way of example, the method could include carrying out an oral glucose tolerance test or a test of resting blood sugar level.

In an embodiment, an oxyntomodulin mimetic of the present invention is administered at adequate dosage to maintain serum levels of the mimetic in patients between 5 picograms and 1000 nanograms per milliliter, preferably between 50 picograms and 500 nanograms, e.g. between 1 and 300 nanograms per milliliter. The serum levels may be measured by any suitable techniques known in the art, such as radioimmunoassay or mass spectrometry. The attending physician may monitor patient response, and may then alter the dosage somewhat to account for individual patient metabolism and response. Where the oxyntomodulin mimetic is formulated for oral administration in the form of a pill or capsule, near simultaneous release is best achieved by administering all of the required dosage as a single pill or capsule. However, the required amount of the oxyntomodulin mimetic may also be divided among two or more tablets or capsules which may be administered together such that they together provide the necessary amount of all ingredients. A "pharmaceutical composition" as used herein includes but is not limited to a complete dosage appropriate to a particular administration to a patient regardless of whether one or more tablets or capsules (or other dosage forms) are recommended at a given administration.

Suitable oral dosage levels for adult humans to be treated may be in the range of 0.05 to 5mg, and more preferably about 0.1 to 2.5mg of the oxyntomodulin mimetic.

Administration of the pharmaceutical compositions described herein can be accomplished regularly, e.g. once or more on a daily or weekly basis; intermittently, e.g. irregularly during a day or week; or cyclically, e.g. regularly for a period of days or weeks followed by a period without administration .

The frequency of dosage treatment of patients may be once daily, preferably from one to four times weekly, preferably one to two times weekly, and most preferably once weekly. Treatment will desirably be maintained over a prolonged period of at least 6 weeks, preferably at least 6 months, preferably at least a year, and optionally for life.

Combination treatments for relevant conditions may be carried out using a suitably formulated oxyntomodulin mimetic according to the present invention alongside separate administration of one or more other therapeutics. Alternatively, a composition may be formulated comprising both an oxyntomodulin mimetic according to the present invention and one or more other therapeutics for combined administration .

Combination therapies according to the present invention include combinations of an oxyntomodulin mimetic in accordance with the present invention and insulin, GLP-2, GLP-1, GIP, or amylin, or generally with other anti- diabetics. Thus combination therapies including co- formulations may be made with insulin sensitizers including biguanides such as Metformin, Buformin and Phenformin, TZD's (PPAR) such as Balaglitazone, Pioglitazone, Rivoglitazone, Rosiglitazone and Troglitazone, dual PPAR agonists such as Aleglitazar, Muraglitazar and Tesaglitazar, or secretagogues including sulphonylureas such as Carbutamide,

Chloropropamide, Gliclazide, Tolbutamide, Tolazamide, Glipizide, Glibenclamide, Glyburide, Gliquidone, Glyclopyramide and Glimepriride, Meglitinides/glinides (K+) such as Nateglinide, Repaglinide and Mitiglinide, GLP-1 analogs such as Exenatide, Lixisenatide, Liraglutide, Semaglutide, dulaglutide and Albiglutide, DPP-4 inhibitors such as Alogliptin, Linagliptin, Saxagliptin, Sitagliptin and Vildagliptin, insulin analogs or special formulations such as (fast acting) Insulin lispro, Insulin aspart, Insulin glulisine, (long acting) Insulin glargine, Insulin detemir), inhalable insulin - Exubra and NPH insulin, and others including alpha-glucosidase inhibitors such as Acarbose, Miglitol and Voglibose, amylin analogues such as Pramlintide, Dual Amylin and Calcitonin receptor agonists (DACRAs), SGLT2 inhibitors such as Dapagliflozin, Empagliflozin,

Remogliflozin and Sergliflozin as well as miscellaneous ones including Benfluorex and Tolrestat.

Further combinations include co-administration or co- formulation with leptins. Leptin resistance is a well- established component of type 2 diabetes; however, injections of leptin have so far failed to improve upon this condition. In contrast, there is evidence supporting that amylin, and thereby molecules with amylin-like abilities are able to improve leptin sensitivity. Amylin/leptin combination has been shown to have a synergistic effect on body weight and food intake, and also insulin resistance.

A further preferred combination therapy includes co- formulation or co-administration of the oxyntomodulin mimetics of the invention with one or more weight loss drugs. Such weight loss drugs include, but are not limited to, lipase inhibitors (e.g. pancreatic lipase inhibitors, such as Orlistat), appetite suppressing amphetamine derivatives (e.g. Phentermine), Topiramate, Qysmia® (Phentermine/Topiramate combination), 5-HT_2c receptor agonists (e.g. Locaserin), Contrave® (naltrexone/bupropion combination), glucagon-like peptide-1 [GLP-1] analogues and derivatives (e.g.

Liraglutide, semaglutide), sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) inhibitors (e.g. sarcolipin), Fibroblast growth factor 21 [FGF-21] receptor agonists (e.g. analogs of FGF-21), and b3 adreno receptor agonists (e.g. Mirabegron). Such combinations may be used to treat an overweight condition, such as obesity.

A further combination therapy includes co-formulation or co- administration of the oxyntomodulin mimetics of the invention with one or more pharmacological therapies for NAFLD/NASH. Pharmacological therapies for NAFLD/NASH include Obethicolic acid (an FXR receptor agonist), Elafibranor (a PPAR a/d agonist), Cenicriviroc (a CCR2/CCR5 receptor antagonist), Selonsertib (an ASK-1 inhibitor), Aramchol (a synthetic lipid molecule), Emricasan (a Pancaspase inhibitor), NGM282 (a recombinant FGF19 agonist), BMS-986036 (a recombinant FGF21 agonist), GS-0976 (an Acetyl-CoA carboxylase inbibitor), MGL- 3196 (a thyroid hormone receptor b-agonist) and Liraglutide (a GLP-1 analog) (Perazzo & Dufour, 2017).

Examples

Various embodiments are described and disclosed in the following Examples, which are set forth to aid in the understanding of the present disclosure and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Methodology The approach used by the inventors to identify novel oxyntomodulins suitable for development was based on natural variations found in the animal kingdom. The inventors sought to identify oxyntomodulin mimetics with improved properties or at least to provide alternative sequences improving on the properties of oxyntomodulins, particularly in respect of the duration of action.

In order to achieve these goals, an in vitro screening was initiated. Firstly, tail differences were investigated by the screening of peptides consisting of human glucagon fused to different animal tails. Subsequently, the best tails were chosen for further screening. This consisted of testing different animal glucagons fused to the tails with the highest potency from the initial screening. Further optimization led to the development of three lead candidates; referred to herein as "OXM-064", "OXM-073", and "OXM-079". Post in vitro oxyntomodulin mimetic screening, an in vivo screening was initiated. Here, the focus was on finding the best acylation site and linker type in order to achieve a peptide with high in vivo potency.

Methods :

Peptide therapy:

Synthetic oxyntomodulin mimetics (Synpeptide, Shanghai China) were dissolved in Phosphate-buffered Saline (in vitro experimentation,) or 0.9 % NaCl (in vivo experimentation). The doses chosen for oxyntomodulin mimetics administration were based on previous studies found in the literature with dual agonists of the GLP1R/GCGR.

In vitro peptide screening:

The potency of the oxyntomodulin mimetics at the GCGR and GLP1R were determined using the PathHunter® b-Arrestin GPCR assay. Cell lines heterologously expressing the GLP1R (CHO-K1 GLP1R DiscoveRx: Cat. No.: 93-0300C2) and GCGR (CHO-K1 GCGR, DiscoveRx: Cat. No.: 93-0241C2) were used. All experiments were conducted using 2500 cells per well in 10 pL cell-type in Gibco's Ham's F-12 Nutrient Mix, cat. 21765-037 from Invitrogen with the addition of 10 % FBS (fetal bovine serum), 400 pg/mL Hygromycin B, 800 pg/mL Geneticin, and Penicillin/Streptomycin 1 unit. To quantify the GPCR-mediated b-arrestin recruitment, the PathHunter® Detection Kit (93— 0001, DiscoverX) was used and assay performed according to the manufacturer's instructions. In vitro screening of peptides was performed with ligand concentrations starting from either 5 mM or 20 mM.

Animal studies:

All animal procedures were performed in accordance with guidelines from the Animal Welfare Division of the Danish Ministry of justice under the institutional license issued to Nordic Bioscience (2016-15-0201-00910). Female C57BL/6JOlaHsd mice (Envigo, Netherlands) were obtained 12 weeks of age and housed quad-wise under controlled temperature (21-22°C) on a normal 12-hour light-dark cycle (lights on 1900) with unrestricted access to water and food in a standard TYPE III H cage. The mice were fed a High-Fat-diet (60 % kcal fat) and allowed ad libitum access to food and water. Seven studies were in C57BL/6 mice. Mice were matched according to body weight.

Acute study 1 (Example 8) - [Figure 5]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 21.7 grams (~16 weeks old) were used to evaluate the effects of OXM-102 (A16.15) on acute reductions in body weight during a 96-hour evaluation study. The mice were randomized by weight into four groups, (n=12/group), Vehicle (0.9 % NaCl), OXM-102 (A16.15) (10 nmol/kg, 30 nmol/kg and 100 nmol/kg).

They were fasted overnight and then treated with a single dose of OXM-102 (A16.15) at different concentrations or Vehicle (0.9 % NaCl), in the morning using subcutaneous administration. The body weight was monitored in the following intervals every 24 hours.

Acute study 2 (Example 9) - [Figure 6]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 40 grams (~20 weeks old) were used to evaluate the effects of the oxyntomodulin mimetics on acute reductions in body weight during a 96-hour evaluation study. The mice were randomized by weight into six groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-102 (A16.15) 50 nmol/kg, OXM-064 (A16.12) 50 nmol/kg,

OXM-064 (A16.13) 50 nmol/kg, OXM-064 (A13.15) 50 nmol/kg and

OXM-064 (A17.15) 50 nmol/kg. They were fasted overnight and then treated with a single dose of Vehicle (0.9 % NaCl), OXM- 102 (A16.15), OXM-064 (A16.12), OXM-064 (A16.13), OXM-064

(A13.15) or OXM-064 (A17.15) in the morning using subcutaneous administration. The body weight was monitored in the following intervals every 24 hours.

Acute study 3 (Example 10) - [Figure 7]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 44 grams (~28 weeks old) were used to evaluate the effects of the oxyntomodulin mimetics on acute reductions in food intake and body weight loss in vivo during a 96-hour evaluation study. The mice were randomized by weight into three groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-102 (A16.15) 100 nmol/kg and OXM-102 (A16.17) 100 nmol/kg. They were fasted overnight and then treated with a single dose of Vehicle (0.9 % NaCl), OXM-102 (A16.15) 100 nmol/kg or OXM-102 (A16.17) in the morning using subcutaneous administration. The food intake and body weight were monitored in the following intervals every 24 hours.

Acute study 4 (Example 11) - [Figure 8]: Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 50 grams (~41 weeks old) were used to evaluate the effects of the oxyntomodulin mimetics on acute reductions in body weight loss during a 96-hour evaluation study. The mice were randomized by weight into three groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-079 (A16.17) 50 nmol/kg and OXM-102 (A16.17) 75 nmol/kg. They were fasted overnight and then treated with a single dose of Vehicle (0.9 % NaCl), OXM-079 (A16.17) or OXM-102 (A16.17) in the morning using subcutaneous administration. The body weight was monitored in the following intervals every 24 hours.

Acute study 5 (Example 12) - [Figure 9]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 50 grams (~38 weeks old) were used to evaluate the effects of the oxyntomodulin mimetics on acute reductions in body weight loss during a 96-hour evaluation study. The mice were randomized by weight into five groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-079 (A16.14) 200 nmol/kg, OXM-079 (A16.16) 200 nmol/kg, OXM-079 (A16.17) 200 nmol/kg and OXM-102 (A16.17) 200 nmol/kg. They were fasted overnight and then treated with a single dose of Vehicle (0.9 % NaCl), OXM-079 (A16.14) 200 nmol/kg, OXM-079 (A16.16) 200 nmol/kg, OXM-079 (A16.17) 200 nmol/kg or OXM-102 (A16.17) 200 nmol/kg in the morning using subcutaneous administration. The body weight was monitored in the following intervals every 24 hours.

Chronic study 1 (Example 13) - [Figure 10]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 43.5 grams (~35 weeks old) were used to evaluate the effects of OXM-102 (A16.15) 30 nmol/kg and OXM-06430 nmol/kg on chronic reductions in food intake, body weight loss and glucose tolerance during a 20 days evaluation study. The mice were randomized by weight into three groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-102 (A16.15) 30 nmol/kg and OXM-064

(A16.12) 30 nmol/kg. Mice were subcutaneously dosed every third day during the study. An oral glucose tolerance test was performed at day 20 of the study. One day prior to the glucose challenge, mice were subcutaneously treated with Vehicle (0.9 % NaCl), OXM-102 (A16.15) 30 nmol/kg or OXM-064

(A16.12) 30 nmol/kg, followed by a 24 hour fast. The following morning, a bolus consisting of 2 g glucose/ kg body weight was orally given to the mice. Blood glucose was measured at time 0 min, 15 mins, 30 mins, 60 min, 120 mins, and 180 mins.

Chronic study 2 (Example 14) - [Figure 11]:

Female C57BL/6JOLAHSD mice from Envigo Laboratories weighing 35.5 grams (~27 weeks old) were used to evaluate the effects of OXM-102 (A16.15) 30 nmol/kg and 100 nmol/kg on chronic reductions in food intake, body weight loss and glucose tolerance during a 23 days evaluation study. The mice were randomized by weight into five groups, (n=8/group), Vehicle (0.9 % NaCl), OXM-102 (A16.15) 30 nmol/kg, OXM-102 (A16.15)

100 nmol/kg and pair-fed to OXM-102(A16.15) 30 nmol/kg and 100 nmol/kg. An oral glucose tolerance test was performed at day 23 of the study. One day prior to the glucose challenge, mice were subcutaneously treated Vehicle (0.9 % NaCl), OXM- 102 (A16.15) 30 nmol/kg, OXM-102 (A16.15) 100 nmol/kg or pair-fed to OXM-102(A16.15) 30 nmol/kg and 100 nmol/kg, followed by a 24 hour fast. The following morning, a bolus consisting of 2 g glucose/ kg body weight was orally given to the mice. Blood glucose was measured at time 0 min, 15 mins, 30 mins, 60 min, 120 mins, and 180 mins. Furthermore, inguinal and perirenal adipose tissue was collected and weighed at termination. Fibrillation assay (Example 15) - [Figure 12]:

Thioflavin T (T3516, Sigma), Assay stock thioflavin was prepared as a 10 mM solution in 5 mM sodium phosphate pH 7.2. Aliquots was stored, protected from light, at -20 °C. Stock thioflavin was thawed and diluted just prior to use. Oxyntomodulin mimetics were dissolved in phosphate buffered saline. For the oxyntomodulin mimetics, the final peptide concentration in the wells was 200 mM, and the final thioflavin concentration was 4 mM. Thioflavin was added last (1OpL).

In the presence of fibrils, thioflavin has an excitation maximum at 450 nm and enhanced emission at 480 nm, whereas thioflavin is essentially non-fluorescent at these wavelengths when not bound to amyloid fibrils. Thus, thioflavin in combination with a fluorescent plate reader is an ideal tool for screening large numbers of in vitro samples for the presence of amyloid fibrils.

The thioflavin assay used for the oxyntomodulin mimetics was a modification of the procedure described by (Nielsen et al., 2001) for measuring insulin fibrillation. Fibrillation screening assays are conducted in 384-well plates (Greiner Bio-One, 784080) in sample triplicates with a final volume of 20 pL. The plate is sealed using an optical adhesive film to prevent sample evaporation over the course of the assay. The plate is loaded into a fluorescent plate reader, such as a SpectraMax with SoftMax Pro 7.0.2 software, and the template set to 37 °C with excitation wavelength at 450 nm and emission wavelength at 480 nm. The plate reader should measure fluorescence every hour for 18 hours with a five- second plate shake before the first read and a three-second plate shake before all other reads. In other words, the plate is read after the following incubation times; 0, 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 hours.

The relative fluorescence units (RFU) are shown as a function of time. Fibrillation is determined as an increase in RFU over baseline.

Example 1 : Short term in vitro activation

Human oxyntomodulin has a short plasma half-life (Schjoldager et al., 1988), the circulatory half-life of oxyntomodulin being approximately 11-12 minutes (Kervran, Dubrasquet, Blache, Martinez, & Bataille, 1990). In vivo studies have shown the involvement of dipeptidyl peptidase-4 and neutral endopeptidase 24.11 in the breakdown of oxyntomodulin (Hupe- Sodmann et al., 1995; Mentlein, Gallwitz, & Schmidt, 1993).

To protect against proteolytic cleavage, a stabilized human oxyntomodulin was prepared by substituting a serine residue at position 2 with an a-aminoisobutyric acid. This stabilized human oxyntomodulin was named OXM-OOO and was used as an in vitro control for potency. The sequence of OXM-OOO is set out below: - - -

(SEQ ID NO: 41)

Various oxyntomoduline mimetics were prepared by extending the stabilized human glucagon sequence (i.e. the first 29 amino acids of OXM-OOO) at the C-terminus with a tail sequence from several species, to investigate the effect of different oxyntomodulin C-terminal tails on potency. An overview of the peptides tested is shown in Table 1. The potency of the oxyntomodulin mimetics is listed in Table 2 and Table 3.

Table 1 Oxyntomodulin mimetics, consisting of Stabilized Human glucagon with animal C-terminal extensions.

Table 2 The potency of OXM-002-OXM-010 on the GLP1R. EC50 ± SD levels are shown.

Table 3 The potency of OXM-002-OXM-010 on the GCGR. EC50 ± SD levels are shown.

In vitro potency screening of OXM-002 to OXM-OIO on both GLP1R and GCGR indicated that OXM-003, OXM-004, OXM-005, and OXM-009 were most potent.

Oxyntomodulin mimetics screening indicated that stabilized human glucagon merged to a C-terminus of a chicken, bull Frog, angler, and a dog fish led to an increased In vitro potency at both the GLP1R and GCGR. The aim of this initial peptide screening was to investigate natural oxyntomodulins in order to identify novel oxyntomodulin mimetics based on the animal kingdom. Furthermore, the focus was on providing peptides having improved potency at both the GLP1R and GCGR. Therefore, a series of further peptides were synthesized, consisting of protease stabilized glucagon cores merged to the C-terminus tails of OXM-003, OXM-004, OXM-005, and OXM-009, and then screened.

Unique species glucagon from an Alligator gar, an Anglerfish, a Zebra fish, an Elephant shark, a spotted ratfish and a common Dogfish were modified by the addition of a Chicken, a Bullfrog, an Anglerfish or a common Dogfish tail.

Furthermore, an a-aminoisobutyric acid was again introduced at position 2 of the peptides in order to protect the peptides from proteolytic cleavage.

Unique species glucagon:

1) Alligator gar:

2) Anglerfish 1:

3) Anglerfish 2:

4) Zebra fish:

5) Elephant Shark:

6) Spotted Ratfish:

7) Common Dogfish:

8) Degu:

Synthesized oxyntomodulin mimetic sequences for peptide optimization:

OXM-Oil

OXM-012

OXM-013

OXM-015

OXM-036

OXM-037

OXM-038

OXM-039

OXM-041

OXM-042

Take home message from the screening of OXM-002 to OXM-042:

The initial screening of OXM-002 to OXM-OIO demonstrated that it was possible to add different natural C-terminal extensions to a human glucagon core while maintaining a functional agonist at both the GLP1R and GCGR.

Many natural glucagon cores have a glutamic acid (Glu, E) residue at position 3, whereas human glucagon has a Glutamine (Gin, Q). Most of the synthesized peptides naturally had an E residue incorporated at position 3 while OXM-011, OXM-018, OXM-019, OXM-026, OXM-027 OXM-034, OXM-035, and OXM-042 had an Q residue introduced at position 3. Interestingly, the Q->E substitution abolishes GCGR activation while retaining GLP1R activation.

In the human oxyntomodulin amino acid sequence, position 7 and 8 are a threonine (Thr, T) residue followed by a serine (Ser, S) residue. In many glucagon cores of different species, often a conservative amino acid change at position 7 or 8 is found when comparing to the human glucagon sequence. In glucagon of different animals, an S residue is often found at position 7 (OXM-015, OXM-023, OXM-031, and OXM-039) while an asparagine (Asn, N) residue is found at position 8 (OXM- 011, OXM-019, OXM-027, and OXM-035). A single conservative amino acid change at position 7 or 8 did not affect GLP1R activation. However, when both amino acids at positions 7 and 8 are conservatively changed (to S and N respectively), GLP1R receptor activation is lost (OXM-012, OXM-013, OXM-014, OXM- 021, OXM-022, OXM-028, OXM-029, OXM-030, OXM-036, OXM-037 and OXM-38).

Throughout the screening of oxyntomodulin mimetics, glucagon cores varied across the animal kingdom. In the present screening, oxyntomodulin mimetics with an E at position 3 were often used. The inventors sought to find animal oxyntomodulin mimetics which differed from native oxyntomodulin in their sequence similarities. For that reason, all the natural changes were kept. Unfortunately, the Q->E change was not found to improve the properties of the oxyntomodulin mimetics. There were too many amino acid changes in the individual oxyntomodulin mimetics which made the screening more complicated.

In conclusion, the inventors did not manage to discover a dual agonist while screening these peptides. The inventors did, however, increase their understanding of how oxyntomodulin mimetics should be designed.

Is it possible to identify an animal oxyntomodulin mimetics with activity at both the human GLP1R and GCGR?

Dual activity of Oxyntomodulin mimetics was not found when investigating the the above-described oxyntomodulin mimetics. Therefore, it was decided to minimize the number of amino acid changes to the human glucagon core sequence of the oxyntomodulin mimetics, and design the oxyntomodulin mimetics differently in order to achieve dual activity.

A series of further oxyntomodulin mimetics were synthesized, in the pursuit of a potent dual agonist of GLP1R and GCGR. These oxyntomodulin mimetics consisted of different glucagon cores fused to different C-terminal extensions, with each of the glucagon cores being based on the stabilized human glucagon core sequence to which further limited amino acid substitutions had been made. Of these further oxyntomodulin mimetics that were investigated, the most potent dual GLP1R/GCGR agonists were the peptides OXM-064, OXM-073 and OXM-079, having the sequences listed below.

OXM-064 :

OXM-073 :

OXM-079 :

These three lead oxyntomodulin mimetics each comprise a glucagon core sequence based on the human glucagon core (but having the further modifications detailed below), C- terminally fused to a Chicken oxyntomodulin tail. To stabilize these peptides, a-aminoisobutyric acid was in each case introduced at both positions 2 and 16 of the human glucagon core sequence. In the first oxyntomodulin mimetic (OXM-064), a Lysine (K) residue was introduced at position 20 (this change being based on Ghost shark glucagon sequence).

In the second oxyntomodulin mimetic (OXM-073), a Histidine (H) residue at position 13, a Tyrosine (Y) residue at position 18, and a glutamic acid (E) residue at position 21 were introduced (these changes being based on the glucagon cores of a Chinchilla, a King Cobra and a Sea Lamprey). In the third oxyntomodulin mimetic (OXM-079), an Arginine (R) residue was introduced at position 20 (as present in the King Cobra glucagon core, and also due to inspiration from GLP1).

The in vitro potencies of OXM-064, OXM-073, and OXM-079 on the GLP1R and GCGR are listed in Table 4 and Table 5.

Table 4 The potency of OXM-064. OXM-073. OXM-079. and OXM-OOO on the GLP1R. EC50 ± SD levels are shown

Table 5 The potency of OXM-064. OXM-073. OXM-079. and OXM-OOO on the GCGR. EC50 ± SD levels are shown Example 2: The lead oxyntomodulin mimetics are more potent over 24 hours compared to stabilized human oxyntomodulin in vitro

Cells were stimulated with 200 nM of OXM-064, OXM-073, or OXM-079. b-arrestin recruitment was measured at baseline, after 3 hours, 6 hours, and after 24 hours. The results are shown in Figure 1.

OXM-064 was found to be more potent than OXM-OOO at the GLP1R while the potency at the GCGR was approximately similar to OXM-OOO. OXM-073 exhibited a similar potency as OXM-OOO on the GLP1R. However, at the GCGR, OXM-073 was found to be more potent than OXM-OOO. Interestingly, OXM-079 was found to be superior to OXM-OOO on both the GLP1R and GCGR.

Example 3: OXM-103 is more potent than OXM-OOO. However, OXM- 079 is superior to OXM-103 in vitro.

The C-terminal tail of OXM-079 (i.e. amino acids 30-37) differs by 62.5 % compared to OXM-OOO, whereas the glucagon core of OXM-079 (amino acids 1-29) only differs by 6.9 % compared to OXM-OOO. In order to understand the impact of this 6.9 % dissimilarity in terms of GCGR and GLP1R potency, OXM-103 was synthesized. OXM-103 consists essentially of the glucagon core of OXM-079 and an OXM-OOO C-terminal tail. In vitro screening of OXM-103 clearly demonstrated an increased GLP1R potency compared to OXM-OOO, while GCGR potency was unchanged compared to OXM-OOO. The amino acid sequence of OXM-103 from 1-29 thus increases the GLP1R potency of this peptide by 10.8-fold as compared to OXM-OOO.

Whereas in OXM-OOO the peptide is stabilized by the substitution of the serine residue at position 2 of native human oxyntomodulin with an a-aminoisobutyric acid, in OXM- 079 the peptide is further stabilized by introduction of an a-aminoisobutyric acid at position 16 also. In order to make the comparison more even, in OXM-103 the a-aminoisobutyric acid at position 16 was changed back to a serine residue. For ease of comparison, the full sequences of OXM-079, OXM-OOO and OXM-103 are shown below (with the amino acids that differ from human oxyntomodulin being highlighted).

OXM-079

OXM-000

OXM-103

To answer the question of whether OXM-079 is superior to OXM- 103, a comparison of the EC50 levels at both the GLP1R and GCGR was performed. The potency of OXM-079 is listed in Table 4 (GLP1R potency) and Table 5 (GCGR potency). Interestingly, the OXM-079 is 2.9-fold more potent at the

GLP1R compared to OXM-103. Furthermore, OXM-079 is superior to OXM-103 at the GCGR by 2.6-fold.

Table 6 The potency of OXM-OOO. and OXM-103 at both the GLP1R and GCGR. EC50 ± SD levels are shown.

Example 4: How does tail length affect the potency of the lead oxyntomodulin mimetics in vitro.

The potency of OXM-079 was found to be superior to OXM-OOO at GLP1R and GCGR. To investigate how tail length can affect potency, a series of oxyntomodulin mimetics with truncated tails were made based on OXM-079. The C-terminal chicken octapeptide sequence is KRNGQQGQ. Seven modified OXM-079 variants were synthesized; OXM-079, OXM-094, OXM-095, OXM- 096, OXM-097 and OXM-099

The results are shown below and in Figure 2. Interestingly, all the truncated versions of OXM-079 were found to be superior to OXM-OOO on both receptors. The full chicken tail (OXM-079) resulted in the most potent GCGR/GLP1R dual agonist, but the truncated tails present in OXM-097 and OXM- 100 still resulted in strong activation of both receptors.

Example 5: is it necessary to modify the lead oxyntomodulin mimetics by amidation of the C-terminal?

Many biologically active peptides and peptide hormones are a- amidated at their C-terminus. which is essential for their full biological activities (K. Kim & Seong, 2001). The utility of this modification has included a role in protecting peptides from enzymatic degradation (half-life) and increasing binding affinity.

To investigate the effect of a C-terminal amidation, a C- termal amidated version of OXM-64 was generated and tested. C-terminal amidation of OXM-064 (OXM-102) did not affect OXM-

064 performance. The GLP1R and GCGR potency were equally high. The results are shown in Figure 3.

OXM-064 : HXQGTFTSDYSKYLDXRRAKDFVQWLMNTKRNGQQGQ (SEQ ID NO: 29)

OXM-102 : HXQGTFTSDYSKYLDXRRAKDFVQWLMNTKRNGQQGQ-NH2 (SEQ ID NO: 93)

Example 6: Can conservative substitutions be made to the glutamine (Q) residues in the chicken oxyntomodulin tail?

To investigate the effects of substituting the glutamine (Q) residues in the chicken oxyntomodulin tail with glutamic acid (E) residues, a C-terminal amidated version of OXM-079 having E for Q substitutions at amino acid positions 34, 35 and 37 (OXM-105) was synthesized, and tested in comparison to OXM- 079 and a C-terminal amidated version of OXM-079 (OXM-104). The table below shows the potency of OXM-079, OXM-104 and OXM-105 on both the GLP1R and GCGR (EC50 ± SD levels are shown based on three or more independent runs).

Sequences of oxyntomodulin mimetics:

As can be seen from the above, compared to OXM-079: OXM-104 was 2.5-fold more potent at the GLP1R and 2.8-fold more potent at the GCGR, while OXM-105 was equally potent at the GLP1R and 3.3 fold more potent at the GCGR.

Thus, substituting all three glutamine (Q) residues in the chicken oxyntomodulin tail of OXM-079 with glutamic acid (E) residues resulted in an oxyntomodulin mimetic that was still a potent GLP1R/GCGR dual agonist.

Example 7: Does acylation of the lead oxyntomodulin mimetics work in vitro?

As noted above, in the lead oxyntomodulin mimetics, the serine residues at positions 2 and 16 are substituted with a- aminoisobutyric acid residues to protect the peptides against proteolytic cleavage.

In this test, the effect on in vivo action was investigated of substituting the a-aminoisobutyric acid at position 16 of OXM-079 with an acylated lysine (K) residue. The idea behind a prolongation of peptide plasma half via acylation of a peptide originates from useful properties found in albumin. The discovery of the structure of albumin led to the finding that multiple binding sites for fatty acids exist (Bhattacharya, Griine, & Curry, 2000). The ability of albumin to solubilize and transport insoluble substrates such as fatty acids has been used in drug discovery (Knudsen & Lau, 2019).

A variant of OXM-079 was synthesized, referred to as OXM-079 (A16.17), having an acylated lysine residue at position 16 of the peptide, the lysine residue being acylated with a C20- diacid conjugated to the side chain ε-amino group of said lysine residue via a YGlu-yGlu linker.

In order to investigate the in vitro functionality of acylated oxyntomodulin mimetics, b-arrestin mediated activation of GLP1R and GCGR by OXM-079 (A16.17) and OXM-079 (non-acylated) were assessed. The cells were cultured in medium with low concentrations (0.1 %) or high (10 %) concentrations of FBS. The dose-response curves of OXM-079 (A16.17) and OXM-079

(non-acylated) are shown in Figure 4. In the presence of 10 % FBS, a clear shift in EC50 levels of OXM-079 (A16.17) was seen both at the GLP1R and GCGR when compared to OXM-079.

This demonstrated the functionality of OXM-079 (A16.17), as receptor activation required more OXM-079 (A16.17) due to albumin binding. Furthermore, this finding was confirmed using a low percentage of FBS (0.1 %). Here, no shift was observed in EC50 levels both at the GLP1R and GCGR. Example 8: During an acute in vivo test, how much weight can High-Fat-Diet fed C57BL/6JOLAHSD Mice lose using OXM-102 (A16.15)?

As described in example 5, OXM-064 was modified by the introduction of a C-terminal amidation (OXM-102). OXM-102 was then modified further by introduction of a lysine residue acylated with an yGlu-yGlu-Cl8 fatty diacid at position 16 in place of the a-aminoisobutyric acid residue (OXM-102 (A16.5), see Table 7 for the full sequence).

This oxyntomodulin mimetic was acutely tested in female C57BL/6JOLAHSD mice in order to assess its potency. Body weight was monitored every 24 hours. The results are shown in Figure 5.

As demonstrated by the results, OXM-102 (A16.15) reduced body weight for up to 48 hours in a dose-dependent manner. Administration of OXM-102 (A16.15) 100 nmol/kg resulted in a

~9 % decrease in body weight. The effect of OXM-102 (A16.15)

100 nmol/kg sustained for 48 hours after which the effect tailed off. OXM-102 (A16.15) 30 nmol/kg resulted in a ~ 7 % body weight loss 24 hours post-dosing. The effect on body weight was decreased by 48 hours and completely faded after 72 hours using OXM-102 (A16.15) 10 nmol/kg.

The mice were dosed with different concentrations of OXM-102 (A16.15) in order to obtain an understanding of the pharmacokinetics profile. As an objective is to achieve a once weekly therapy, drug effect over time is of high relevance. Three days in rodents is approximately a week in humans. As shown in Figure 5, OXM-102 (A16.15) 100 nmol/kg can maintain a reduction in bodyweight for up to 48 hours prior to rebounding. Potentially, such high concentrations of OXM-102 (A16.15) can therefore function in a once weekly setting in man.

Example 9: What is the optimal position to introduce an acylation? Which linker should be used?

The effect of different acylations were investigated, together with the positioning of the acylations.

Several variants of OXM-64 were synthesized, the sequences of which are shown in Table 7. In these peptides, either a C16 fatty acid or C18 fatty diacid was conjugated to a lysine residue via a linker selected from a single yGlu group (OXM- 064 (A16.12)) or a two y-Glu repeat (OXM-102 (A16.15), OXM-

064 (A16.13), OXM-064 (A13.15) and OXM-064 (A17.15)). In OXM- 102 (A16.15), OXM-064 (A16.12) and OXM-064 (A16.13) the acylated lysine residue is located at position 16 of the peptide. In others, the acylated lysine residue is located at at position 13 (OXM-064 (A13.15)) or position 17 (OXM-064

(A17.15)).

This study was conducted in order to assess the potency and efficacy of the above-mentioned oxyntomodulin mimetics. Food intake and body weight were monitored every 24 hours for 96 hours. The percent decrease in body weight and daily food intake values were calculated. The results are shown in Figure 6.

Acylations at postions 16 or 17 using a linker consisting of yGlu-yGlu were found to be the most effective, as administration of OXM-102(16.15) and OXM-064(A17.15) resulted in the most extensive weight loss over 96 hours. The use of a C18 fatty diacid chain was found to be superior to palmitoyl chains, as both OXM-064 (A16.12), and OXM-064 (A16.13) only showed slight reductions in body weight. OXM-102 (A16.15) and OXM-064 (A17.15) were shown to have an effect to reduce body weight for up to 72 hours.

In summary, a yGlu-yGlu linker with a C18 fatty diacid conjugated to a K at position 16 was found to lower body weight the most.

Table 7 Linkers and acylations

Example 10: Does a C-20 fatty chain increase in vivo exposure/effect compared to a C-18 fatty diacid? An increase in chain length and relative lipophilicity of the albumin binder going from C12, C14, C16, C18 to C20-diacid moiety was shown to increase in vivo exsposure during the discovery of semaglutide (Lau et al., 2015). However, backbone substitutions and different linkers can have an effect on the pharmacokinetic properties. In order to simplify the test, it was decided to investigate the effect of a C-18 fatty diacid versus C-20 fatty diacid linked through a yGlu-yGlu linkage to a lysine residue at postion 16 of OXM-102.

In an acute in vivo study OXM-102 (A16.15) [yGlu-yGlu-Cl8 diacid conjugated to lysine at position 16] was compared to OXM-102 (A16.17) [YGlu-yGlu-C20 diacid conjugated to lysine at position 16] at equimolar concentrations. Food intake and body weight were monitored every 24 hours for 96 hours. The percent decrease in body weight and daily food intake values were calculated. The results are shown in Figure 7.

The choice of a C-18 fatty diacid or C-20 fatty diacid did not significantly affect the potency in an acute setting. OXM-102 (A16.15) and OXM-102 (A16.17) lowered the body weight to the same extent - approximately 10 %. Furthermore, the same pattern was seen with food intake.

Example 11: Is the backbone of OXM-079 superior to OXM-102 in vivo?

The peptide backbones of OXM-102 and OXM-079 differ by a single amino acid at position 20. In the sequence of OXM-102, a lysine residue (K) is located at position 20, whereas an arginine residue (R) is present at position 20 in the sequence of OXM-079. In vitro, OXM-079 was found to be more potent than OXM-102 as demonstrated in the previous examples.

A head to head in vivo study was performed, investigating the body weight lowering capacity of OXM-102 vs OXM-079, both of which were acylated with YGlu-yGlu-C20 diacid conjugated to lysine at position 16 of the peptide backbone. The results are shown in Figure 8. In a 96-hours acute study, administration of OXM-102 (A16.17) 75 nmol/kg and OXM-079

(A16.17) 50 nmol/kg clearly demonstrated superiority of OXM- 079 (A16.17).

Throughout the first 24 hours of the study, both OXM-102 (A16.17) and OXM-079 (A16.17) lowered the body weight to the same extent. However, the following hours OXM-102 (A16.17) reached a plateau whereas OXM-079 (A16.17) continued to lower the bodyweight throughout the whole study.

Example 12: How many yGlu spacers is needed for maximum potency in vivo?

The effect of using different length linkers between the e- amino located on the lysine sidechain and the carboxyl group of the fatty acid was investiaged. Three different linker lengths, together with either a C-18 or C-20 fatty diacid, were used, as shown in Table 8.

Table 8 In order to establish the linker length needed for greatest potency, the effect on OXM-079 of using a single (yGlu) linker or a triple (YG1U-YG1U-YG1U) linker together with a C- 18 fatty diacid conjugated to lysine at position 16 of the peptide backbone was investigated and compared to the effect of using a double (YG1U-YG1U) linker together with a C-20 fatty diacid conjugated to lysine at position 16 of the peptide backbone. The potency of these peptides were also compared to OXM-102 (A16.17), i.e. OXM-102 having a yGlu-yGlu

C-20 fatty diacid conjugated to lysine at position 16 of the peptide backbone.

The rationale for using OXM-102 (A16.17) and OXM-079 (A16.17)

(peptides acylated at position 16 with a yGlu-yGlu C-20 fatty diacid) as a comparison to OXM-079 (A16.14) and OXM-079 (A16.16) (peptides acylated at position 16 with, respectively, a yGlu C-18 fatty diacid and a yGlu-yGlu-yGlu C-18 fatty diacid), was that it was already shown in Figure 8 (c.f. Example 10) that no difference was observed in the body weight lowering capacity of OXM-102 when acylated with yGlu- yGlu together with a C-18 or C-20 fatty diacid. Furthermore, the inventors were interested in investigating the maximum effects of OXM-079 (A16.17) and OXM-102 (16.17), when administered at very high concentrations (higher than the moderate concentrations used in the the acute head to head study conducted in Example 11).

An acute in vivo study, was conducted, administering the aforementioned peptides. Body weights were monitored every 24 hours for 96 hours. The percent decrease in body weight was calculated, and the results are shown in Figure 9.

In this test it became evident that acylations using a double yGlu-yGlu linker were preferred as peptides with these acylations (OXM-102 (A16.17) and OXM-079 (A16.17))lowered the bodyweight the most. Acylation using a linker consisting of YGlu-yGlu-yGlu was found to result in a peptide (OXM-079 (A16.16)) having the lowest potency in terms of body weight lowering. Acylation using a linker consisting of a single yGlu resulted in a peptide (OXM-079 (A16.14)) that lowered the body weight to a larger extent than OXM-079 (A16.16). However, it was less potent than OXM-102 (A16.17) and OXM-079

(A16.17).

When dosing with supra pharmacological (200 nmol/kg) doses, no difference was found in the body weight lowering capacity of OXM-079 (A16.17) and OXM-102 (A16.17). However, as previously shown, the backbone of OXM-079 was found to be more potent in vivo at lower doses (c.f. Example 11 and Figure 8).

Example 13: Anti-obesity and metabolic effects of OXM-102 (A16.15)

This study was conducted in order to examine the anti-obesity and metabolic effects of OXM-102 (A16.15) on female

C57BL/6JOLAHSD mice fed a high fat diet. The results are shown in Figure 10.

Repeated administration of OXM-102 (A16.15) resulted in a

11.7 % body weight loss compared to Vehicle (0.9 % NaCl). Longer term administration of equivalent doses of OXM-102 (A16.15) and OXM-064 (A16.12) clearly demonstrated the potential of OXM-102 (A16.15) as anti-obesity agent (c.f.

Figure 10 A and B). The metabolic impact of OXM-102 (A16.15) was clearly seen as mice receiving OXM-102 (A16.15) had an improvement glucose tolerance compared to Vehicle (0.9 %

NaCl) (c.f. Figure 10 C, D and E).

Example 14: Effects of OXM-102 (A16.15) on metabolism independent of energy intake This study was conducted in order to examine the effects of OXM-102 (A16.15) independent of energy intake in vivo. The results are shown in Figure 11.

OXM-102 (A16.15) induced a 18.1 % (30 nmol/kg) and a 29 %

(100 nmol/kg) body weight loss when compared to Vehicle (0.9 % NaCl). Furthermore, the pair-fed to OXM-102 (A16.15) (30 nmol/kg) lost 18.7 % of its bodyweight, while the pair-fed to OXM-102 (A16.15) (100 nmol/kg) lost 22.5 % of its bodyweight when compared to Vehicle (0.9 % NaCl). Interestingly, OXM-102 (A16.15) 100 nmol/kg induced a larger body weight loss compared to the pair-fed to OXM-102 (A16.15) 100 nmol/kg group (c.f. Figure 11 A and B).

During the Oral glucose tolerance test, OXM-102 (A16.15) significantly lowered blood glucose levels (%) in a dose- dependent manner compared to Vehicle (0.9 % NaCl). OXM-102 (A16.15) administration lowered the cumulative food intake dose-dependably, 29.5 % (30 nmol/kg) and 56.6 % 100 nmol/kg when compared to Vehicle (0.9 % NaCl) (c.f. Figure 11 C, D and E). Due to the unexpected efficacy of OXM-102 (A16.15) on body weight, four mice receiving OXM-102 (A16.15) 100 nmol/kg had to be euthanized prior to study end (3 at day 17 and 1 at day 19) due to ethical considerations. This explains the difference in cumulative food intake between the OXM-102 (A16.15) 100 nmol/kg and pair-fed to OXM-102 (A16.15) 100 nmol/kg.

Example 15: Do the oxyntomodulin mimetics fibrillate?

The peptide hormone glucagon has been shown to form many different morphological types of amyloid-like fibrils, depending on solvent conditions (Ghodke et al., 2012). Thioflavin T is a dye widely used for the detection of amyloid fibrils. The extent to which OXM-102 fibrillates under physiological pH (7.2) was investigated, in particular as regards the effects of acylating OXM-102 with different sizes of fatty diacids with a yGlu-yGlu linker. The specific acylations that were investigated are shown in Table 9.

Table 9

During an 18-hours screening of these differently acylated variants of OXM-102, it became evident that fibrillation was no issue at physiological pH. These results are shown in Figure 12.

Example 16: effects in vitro of substitutions at postions 7 and 8 in OXM-079

In each of the three lead oxyntomodulin mimetics (OXM-064, OXM-073 and OXM-079) the amino acids residues at positions 7 and 8 are, respectively, threonine (Thr, T) and a serine (Ser, S), mirroring the residues at these positions in the human oxyntomodulin amino acid sequence. However, as noted above (c.f. Example 1), many other species have conservative amino acid changes at position 7 or 8.

In order to further investiage the role of the amino acid residues at positions 7 and 8, the following variants of OXM- 079 having a substitution at either of both of positions 7 and 8 were synthesized, and the effects of these substitions were then analysed using the receptor cell lines expressing the GLP-1R and the GCGR.

Substituted variants of OXM-079 (positions 7 and

shown underlined):

OXM-108 HXQGTFSTDYSKYLDXRRARDFVQWLMNTKRNGQQGQ (SEQ ID NO: 97)

OXM-109 HXQGTFTNDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 98)

OXM-110 HXQGTFNTDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 99)

OXM-111 HXQGTFSNDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 100)

OXM-112 HXQGTFNSDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 101)

OXM-113 HXQGTFSSDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 102)

OXM-114 HXQGTFTTDYSKYLDXRRARDFVQWLMN TKRNGQQGQ (SEQ ID NO: 103)

Table 11 below lists the results of these tests. As can be seen, the alterations in the sequence going away from a T at position 7 and S at position 8 resulted in inferior activities (as compared to OXM-079) on either one or both of the two target receptors. As such, the TS sequence at postions 7 and 8 was the optimal sequence for activation of both receptors.

113 and OXM-079 on the GLP1R and GCGR. EC50 levels are shown.

All prior teachings acknowledged above are hereby incorporated by reference.

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Claims

CLAIMS:

1. An oxyntomodulin mimetic having the amino acid sequence:

HX2QGTFX₇X8DYSKX13LDX16X17X18AX20X21FVQWLMNTX30 (SEQ ID NO: 21) wherein

X2 = a-aminoisobutyric acid X₇ = T or S

X₈ = S or N, subject to the proviso that where X₇ is S then X₈ is not N X₁₃ = Y or H X₁₆ = a-aminoisobutyric acid or K

X₁₇ = R or K X₁₈ = R or Y

X₂₀ = R, K or Q

X₂₁ = D or E

X₃₀ is an or amino acid sequence or amino acid selected from KRNGX₃₄X₃₅GX₃₇ (SEQ ID NO: 22) , KRNGX₃₄X₃₅G (SEQ ID

NO : 23) , KRNGX₃₄X₃₅ ( SEQ ID NO : 24) , KRNGX₃₄ ( SEQ ID NO :

25), KRNG (SEQ ID NO: 26), KRN, KR and K

X₃₄ , where present, is Q or E

X₃₅ , where present, is Q or E

X₃₇ , where present, is Q or E.

2. An oxyntomodulin mimetic as claimed in claim 1, wherein X₃₀ is KRNGX₃₄X₃₅GX₃₇ (SEQ ID NO: 22), KRNG (SEQ ID NO:

26) or K.

3. An oxyntomodulin mimetic as claimed in claim 1, wherein X₃₀ is KRNGX₃₄X₃₅GX₃₇ (SEQ ID NO: 22).

4. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₂₀ is R or K.

5. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₂₀ is R.

6. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₇ is T and X₈ is S.

7. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₁₃ is Y, X₁₈ is R and X₂₁ is D.

8. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₁₃ is H, X₁₈ is Y and X₂₁ is E.

9. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₁₆ is a-aminoisobutyric acid.

10. An oxyntomodulin mimetic as claimed in any one of claims 1 to 8, wherein X₁₆ is K and wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group.

11. An oxyntomodulin mimetic as claimed in any preceding claim, wherein X₁₇ is R.

12. An oxyntomodulin mimetic as claimed in any one of

Claims 1 to 9, wherein X₁₇ is K and wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group.

13. An oxyntomodulin mimetic as claimed in Claim 10 or 12, wherein said lysine residue is acylated with an acyl group selected from any one of the following: a C₁₈ or longer fatty acid with an optional linker, or a C₁₈ or longer fatty diacid with an optional linker.

14. An oxyntomodulin mimetic as claimed in Claim 13, wherein said acyl group is selected from any one of the following: a C₁₈ to C₃₀ fatty acid with an optional linker, or a C₁₈ to C₃₀ fatty diacid with an optional linker.

15. An oxyntomodulin mimetic as claimed in Claim 13, wherein said acyl group is selected from any one of the following: a C₁₈ to C₂₂ fatty acid with an optional linker, or a C₁₈ to C₂₂ fatty diacid with an optional linker.

16. An oxyntomodulin mimetic as claimed in any one of Claims 13 to 15, where said linker is present and is a Gamma-Glutamic acid based linker.

17. An oxyntomodulin mimetic as claimed in Claim 16, wherein the linker comprises two Gamma-Glutamic acid residues linked together.

18. An oxyntomodulin mimetic as claimed in any one of the preceding claims, wherein the oxyntomodulin mimetic has an amino acid sequence selected from any one of the following sequences:

HAiBQGTFTSDYSKYLDAiBRRAKDFVQWLMNTKRNGQQGQ (OXM-064)

(SEQ ID NO: 29)

wherein AiB is a-aminoisobutyric acid and wherein K_AC is a lysine residue wherein the side chain ε-amino group of said lysine residue is acylated with an acyl group.

19. An oxyntomodulin mimetic as claimed in any one of Claims 1 to 18, formulated for parenteral administration.

20. An oxyntomodulin mimetic peptide as claimed in Claim 19, formulated for injection.

21. An oxyntomodulin mimetic as claimed in any one of Claims 1 to 18, formulated for enteral administration.

22. An oxyntomodulin mimetic as claimed in Claim 21, wherein the oxyntomodulin mimetic is formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide

23. An oxyntomodulin mimetic as claimed in Claim 21, formulated with a carrier for oral administration, wherein the carrier comprises 5-CNAC, SNAD, or SNAC.

24. A pharmaceutical composition comprising an oxyntomodulin mimetic as claimed in any one of claims 1 to 18.

25. An oxyntomodulin mimetic as claimed in any one of claims 1 to 18 for use as a medicament.

26. An oxyntomodulin mimetic as claimed in any one of claims 1 to 18, for use in treating excess bodyweight, excessive food consumption, metabolic syndrome, non- alcoholic steatohepatitis, non-alcoholic fatty liver disease, alcoholic fatty liver disease, diabetes (Type I and/or Type II), obesity, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake.

27. A method of treating excess bodyweight, excessive food consumption, metabolic syndrome, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, alcoholic fatty liver disease, diabetes (Type I and/or Type II), obesity, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake, comprising administering an effective amount of an oxyntomodulin mimetic as claimed in any one of claims 1 to 18 to a patient in need of said treatment.