Azapeptide therapeutics

Abstract

Disclosed are azapeptides, and their use in the treatment of diseases such as diabetes, obesity, Alzheimer's disease, liver disease, substance addiction, traumatic brain injury, chronic kidney disease, inflammation and cardiovascular diseases.

Description

ATTORNEY DOCKET NO.: TUV-19425 AZAPEPTIDE THERAPEUTICS RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No.: 63 / 683,335, filed August 15, 2024; and U.S. Provisional Patent No.: 63 / 724,565, filed November 25, 2024; the contents of each of which are incorporated by reference in their entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM130257, GM142448, DK131842, AG061909 and GM124160 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND

[0003] The twin epidemic of type 2 diabetes (T2D) and obesity, termed “diabesity”, is a global public health problem. Current projections suggest that 10% of the world population will suffer from T2D within the next two decades. Obesity (as defined by body mass index 30) has also seen a significant increase, with 42% of the current US population classified as such, and another 30% deemed overweight according to the Centers for Disease Control (CDC). The pursuit of safe and effective treatments for T2D and obesity has prompted investigation of peptides secreted in the postprandial state. These molecules include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which both regulate glucose homeostasis and related physiological functions. SUMMARY

[0004] In some aspects, the present invention provides a peptide comprising an aza-amino acid residue (Aza); wherein the peptide is selected from the group consisting of Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP-2), Glucose-dependent insulinotropic polypeptide (GIP), Glucagon (GCG), Growth hormone releasing hormone (GHRH), FGF2, FGF21, Tyr-melanostatin, Endomorphin-2, Enterostatin, -casmorphin, Trypsinogen pro- peptide, Corticotropin-like intermediate lobe peptide, Gastrin-release peptide, Aprotinin, GCP-2, MDC, MCP-2, Eotaxin, IP-10, Insulin-like growth factor-1, Interleukin-2, Interleukin-1 ,1-Microglobulin, PHM, GRH-(1-29), GRH-(1-44), Oxyntomodulin, Secretin (SCT), Vasoactive Intestinal Peptide (VIP), Neuropeptide Y (NPY), Met-enkephalin, Pancreatic Polypeptide (PP), Peptide YY (PYY 1-36 and 3-36 form), Exenatide, Substance P, Parathyroid hormone (hPTH 1-34), BNP, Liraglutide, Tirzepatide, Semaglutide, Retratrutide, - 1 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425Taspoglutide, Lixisenatide, Albiglutide, Dulaglutide, Stromal cell-derived factor 1 ( and ),BI-456906 MAR423, CCL5, CCL11, Pituitary Adenylate Cyclase-Activating Polypeptide, NNC0090-2746, and Ecnoglutide peptides having at least 85% sequence identity to any one of them; and an amino acid residue of the peptide is replaced with the aza-amino acid residue (Aza).

[0005] In some aspects, the present invention provides a method of: a) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, and / or obesity; b) reducing body weight and / or food intake, and / or inducing satiety; c) treating or preventing Alzheimer's disease, nonalcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated liver disease (MASLD), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; d) treating chronic kidney disease, traumatic brain injury, alcohol addiction, and / or substance addiction; e) treating or preventing emesis; f) treating or preventing effects of aging; g) support of islet transplant survival in Type 1 diabetes; and h) treating or preventing arthritis and related diseases, including but not limited to: Osteoarthritis, Rheumatoid arthritis, Gout, Ankylosing spondylitis, Psoriatic arthritis, Juvenile arthritis, Fibromyalgia, Infectious arthritis, Spondyloarthritis, Sjogren's syndrome, Scleroderma, and Polymyalgia rheumatica, comprising administering to a subject in need thereof an effective amount of a peptide of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 shows the amino acid sequences of GLP-1, GIP, semaglutide, and a balanced dual agonist (DA) of both the GLP-1R and GIPR with unnatural, lipidated, and conserved residues highlighted. The site of DPP4 catalyzed hydrolysis of GIP and GLP-1 is shown in addition to the aza-amino acid modifications (underlined).

[0007] Figure 2 shows representative aza-amino acid containing peptide highlighting key structural differences from L-amino acids. The dihedral angle is approximately ±90° dictated by the lone pair–lone pair repulsion between the two nitrogen (blue) atoms. In addition, the planar urea [N(R2)–C(=O)–NH] moiety enforces further structural constraints and influences the three-dimensional disposition of the R3and R4groups.

[0008] Figures 3A-3H show concentration-response curves from cAMP luciferase reporter assays. A8AzaA GLP-1 (Figure 3A), A8AzaG GLP-1 (Figure 3B), A8AzaP GLP-1 (Figure 3C), and Aib2AzaA semaglutide (Figure 3F) in comparison to native GLP-1 at the GLP-1R. Assays were performed using HEK 293 C34L cells stably transfected with GLP-1R and - 2 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 CRE6x-luciferase. Both peptides engender similar G-protein mediated signaling through theG s pathway.

[0009] Figure 3D shows stimulative activity at the GIPR of Ala2AzaA GIP and native GIP illustrates a minimal potency decrease (< 6-fold), with a slightly higher potency loss observed in ) Ala2AzaG GIP (Figure 3E). A dual agonist (DA) 'DA X2AzaA, X20A' with the Aib2AzaA and Aib20Ala modifications is an equipotent and equiefficacious agonist compared to native ligands at both GLP-1R (Figure 3G) and GIPR (Figure 3H). Errors represent ± SEM.

[0010] Figures 4A-4H show concentration-response agonism curves at the GLP-1R and GIPR from cAMP luciferase reporter assay: GLP-1 (Figure 4A), Ala8AzaA GLP-1 (Figure 4B), Ala8AzaG GLP-1 (Figure 4C), Ala8AzaP GLP-1 (Figure 4D), X2AzaA Semaglutide (Figure 4G), and dual agonist (DA) X2AzaA (Figure 4H), X20Ala pre-incubated with DPP4 or vehicle overnight. Assays were performed using HEK-293 C34L cells stably transfected with GLP-1R and CRE6x-luciferase. Concentration-response curves at the GIPR of (Figure 4E) GIP and (Figure 4F) Ala2AzaA GIP were generated under the same pre-incubation conditions, but with transient transfection of HEK-293T cells. Errors represent ± SEM.

[0011] Figure 5A shows LC-MS analysis of Ala8AzaA GLP-1 and native GLP-1 following their incubation with DPP4.

[0012] Figure 5B shows hydrolysis kinetics of Gly-Pro-pNa catalyzed by DPP4 that was incubated for 30 mins with either vehicle (PBS, pH 8.0, circle), GLP-1 (2 μM, diamond), A8AzaA GLP-1 (2 μM hexagon), A8AzaP GLP-1 (2 μM, square), or linagliptin (1 nM, a known competitive inhibitor, triangle) prior to addition of the substrate. Azapeptides do not seem to affect substrate processing to any significant extent even at 2 μM, suggesting that they do not bind or interact with the DPP4 active site as they are comparable to the buffer control. Errors represent ± SEM in Figure 5B.

[0013] Figure 6 the incubation of GLP-1 in DDP-4. To an Eppendorf tube containing DPP4 (4 nM) in reaction buffer (20 mM TRIS, 100 mM NaCl, 1 mM EDTA, pH 8.0) was added GLP-17-36 amide (12 μM) and the mixture was gently mixed. Aliquots were removed from the reaction mixture at the 0, 45, 87, 110, and 143 second time points and immediately quenched by a 10× volume dilution with 0.2% TFA in water solution. This solution was then vortexed and cooled on dry ice and subjected to LC-MS analysis. The m / z extractions of intact (7-36 amide) and truncated (9-36 amide) were collected, and the rate of hydrolysis was determined through the ratio of intact verses truncated products. The data were fit to a single exponential decay to yield a t1 / 2of 29.2 seconds (n = 1, Prism v10). - 3 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0014] Figure 7A shows a two-dimensional DPP4 active site with a GLP-17–36 amide bound. Negatively charged Glu205and Glu206residues form salt bridge interactions with the N-terminal amine (as the ammonium form) while the His7and Ala8residues of GLP-1 bind to the S2 pocket and the S1 pocket of the receptor, respectively. The hydrolysis resulting in the His-Ala dipeptide and GLP-1 9–36 amide occurs between the P1 and P1’ residues, i.e., the ‘scissile bond’, of GLP-1 by the catalytic Ser630of the enzyme. The AzaA, AzaG, and AzaP residues were all placed at the P1 site of GLP-1 and examined for degradation.

[0015] Figure 7B shows the X-ray structure of diprotin A (Ile-Pro-Ile) bound to the DPP4 active site where the P1 Proline residue, and P1’ isoleucine are residing at the scissile bond with Ser630in close proximity (PDB: 1NU8).

[0016] Figure 7C shows H-His-AzaA-Glu-NMe (left) with Ramachandran plot (right) illustrating its conformational preference compared to the dihedral angles of the P1 proline and P1’ isoleucine residues of diprotin A when bound to DPP4.

[0017] Figures 8A & 8B show biased signaling of GLP-1 azapeptide derivatives at the GLP- 1R. Concentration-response agonism curves at the GLP-1R for (Figure 8A) cAMP-stimulated luciferase production normalized to GLP-1 and (Figure 8B) Arr2 recruitment to the GLP-1R induced by agonist-binding reported through the BRET assay. Modifications at the second position of GLP-1 relative to the N-terminus are most superior in cAMP production potency, but only certain modifications (AzaG and AzaP) can reduce arrestin recruitment. Error is mean ± SEM. DETAILED DESCRIPTION

[0018] The two ‘incretin’ peptide hormones, through the agency of their complementary roles in satiety signaling and accompanying weight loss and glycemic control, are at the very nexus of relief from liabilities arising from T2D and obesity. The stimulation of insulin biosynthesis and secretion by pancreatic -cells takes place in a glucose-dependent manner modulated through the agonism of the cognate receptors of GLP-1 (GLP-1R) and GIP (GIPR). In peripheral tissue, GLP-1 delays gastric emptying in the stomach and induces satiety through signaling via vagal afferents in the brain, and GIP enhances hippocampal progenitor proliferation and regulates free fatty acid levels.

[0019] Stabilized agonists of the GLP-1 receptor (GLP-1R) and dual agonists of GLP-1R and GIP receptor (GIPR) for the management of type 2 diabetes and obesity have generated widespread enthusiasm and have become blockbuster drugs. These therapeutics are refractory to the action of dipeptidyl peptidase-4 (DPP4), that catalyzes rapid removal of the two N- - 4 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 terminal residues of the native peptides, in turn severely diminishing their activity profiles. Here we report that a single atom change from carbon to nitrogen in the backbone of the entire peptide makes them refractory to DPP4 action while still retaining full potency and efficacy at their respective receptors. This was accomplished by use of aza-amino acids, that are bioisosteric replacements for α-amino acids that perturb the structural backbone and local side chain conformations. Molecular dynamics simulations reveal that aza-amino acid can populate the same conformational space that GLP-1 adopts when bound to the GLP-1R. The insertion of an aza-amino acid at the second position from the N-terminus in semaglutide and in a dual agonist of GLP-1R and GIPR further demonstrates its capability as a viable alternative to current DPP4 resistance strategies while offering additional structural variation that may influence downstream signaling.

[0020] A key modulator of the activity of GLP-1 and GIP is dipeptidyl peptidase-4 (DPP4), an ubiquitous serine protease that catalyzes removal of the two N-terminal residues of GLP-1 (His-Ala) and GIP (Tyr-Ala) (Figure 1). The resulting truncated forms have severely diminished activity profiles at their respective receptors. DPP4 exists within the epithelial and endothelial cells of many tissues including the liver. Several approaches have been utilized to make GLP-1 and related peptides more resistant to proteolysis. The most useful of these effortshave been (i) the use of an α / peptide scaffold that changes the backbone of the construct, (ii)incorporation of fluorinated amino acids at strategic positions, (iii) the modification of side chains with saccharides, (iv) use of thioamides as the peptide linkages, (v) the use of 2- aminoisobutyric acid (Aib) at position 8 (second from the N-terminus) and more recently, (vi) the introduction of a new platform that relies on the modification of the N-terminus via alkylation. Substitution of hexafluoroleucine at the P1 or P1’ position of GLP-1 results in partial protection from proteolysis. N-terminal acylation of GLP-1 and GIP renders them refractory to enzyme-catalyzed hydrolysis; however, this approach leads to a significant diminution of agonism at the cognate receptors. We have previously shown that N-terminal alkylations can be a powerful method to simultaneously provide protease resistance and maintain full stimulative activity at both GLP-1R and GIPR. The more commonly used solution is the use of Aib at the P1 position of the substrate that occupies the S1 site of the enzyme active site. Synthetic analogues of GLP-1, and unimolecular dual agonists that exhibit both GLP-1 and GIP activity in the clinic have lately relied on this single modification. Through the use of Aib at position 2 of the peptide, agonists of GLP-1R and GIPR have resulted in leading peptide based clinical compounds in management of T2D and obesity (Figure 1). With the - 5 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 burgeoning interest in this field, the arsenal of chemical strategies that equip agonists with DPP4 resistance, and simultaneously do not interfere with receptor activation must be expanded. In this work, the replacement of canonical α–amino acids with an aza-amino acid (Figure 2), which substitutes a nitrogen atom in place of the Cα atom, was pursued as a means of simultaneously preserving full efficacy and potency, modulating structural characteristics of the ligand–receptor complex, and to prevent the peptide from being inactivated by the hydrolytic action of the frontline protease.

[0021] An aza-amino acid-containing peptide (an 'azapeptide') features a semicarbazide functionality that perturbs electronic structure, and both the backbone and side chain conformations relative to the parent peptide. In addition, there is an observed a loss of configurational permanence effectuated by the change from a tetrahedral α-carbon to a trivalently substituted nitrogen. This dynamic chirality thus expands the three dimensional disposition of chemical functionality that the molecule can display. Aza-amino acids have strong conformational preferences accompanying an additional lone pair–lone pair repulsion, and the coplanar nature of the urea moiety imposed through extended conjugation (Figure 2, left). Similar modifications have been used to create 1,3,4-benzotriazepin-2-one tetrapeptide mimicking receptor agonists with selective signaling pathway activation profiles, making them ‘biased’ agonists. While Aib has a preference for -helical structures, aza-amino acids induce -turn conformations, as judged by computation, X-ray crystallography, and NMR spectroscopy. Compounds that can occupy and help populate different regions of the available conformational space may trigger 'bias' in the signaling pathways associated with GPCR activation. The conformational space preferred by aza-amino acids is different from L-amino acids, making them a compelling addition to the toolbox to prevent enzymatic recognition of peptide substrates. Azapeptides, therefore, represent a new structural motif that can both be useful in conferring protease protection and additionally, because of conformational preferences may dictate pharmacological efficiency and outcome.

[0022] The residues of interest in GLP-1 and GIP chosen for aza-amino acid replacement were those adjacent to the scissile bond in the ligand that are subject to enzyme catalyzed hydrolysis. Accordingly, alanine at position P1 (second from the N-terminus in the peptide), and glutamic acid at P1’ (third from N-terminus) that are present in both GLP-1 and GIP were first targeted for modification. As the second position from the N-terminus of these peptides is the most common manipulation point for abrogating DPP4 action, we explored three different aza- amino acids at this site. Aza-alanine (AzaA) was chosen as the closest structural proxy to the - 6 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 canonical residue alanine, and the insertion of aza-glycine (AzaG) and aza-proline (AzaP) were explored as substitutions that yield more and less flexible structures, respectively. The glutamic acid at position three (P1’ as a substrate) of GLP-1 and GIP was replaced with aza-glutamic acid (AzaE) with the intent to preserve side chain:receptor interactions (Figure 1). In addition, the glycine at position 10 of GLP-1 was replaced with AzaG to assess the influence of a turn- inducing residue on G-protein signaling. Modifications at this position have previously established that both helix- and turn-promoting residues affect this pathway.

[0023] Azapeptides were assembled using standard solid-phase Fmoc peptide synthesis with substitutions based on the side chain of interest as discussed vide supra. This first necessitated the synthesis of the individual aza-amino acids that were chemically assembled using literature procedures which were altered slightly for efficiency (see Supporting Information). Briefly, the use of selective nitrogen differentiation on protected hydrazine was employed and followed by addition of the carbonyl donor by either using a phosgene equivalent (for AzaA and AzaP), 4- nitrophenyl chloroformate (for AzaE), or disuccinimidyl carbonate (for AzaG) as the activating agent. Boc-methylhydrazine was used to generate Fmoc-AzaA using a slightly modified version of a reported procedure. AzaG was obtained as the Fmoc-protected hydrazine, and AzaP has a well-documented synthetic route. The Fmoc-protected AzaE was synthesized using a combination of procedures. These compounds were reacted with carbonyl-donor moieties to in situ generate the activated Fmoc-aza amino acid, that was transferred directly to resin.

[0024] We designed our azapeptides for testing on the two important incretin hormone receptors, the GLP-1R and the GIPR. In addition to the native ligands GLP-1 (for GLP-1R) and GIP (GIPR), we also tested our strategy on semaglutide, a clinical drug that is active on the GLP-1R, and a previously described unimolecular dual agonist (NNC0090-2746 from Novo Nordisk, termed ‘DA’ in this manuscript) of both GLP-1R and GIPR (Figure 1).

[0025] GLP-1 and GIP are both extremely selective and potent agonists of their cognate receptors, which they agonize at single digit pM concentrations (Table 1). Since the frontline protease, DPP4, removes the two N-terminal amino acid residues from each of the peptide ligands that results in severe diminution of activity (>99.9%), we first investigated the introduction of aza-amino acids at position two from the N-terminus of both GLP-1 and GIP and the capacity of the resulting constructs for robust agonism. To obtain potencies of the peptide, we utilized a concentration responsive cell-based bioassay that measures the ligand’s ability to stimulate the receptor and activate adenylyl cyclase to result in cAMP production, that is linked biosynthesis of luciferase. For measuring GLP-1R agonism, human embryonic kidney-293 (HEK-293) cells stably transfected with GLP-1R and the CRE6X-luciferase - 7 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 reporter were used. GIPR agonism was assessed with HEK-293T cells transiently transfected with plasmids coding for the GIPR, reporter, and -galactosidase to account for transfection variability. Table 1. Activities of GLP-1, GIP and their analogues on receptors. PeptideaEC50(pM)b± SEM pEC50bncGLP-1R GLP-1 (native) 1.6 ± 0.14 11.8 3 A8AzaA GLP-1 2.4 ± 0.38 11.6 3 A8AzaG GLP-1 28 ± 3.6 10.6 3 A8AzaP GLP-1 96 ± 27 10.0 3 E9AzaE GLP-1 1200 ± 250 8.9 3 G10AzaG GLP-1 22 ± 2.0 10.7 3 DA X2AzaA, X20A 0.8 ± 0.19 12.1 3 Semaglutide 3.0 ± 0.27 11.5 3 X2AzaA Semaglutide 11 ± 1.0 11.0 3 GIP, Native 11.2 3 A2AzaA GIP 10.4 3 A2AzaG GIP 9.9 4 DA X2AzaA, X20A11.6 3 aIdentity of the peptides that were evaluated (Figure 1) for agonism at GLP-1R and GIPR using the luciferase reporter system in HEK293 cells.bEC50is the concentration of peptide required for half-maximal activity at the target receptor. pEC50= –log(EC50) ± SEM of independent experiments.cNumber (n) of independent experiments.

[0026] We first examined receptor stimulative properties of A8AzaA GLP-1, and remarkably to our pleasant surprise, this variant was extremely potent at the GLP-1R (EC50 = 2.4 pM; Figure 3A and Table 1) and was essentially equipotent and equiefficacious as native GLP-1. This outcome was both unexpected and dramatic as azapeptides like to adopt β-turn conformations, and the co-crystal structure of several ligands and GLP-1 or GIP show that residues at this position when bound to the receptor usually populate the α-helical region. Further structural perturbations of the Ala8 site of GLP-1 with more and less structurally restricted aza-residues, AzaG and AzaP, resulted in observations that were more in line with expectations based on known azapeptide conformational preferences. When Ala8 was substituted with AzaG in GLP-1, it suffered a 15+ fold loss in potency (Figure 3B and Table 1), and the more rigidifying A8AzaP variant led to a more significant 60-fold decrease in potency when compared to native GLP-1 (Figure 3C). These data suggest that the aza-residue that was the closest surrogate in terms of both size and shape was well accommodated by the receptor while others with access to larger (Ala8AzaG) or smaller (Ala8AzaP) conformational space were less effective agonists at the GLP-1R. - 8 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0027] We then interrogated the C-terminal side (P1’ residue) of the scissile bond (DPP4 cleavage) by replacing the glutamic acid residue (third from the N-terminus, E9) with AzaE. This residue has previously been inspected using alanine scanning (Glu9Ala variant, 30-fold loss in binding as judged by IC50 values, but equivalent adenylyl cyclase activity), or substitution by tert-Leu or β-dimethyl-Asp (equivalent cAMP production as native GLP-1). These findings encouraged us to incorporate AzaE at the P1’ position (third residue from the N-terminus) of GLP-1. Unexpectedly, the E9AzaE GLP-1 analogue suffered a dramatic 250- fold potency loss. These data suggest that the E9 residue occupies conformational space that the aza-amino acid residue is unable to adopt. Given the unexpected and surprising result of being able to modify position two, we also explored substitution at position 10 (fourth from the N-terminus) that is the P2’ site of the substrate with respect to DPP4. In this instance, alanine scanning resulted in greater than 103–fold diminished adenylyl cyclase activity, and Gellman and co-workers have shown that replacement with an ACPC-(R,R–X) residue results in a significant loss in receptor stimulation. Upon examining the G10AzaG analogue using the luciferase assay, we found that it suffers a 19-fold decline in potency compared to native GLP- 1. These data point to the relative promiscuity of position two from N-terminus with regard to substitutions on GLP-1 and that it can also accommodate aza-amino acids to various degrees with a preference for AzaA (Table 1). As mentioned previously, it was somewhat surprising to find that aza-amino acids could be used as they have strong (φ, ψ) dihedral angle preferences dictated by the diacyl hydrazine and the urea moieties. This prompted us to computationally investigate what solution backbone structures are favored in Ac-Xaa-NMe and His-Xaa-Glu- NMe, with Xaa being Ala, Aib, AzaA, AzaG, or AzaP, and whether they can occupy conformational space as those populated by natural amino acids in the GLP-1:GLP-1R complex (PDBID: 6X18), GIP:GIPR complex (PDBID: 7RA3), Semaglutide:GLP-1R complex (PDBID: 7KI0), tirzepatide:GLP-1R complex (PDBID: 7RGP), and tirzepatide:GIPR complex (PDBID: 7RBT) (Tables S3 and S4, Supporting Information).

[0028] Molecular dynamics simulations of the construct Ac-Ala-NMe showed that the Ala residue in this minimal construct adopted multiple conformations, e.g., in the β-sheet, Polyproline II (PPII), and αR regions. As Aib, AzaA, AzaG, and AzaP are achiral, the Ramachandran plots of these amino acids in the dipeptide simulations were center-symmetric. For Aib, these conformations include a major population in the αR conformation and a minor population in the PPII conformation (and accompanying mirror conformations). On the other hand, AzaA favors the conformation around (–130°, 10°) along with a minor conformation - 9 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 around (–130°, –175°) (and the mirror conformations). AzaG predominantly adopts an extended conformation around (–155°, –170°) and the mirror conformation. AzaP exhibits two main conformations: a shifted PPII-like conformation around (–65°, –175°) and a conformation around (–75°, 10°) (and the mirror conformations). The broad distribution of Ala nearly encompasses the distributions of Aib, AzaA, AzaG, and AzaP in the negative φ region.

[0029] The Ramachandran plots for Xaa in His-Xaa-Glu-NMe showed that the addition of neighboring amino acids appears to favor the positioning of Ala and Aib in the -helix region. The (φ, ψ) values for Ala and Aib in these simulations generally align with those observed in X-ray co-crystal structures. It is evident that AzaA prefers (–130°, 10°) and AzaP (–75°, 10°). On the other hand, AzaG continues to prefer an extended conformation.

[0030] Co-crystal structures show that GLP-1 when bound to the GLP-1R is predominantly in an -helical conformation. However, closer to the N-terminus of the ligand, there is a region that is unstructured, and we postulated this section may be able to accommodate aza-amino acids and still engage with the receptor productively. In order to interrogate whether aza-amino acids can adopt conformational space that GLP-1 occupies in the ligand:receptor complex, we undertook molecular dynamics simulations of the native (GLP-1:GLP-1R) and modified complexes that substituted aza-amino acids at position 8 (second from the N-terminus) in native GLP-1, i.e., GLP-1A8AzaA, GLP-1A8AzaG, or GLP-1A8AzaP bound to GLP-1R. Ala8 of GLP-1 was found mostly in regions of -helicity, but also populated stable conformational regions that the aza-amino acid readily adopts. This discovery was surprising, as it was thought that Ala8 would be confined to strictly -helical regions similar to the Aib8 containing GLP-1 observed in the simulation of the receptor:ligand complex. We further note that while the AzaA residue in GLP-1A8AzaA:GLP-1R populated backbone dihedrals around (–130°, 0°), close to the dihedrals observed in Ala8 in GLP-1:GLP-1R, the AzaG in GLP-1A8AzaG:GLP-1R populated backbone dihedrals around (–150°, 0°) and the AzaP in GLP-1A8AzaP:GLP-1R populated backbone dihedrals around (–60°, 0°).

[0031] We also examined the crucial interactions involving His7 and residue 8 (Res8) of the GLP-1 ligands with the GLP-1R to assess the influence of these substitutions. These include the interactions of the terminal α-ammonium group (–NH3+) of His7 with the side chain of Glu387(receptor), the NH3+ of His7 with the side chain of Glu364(receptor), the side chain of His7 with the side chain of Trp306(receptor), the side chain of Res8 with the side chain of Leu384(receptor), and the side chain of Res8 with the side chain of Leu388(receptor) as shown in. - 10 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0032] The A8AzaA variant had similar interactions of comparable strength as those of the native peptide and GLP-1A8Aib with GLP-1R. On the other hand, analysis of the dynamics of the GLP-1A8AzaG:GLP-1R complex revealed less frequent interaction between AzaG8 and Leu388(receptor). In the case of the GLP-1A8AzaP:GLP-1R complex, interactions between His7 and Trp306(receptor) were relatively sparse.

[0033] Examination of the cryoEM structures of the GIP:GIPR complex also reveals an - helical region for GIP reminiscent of the GLP-1:GLP-1R case, and a familiar unstructured N- terminus. This observation and the success of the aza-amino acid containing GLP-1 analogues encouraged us to make azapeptide derivatives of GIP. We first tested A2AzaA variant of GIP and found it suffered a minor loss in potency (5.8-fold) which being equally efficacious. On the other hand, an A2AzaG derivative of GIP underwent a more significant decrease in potency (12-fold) (Figures 3D & 3E). We conclude from these observations that the native GIP:GIPR complex is more sensitive and less tolerant to subtle changes in backbone structure than it was in the case of GLP-1:GLP-1R combination. We then shifted our attention to making aza-amino acid variants of semaglutide (Ozempic) that has changed the landscape of T2D management and weight loss therapies. Popular in the public imagination and extremely effective as a clinical compound, semaglutide contains Aib at position 2 and we replaced that with an AzaA as we had previously with GLP-1. Semaglutide also linker attached to a C18 diacid on a Lys side chain. This new azapeptide variant of semaglutide was equipotent and as efficacious as the parent drug and native GLP-1 (Figure 3F).

[0034] The extent of aza-amino acid tolerance was further explored to expand the template inventory and document the generalizability of our approach. Unimolecular dual agonists (DAs) have recently emerged as potent compounds that enable higher percentages of weight loss, and we used a previously described template, NNC0090-2746, termed “DA” in this paper. It is also known that the binding mode of some dual and triple agonists are slightly distinct especially at the N-terminus of the peptide when in complex with the receptor. DA is an unimolecular dual agonist compound that has been shown to be balanced, in that it is equally potent at both the GLP-1R and GIPR as compared to the respective native ligands. We introduced two modifications in DA, first 2-aminoisobutyric acid (X) at position 2 was changed to AzaA, and the X residue at position 20 was replaced with alanine (X2AzaA, X20A). A dual substitution of X at positions 2 and 20 to alanine (X2A, X20A) with all other structural characteristics of DA was equipotent at GIPR and GLP-1R compared to native ligands (unpublished data), and therefore we envisioned that it could tolerate the aza-amino acid modification at position 2. Our findings show that this new dual agonist azapeptide maintains - 11 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 the precedented balance at GLP-1R and GIPR with essentially equivalent potencies to the native ligands (Figures 3G & 3H). We note that while an A2AzaA modification of GIP resulted in a comparative decline in potency at GIPR, and the A8AzaA modification of GLP-1 demonstrated equal potency at GLP-1R, a mutation to this dual agonist resulted in no shift in receptor balance or potency loss compared to native ligand at either receptor. This is presumably due to the subtle structural differences in the way the dual agonist and GIP bind to the GIPR.

[0035] Aza-modified GLP-1R and GIPR agonists were further tested for their ability to be refractive to DPP4-catalyzed proteolysis and inactivation (Table 2). We used two complementary methods to demonstrate this. First, we carried out an LC ESI-MS experiment where the azapeptide was incubated with DPP4 in an aqueous buffer, and the reaction mixture examined after 18 hours to assess the extent of proteolysis. The A8AzaA GLP-1 analogue was found to be completely resistant to DPP4 action and remained intact, while native GLP-1 was almost quantitatively degraded with no detectable full-length peptide remaining (Figure 5A). We then employed the luciferase-based cAMP cellular assay to assess whether the protease can inactivate the azapeptide ligands. This latter method relies on the observation that the truncated peptides suffer a 103-fold loss in potency. Briefly, peptides were incubated overnight with DPP4 and then the reaction mixture directly applied to cells and the concentration response assay conducted as previously. A shift in potency after such an experiment reveals the extent of cleavage. This method is sensitive enough to detect even 0.5% of the peptide remaining, whereas the LC ESI-MS experiments are more sensitive only a smaller fraction of the peptides( 80%) is degraded. For instance, if one half of the peptide were degraded, a potency loss ofonly 2-fold would be seen (within error of cellular assays, but easily discernible in LC-MS) whereas a 99.5% effective cleavage would result in a ~500-fold decrease in potency that would easily be detected by the cellular assay, but not so by LC-MS experiments. GLP-1R agonists were first tested including the native, A8AzaA, A8AzaG, A8AzaP GLP-1, X2AzaA semaglutide, and DA X2AzaA X20A (Figures 4A-D, figure 4G & 4H). GIPR agonism of native GIP and the most potent azapeptide variant A2AzaA GIP were tested (Figures 5E & 5F). All azapeptide agonists incubated with DPP4 showed no significant difference from azapeptide agonists incubated in the vehicle prior to the cell assay. These data demonstrate that agonists containing aza-amino acids at the second position are not degraded by DPP4.

[0036] Three plausible reasons could explain the refractory nature of the azapeptides towardsDPP4. First, the degradation may be kinetically slow ( 18 hours); second, because of structuraldifferences, it is possible that the scissile bond is improperly aligned in the enzyme active site; - 12 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 or finally, due to the conformational preferences of azapeptides, they may not bind the enzyme active site at all. Incubation of native GLP-1 with DPP4 followed by quenching and LC-MS analysis showed that GLP-1 has an approximate half-life of 30 seconds under these conditions (Figure 7). There is precedent for compounds such as diprotin A to be hydrolyzed at a slower rate, but a more likely scenario was that the azapeptide was a competitive inhibitor or was incapable of occupying the enzyme active site. Peptides containing aza-amino acids at the P1 position of the substrate have been documented to resist degradation. In addition, azapeptides (P1 position) have also been able to known to be noncovalent competitive inhibitors, such as in the case of the hepatitis C virus serine protease. In order to address which of these two options is operational in this context, we sought to interrogate whether our analogues act as competitive inhibitors, or are just plainly not recognized by DPP4. Table 2. Stability against DPP-4 catalyzed hydrolysis of native and aza-amino acid containing peptidesa.GLP-1, Native 12 ± 0.07 2.2 4 8.8 ± 0.041.5 × 103 4 680A8AzaA GLP-1 11 ± 0.05 4.7 3 11 ± 0.10 4.6 3 0.98 A8AzaG GLP-1 11 ± 0.08 20 3 11 ± 0.06 20 3 1.0 A8AzaP GLP-1 9.8 ± 0.05 160 4 9.5 ± 0.10 310 4 1.9 G10AzaG GLP-1 10 7.9 1 8.17.4 × 103 1 940Semaglutide 11 ± 0.07 19 3 11 ± 0.06 16 3 0.84 X2AzaA 10 ± 0.11 52 3 10 ± 0.06 42 3 0.81 Semaglutide X2AzaA X20A DA 13 ± 0.29 0.29 3 13 ± 0.09 0.3 3 1.0 GIPR GIP 11 ± 0. 17 6.1 3 8.9 ± 0.112.4 × 103 3 390A2AzaA GIP 9.7 ± 0.08 210 3 9.7 ± 0.19 210 3 1.0 aPotencies determined using HEK293 cells expressing GLP-1R or GIPR with the luciferase reporter system. Results are separated by the targeted receptor. Peptides were incubated at 37 °C overnight with and without DPP4 before their incubation with transfected cells.bEC50= [peptide] required for half maximal activity of the targeted receptor. pEC50= −log(EC50) ± SEM of independent experiments where applicable.cNumber of independent experiments.dRatio = (EC50with DPP4) / (EC50without DPP4).

[0037] In order to distinguish between these two possibilities, we tested the ability of the azapeptides to inhibit the cleavage (hydrolysis) of a known chromogenic substrate, Gly-Pro-p- nitroanilide (Gly-Pro-pNA). We measured the rate of Gly-Pro-pNa (added last) by p- nitroaniline production with prior incubation of DPP4 with either (i) native ligand (GLP-1), (ii) - 13 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 azapeptide variant (A8AzaA, A8AzaP), (iii) linagliptin, a known competitive inhibitor, or (iv) vehicle (Figure 5B). No appreciable differences were observed between the aza-analogs, GLP- 1 (which was presumed in its truncated form by the time of substrate addition), and vehicle. In contrast, linagliptin severely diminished enzyme action.

[0038] These data once again highlight the aversion of the azapeptide variants of GLP-1 and GIP to bind the DPP4 active site to any significant extent. DPP4 has a particular liking for proline at the second position (P1) and one may advance the idea that the aza-analog A8AzaP, the closest in structure to proline, may have affinity to the active site. This idea is negated in the experiment shown in Figure 5B as A8AzaP failed to show an inhibitory effect, and was similar to the vehicle (Figures 7A-7C). A single atom modification (O --> S) reported in GLP- 1 by Petersson and co-workers also conferred stability against DPP4 and comparable cAMP production; although it required changing the N-terminal His to Phe in GLP-1 because of intramolecular cyclization.

[0039] Analogues of GLP-1 and GIP containing aza-alanine residue at the second position from the N-terminus are refractory to DPP4 catalyzed hydrolysis and inactivation. Simultaneously these azapeptides suffer minimal to no loss of potency or efficacy at their cognate receptors. Rapid renal clearance also affects longevity in vivo, and while backbone modifications do not redress this concern, the compounds described in this paper are compatible with albumin binding lipid modifications that allay this liability. The superb activity profiles exhibited by the X2AzaA analogues of semaglutide and a dual agonist of the GLP- 1R / GIPR, both exhibiting improved renal clearance profiles, further underscores the utility and potential of aza-amino acids to expand the universe of non-canonical amino acids employed in peptide therapeutics in the realm of T2D and obesity management. Moreover, these data help to illustrate an already known feature of aza-amino acids, in that they resist enzymatic degradation, and our study expands this space to the previously unreported serine proteases, the largest class of hydrolases. The utility of aza-amino acids may also show promise in exploring their relative pharmacological profile on Class B G-protein Coupled Receptors (GPCRs) compared to native ligands for class B GPCRs. Within this class, receptors and the downstream signaling they engender is dependent on subtle variations in the structural and conformational attributes of the agonist. This outcome is of clear and present interest as a means of modulating other signaling pathways such as -arrestin 1 and 2 recruitment and concomitant receptor internalization. Signaling triggered by these proteins has differential effects and has been postulated to play a role in determining efficacy of therapeutic outcomes. Studies have already shown that a 'bias' in cAMP signaling for GLP-1R agonism can be encouraged by - 14 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 single or minimal modification with structurally rigid residues, and therefore the aza-amino acids may serve as another avenue for all templates to not only provide protection against the frontline protease, namely DPP4, but also influence signaling pathways.

[0040] To this effect, aza-glycine modifications applied to GLP-1 at the second and fourth position relative to the N-terminus produce a significant degree of bias while retaining modest cAMP stimulation (Figure 8). It appears that the near-native structure of aza-alanine does not impart significant bias, as Arr2 recruitment to the GLP-1R was nearly equivalent to GLP-1- induced recruitment. Instances where the compound was considered a weak agonist at the GLP- 1R for cAMP stimulation, such as A8AzaP GLP-1 and E9AzaE GLP-1, resulted in minimal to no Arr2 recruitment (< 20% maximal recruitment) (Figure 8). These data outline that aza- amino acids engender varying degrees of biased agonism and can be leveraged as a tool for receptor signaling modulation.

[0041] In certain aspects, disclosed herein are peptides comprising an aza-amino acid residue (Aza); wherein the peptide is selected from the group consisting of Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP-2), Glucose-dependent insulinotropic polypeptide (GIP), Glucagon (GCG), Growth hormone releasing hormone (GHRH), FGF2, FGF21, Tyr- melanostatin, Endomorphin-2, Enterostatin, -casmorphin, Trypsinogen pro-peptide, Corticotropin-like intermediate lobe peptide, Gastrin-release peptide, Aprotinin, GCP-2, MDC,MCP-2, Eotaxin, IP-10, Insulin-like growth factor-1, Interleukin-2, Interleukin-1 , -Microglobulin, PHM, GRH-(1-29), GRH-(1-44), Oxyntomodulin, Secretin (SCT), Vasoactive Intestinal Peptide (VIP), Neuropeptide Y (NPY), Met-enkephalin, Pancreatic Polypeptide (PP), Peptide YY (PYY 1-36 and 3-36 form), Exenatide, Substance P, Parathyroid hormone (hPTH 1-34), BNP, Liraglutide, Tirzepatide, Semaglutide, Retratrutide, Taspoglutide, Lixisenatide,Albiglutide, Dulaglutide, Stromal cell-derived factor 1 ( and ), BI-456906 MAR423, CCL5,CCL11, Pituitary Adenylate Cyclase-Activating Polypeptide, NNC0090-2746, and Ecnoglutide peptides having at least 85% sequence identity to any one of them; and an amino acid residue of the peptide is replaced with the aza-amino acid residue (Aza).

[0042] In certain embodiments, the aza-amino acid residue (Aza) is:- 15 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 16 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 17 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 18 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 19 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 20 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 21 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 22 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 23 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 24 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 25 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 26 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 27 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0043] In certain emboidments, the aza-amino acid residue (Aza) is selected from aza-glycine (AzaG), aza-alanine (AzaA), aza-proline (AzaP) and aza-glutamic acid (AzaE).

[0044] In certain embodiments, a 2-aminoisobutyric acid (X), -methyl-L-Leucine (meL), alanine (A), glutamic acid (E), glycine (G), leucine (L), lysine (K), phenylalanine (F), isoleucine (I), aspartic acid (D), arginine (R), or proline (P) is replaced with the aza-amino acid residue (Aza). In certain embodiments, the peptide is selected from the group consisting of: H*A*E*G*TFTSDVSSYL*EG*QAAK*EF*I*AWL*VK*G*R-NH2, H*A*E*G*TFTSDVSSYL*EG*QAAK*EF*I*AWL*VK*G*RG-OH, H*A*D*G*SFSDEMNTI*L*DNL*AARDF*INWL*IQTK*ITD, Y*A*E*G*TFISDYSI*AMDK*IHQQDFVNWL*L*AQK*G*K*K*NDWK*HNI*TQ, HS*Q*G*TFTSDYSK*YL*DSR*R*AQDFVQWL*MNT, YA*DAI*FTNSYR*K*VL*G*QL*SAR*K*L*L*QDIMSR*QQG*ESNQER*G*AR*AR* L, HS*Q*G*TFTSDYSK*YL*DSR*R*AQDFVQWL*MNTK*R*NR*NNI*A, HS*D*G*TFTSEL*SR*L*R*DSAR*L*NR*L*L*NGL*V, HD*A*VFTDNYTR*L*R*K*NMAVK*K*YL*NSI*L*N, YP*SK*P*DNP*G*EDAP*AEDL*AR*YYSAL*R*HYI*NL*I*TR*QR*Y, - 28 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 Y*G*G*F*M, AP*L*EP*VYP*G*DNATP*EQMAQYAADL*R*R*YI*NML*TR*P*R*Y, YP*IKP*EAP*G*EDASPEEL*NR*YYASL*R*HYL*NL*VTR*QR*Y–NH2, H*G*E*G*TFTSDLSK*QME*E*E*AVR*LF*IEW*L*K*NG*G*P*SSG*AP*P*P*S– NH2, R*P*K*P*QQFFG*L*M, YP*SK*P*DNP*G*EDAP*AEDMAR*YYSAL*R*HYI*NL*I*TR*QR*Y, HA*E*G*TFTSDVSSYL*EG*QAA(X)*EF*I*AWL*VR*G*R*G wherein X attached to the -amine of lysine is:, YAib*E*G*TFTSDYSI*Aib*L*DKI*AQ(X)AFVQWL*I*A G*G*P*SSG*AP*P*P*S–NH2where X attached to the -amine of lysine is:, HAib*E*G*TFTSDVSSYL*EG*QAA(X)EFI*AWL*VR*G*R*G wherein X attached to the -amine of lysine is:, YAib*Q*G*TFTSDYSImeL*LDK(X)AQAib*AFIEYL*L*G*G*P*SSG*AP*P*P*S–NH2where X attached to the -amine of lysine is: - 29 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425HAib*E*G*T*FTSDVSSYL*EGQAAK*EFIAWL*VK*Aib*R–NH2, HG*E*G*TFTSDL*SK*QMEEEAVR*L*FI*EWL*K*NGGP*SSGAPPSK*K*K*K*K*K* –NH2, H*S*Q*G*TFTSDLSK*Y*L*E*E*E*AVR*E*F*IAW*LKNGGPSR*HY*LNLVTR*QR* Y*-NH2, H*Aib*H*G*TFTSDY*SIY*LE(X)Y*AAib*EF*VQW*LLE*GGPSSGAPPPS-NH2where X attached to the -amine of lysine is:H*Aib*E*G*TFTSDV*SSY*LEG*QALR*HY*INW*LTR*QR*Y*-NH2, and H*Aib*Q*G*TFTSD(X)SK*Y*LD*E*RAA*Q*DF*VQW*LLD*G*G*P*SSG*AP*P*P*S –NH2where X attached to the -amine of lysine is:or a peptide having at least 85% sequence identity to any one of them; wherein at least one amino acid residue marked with * is replaced with the aza-amino acid.

[0045] In certain embodiments, the peptide has at least 90% sequence identity to GLP-1, GIP or semaglutide. In certain embodiments, the peptide has at least 95% sequence identity to GLP- 1, GIP or semaglutide. In certain embodiments, the peptide is GLP-1, GIP or semaglutide. - 30 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0046] In certain embodiments, the peptide is selected from A8Aza GLP-1, E9Aza GLP-1 and G10Aza GLP-1. In certain embodiments, the peptide is selected from A8AzaA GLP-1, A8AzaG GLP-1, A8AzaP GLP-1, E9AzaE GLP-1 and G10AzaG GLP-1. In certain embodiments, the peptide is A2Aza GIP. In certain embodiments, the peptide is A2AzaA GIP. In certain embodiments, the peptide is X2Aza semaglutide. In certain embodiments, the peptide is X2AzaA semaglutide. In certain embodiments, the peptide is X2Aza NNC0090-2746. In certain embodiments, the peptide is X2AzaA NNC0090-2746.

[0047] In further aspects, disclosed herein are methods of: a) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non- insulin dependent diabetes, and / or obesity; b) reducing body weight and / or food intake, and / or inducing satiety; c) treating or preventing Alzheimer's disease, nonalcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated liver disease (MASLD), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; d) treating chronic kidney disease, traumatic brain injury, alcohol addiction, and / or substance addiction; e) treating or preventing emesis; f) treating or preventing effects of aging; g) support of islet transplant survival in Type 1 diabetes; and h) treating or preventing arthritis and related diseases, including but not limited to: Osteoarthritis, Rheumatoid arthritis, Gout, Ankylosing spondylitis, Psoriatic arthritis, Juvenile arthritis, Fibromyalgia, Infectious arthritis, Spondyloarthritis, Sjogren's syndrome, Scleroderma, and Polymyalgia rheumatica. comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0048] In yet further aspects, disclosed herein are methods of treating cardiovascular disease and / or hypertension, comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0049] In yet further aspects, disclosed herein are methods of treating a neurodegenerative disease (e.g., Alzheimer’s, Parkinsons, other forms of dementia, traumatic brain injury), comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein. - 31 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0050] In yet further aspects, disclosed herein are methods of treating alcohol use disorder, substance use disorder or smoking addiction, comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0051] In yet further aspects, disclosed herein are methods of treating inflammation, comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0052] In yet further aspects, disclosed herein are methods of treating diabetes, comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0053] In yet further aspects, disclosed herein are methods of treating obesity, comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein.

[0054] In certain embodiments, the subjects referenced herein were not responsive to or did not adequately tolerate a previous course of therapy with an incretin mono- or dual- or tri- agonist, wherein the azapeptide replaces non-tolerated or ineffective incretin-based mono- or dual agonist therapy. Definitions

[0055] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0056] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0057] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985). - 32 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0058] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0059] It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0060] As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O- alkyl, -OP(O)(O-alkyl)2 or –CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0061] Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0062] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to C1-C6 straight-chain alkyl groups or C1-C6 branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, - 33 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted.

[0063] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0064] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0065] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0066] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0067] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0068] The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

[0069] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.

[0070] The term “Cx-y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. C0alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C1-6alkyl group, for example, contains from one to six carbon atoms in the chain.

[0071] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0072] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0073] The term “amide”, as used herein, refers to a group - 34 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 O R9N R10,

[0074] wherein R9 and R10 each independently represent a hydrogen or hydrocarbyl group, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0075] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by,

[0076] wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0077] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0078] The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0079] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0080] The term “carbamate” is art-recognized and refers to a group,

[0081] wherein R9 and R10 independently represent hydrogen or a hydrocarbyl group.

[0082] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group. - 35 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0083] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct- 3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H- indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0084] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0085] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0086] The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0087] The term “ester”, as used herein, refers to a group -C(O)OR9 wherein R9 represents a hydrocarbyl group.

[0088] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl- O-alkyl.

[0089] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0090] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[0091] The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered - 36 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0092] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0093] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0094] The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0095] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =O substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0096] The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0097] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain - 37 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0098] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0099] The term “sulfate” is art-recognized and refers to the group –OSO3H, or a pharmaceutically acceptable salt thereof.

[0100] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae,

[0101] wherein R9 and R10 independently represents hydrogen or hydrocarbyl.

[0102] The term “sulfoxide” is art-recognized and refers to the group–S(O)-.

[0103] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0104] The term “sulfone” is art-recognized and refers to the group –S(O)2-.

[0105] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have - 38 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0106] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0107] The term “thioester”, as used herein, refers to a group -C(O)SR9 or –SC(O)R9.

[0108] wherein R9 represents a hydrocarbyl.

[0109] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0110] The term “urea” is art-recognized and may be represented by the general formula O R10NNR9R9,

[0111] wherein R9and R10independently represent hydrogen or a hydrocarbyl.

[0112] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0113] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0114] “Salt” is used herein to refer to an acid addition salt or a basic addition salt.

[0115] Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms - 39 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0116] Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

[0117] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0118] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0119] “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid , 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid , gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion , an alkaline earth ion , or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of nontoxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. - 40 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0120] The term “pharmaceutically acceptable cation” refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e. g., Berge, et al., J. Pharm. Sci. 66 (1):1-79 (January 77).

[0121] “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.

[0122] “Pharmaceutically acceptable metabolically cleavable group” refers to a group which is cleaved in vivo to yield the parent molecule of the structural formula indicated herein. Examples of metabolically cleavable groups include -COR, -COOR, -CONRR and -CH2OR radicals, where R is selected independently at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.

[0123] “Prodrugs” refers to compounds, including derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N- alkylmorpholine esters and the like. Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp.7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are particular prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkylesters or (alkoxycarbonyl)oxy)alkylesters. Particularly the C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, C7-C12substituted aryl, and C7-C12arylalkyl esters of the compounds of the invention.

[0124] “Solvate” refers to forms of the compound that are associated with a solvent or water (also referred to as “hydrate”), usually by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g., in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as - 41 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0125] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle aged adult or senior adult) and / or a non- human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms “human,” “patient,” and “subject” are used interchangeably herein.

[0126] An “effective amount” means the amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to effect such treatment or prevention. The “effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A “therapeutically effective amount” refers to the effective amount for therapeutic treatment. A “prophylatically effective amount” refers to the effective amount for prophylactic treatment.

[0127] “Preventing” or “prevention” or “prophylactic treatment” refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject not yet exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[0128] The term “prophylaxis” is related to “prevention,” and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization, and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0129] “Treating” or “treatment” or “therapeutic treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” - 42 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, “treating” or “treatment” relates to slowing the progression of the disease.

[0130] As used herein, the term “isotopic variant” refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an “isotopic variant” of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium (2H or D), carbon-13 (13C), nitrogen-15 (15N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be “2H / D, any carbon may be13C, or any nitrogen may be15N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and / or substrate tissue distribution studies. The radio-active isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as11C,18F,15O and13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.

[0131] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.”

[0132] Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R - and S - sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+)- or (-)- isomers, respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”. - 43 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0133] “Tautomers” refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of it electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro-forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0134] As used herein a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0135] As used herein and unless otherwise indicated, the term “enantiomerically pure R- compound” refers to at least about 95% by weight R-compound and at most about 5% by weight S-compound, at least about 99% by weight R-compound and at most about 1% by weight S-compound, or at least about 99.9 % by weight R-compound and at most about 0.1% by weight S-compound. In certain embodiments, the weights are based upon total weight of compound.

[0136] As used herein and unless otherwise indicated, the term “enantiomerically pure S- compound” or “S-compound” refers to at least about 95% by weight S-compound and at most about 5% by weight R-compound, at least about 99% by weight S-compound and at most about 1% by weight R-compound or at least about 99.9% by weight S-compound and at most about 0.1% by weight R-compound. In certain embodiments, the weights are based upon total weight of compound.

[0137] In the compositions provided herein, an enantiomerically pure compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about - 44 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 10% enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound. For example, a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S- compound. In certain embodiments, the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

[0138] The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.

[0139] Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

[0140] One having ordinary skill in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non-aromatic, is determined by the size of the ring, the degree of unsaturation and the valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.

[0141] Abbreviations - Glucagon like peptide 1 (GLP 1), Glucose-dependent insulinotropicpolypeptide (GIP), 2-aminoisobutyric acid (Aib, X), type 2 diabetes (T2D), Dual agonist (DA), World Health Organization (WHO). EXAMPLES

[0142] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the compounds, compositions, materials, device, and methods provided herein and are not to be construed in any way as limiting their scope. Preparation and characterization of exemplary peptides General

[0143] NMR spectra were recorded on a Bruker Advance III 500 MHz NMR spectrometer. Chemical shifts are reported in ppm relative to residual CHCl3in CDCl3solvent unless - 45 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 specified otherwise. Mass spectra (MS) were obtained using a Finnigan LTQ ESI TOF Mass Spectrometer. Liquid chromatography mass spectrometry (LC-MS) was performed using an Agilent 6230 LC ESI-MS. All C-terminally amidated peptides were synthesized on Rink amide resin (Novabiochem, preloaded with 0.66 mmol NH2eq. / g), GIP analogs were synthesized on 2-chlorotrityl resin preloaded with H-Gln(Trt) (Novabiochem, 0.79 NH2eq. / g). All Fmoc- protected L-amino acids, O-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate (HATU), piperidine, triphosgene, disuccinimidyl carbonate (DSC), and 4-nitrophenyl chloroformate, and Fmoc-chloroformate were purchased from Chem-Impex. Solvents used were obtained from Fisher Scientific. Sodium hydride and 1,3-dibromopropionate were purchased from Thermo Scientific. Cbz chloroformate, hydrazine monohydrate, N,N-diisopropylethylamine were purchased from TCI America, Biograde trifluoroacetic acid was procured from Halocarbon, and 1-Boc-1-methylhydrazine was purchased from Ambeed. Human DPP4 was purchased from Pro-spec with a specific activity of 200 mU, where one unit will hydrolyze 1 μmole of p-nitroaniline per minute at pH 8.0 at 37 °C using 1 mM of Gly-Pro p-nitroanilde as a substrate. Protocols for coupling compounds 2 and 7 to resin were identical aside from the number of equivalents of DIPEA. All protocols outlined for coupling compound 6, 7, and Fmoc-hydrazine to peptide (GLP-1 fragment) on resin were identical to other peptides on resin unless otherwise specified. Solid-phase peptide synthesis

[0144] Peptide backbone elongation was achieved utilizing a fast-flow method for rapid assembly as developed by Pentelute and colleagues. Standard peptide coupling was performed in dry DMF with 4 eq of amino acid, 3.6 eq of HATU (0.36 M), and 8 eq of DIPEA (relative to –NH2eqs. on resin). Coupling was complete in 45 minutes of shaking resin. The deprotection of Fmoc was achieved by reacting resin with 20% piperidine in DMF for 5 minutes, and then with drained solution a fresh batch of 20% piperidine in DMF was added for an additional 15 minutes. Lipid side chain appendages were incorporated into peptides on resin from Sicinski, K. M.; Sürmeli, D.; Du, J.; Raman, V. S.; Montanari, V.; Lee, M.; Harwood, B. N.; Kopin, A. S.; Beinborn, M.; Kumar, K. Journal of Medicinal Chemistry 2024, 67, 4998- 5010. Peptide Purification

[0145] Following reaction, the resin was washed with DMF (3 x 5 mL), CH2Cl2(3 x 5 mL), and MeOH (3 x 5 mL) and then dried in vacuo. Peptides were cleaved from the resin in a cleavage cocktail of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and H2O (95:2.5:2.5). After 90 minutes, the TFA was removed by rotary evaporation, and the peptides - 46 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 were precipitated in cold ether. Crude peptides were lyophilized and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) with solvent A (99:1:0.1, H2O:acetonitrile:TFA) and solvent B (10:90:0.07, H2O:acetonitrile:TFA) pre-warmed to 50 °C. A semiprep C18 column (Higgins Analytical, C18 Proto 200, 250 × 10 mm, 5 micron) was used for purification at a flow rate of 2.5 mL / min. Purification was conducted at a 30-50% B gradient for 30 minutes for all GLP-1 analogs, 25-45% B for all GIP analogs, and 40-60% B for DA X2AzaA, X20A and X2AzaA semaglutide. The purity (>95%) was analyzed by analytical C18 column (Higgins Analytical, C18 Proto 200, 250 × 4.6 mm, 5 micron) at a flow rate of 1.0 mL / min monitoring an absorbance at 230 nm unless otherwise specified. The presence of the peptides was confirmed via electrospray ionization mass spectroscopy (ESI- MS) with consistent ESI parameters (Ispray voltage, 4.52 kV; spray current, 0.17 μA; capillary voltage, 23 V; capillary temp 275 °C; tube lens, 89.7 V; N2carrier gas). Concentrations of peptides were determined by tyrosine or tryptophan absorbance at 280 nm in neat DMSO. cAMP Luciferase Reporter Assay for GLP-1R agonism

[0146] HEK293 C34L cells stably transfected with GLP-1R and CRE6x-luciferase were plated into 96-wells with a 0.2K population per well with an additional amount of 4.8K HEK293 QB1 bystander cells (5K total cell population per well). Concentration-response curves were generated by the incubation of peptide for a 4-hour period followed by addition of steadylite plus®reagent and an immediate analysis of chemiluminescence. cAMP Luciferase Reporter Assay for GIPR agonism

[0147] Transient transfection protocols have been outlined in previously described work. Briefly, HEK293T cells were plated into 96-wells with a 10K population per well and were transfected with GIPR, CRE6x-luciferase, and -galactosidase vectors. Concentration-response curves were generated by the incubation of peptide for a 4-hour period. Cell lysis and the introduction of D-luciferin were performed simultaneously through the addition of steadylite plus®reagent followed by immediate analysis of chemiluminescence. Subsequent correctionof the transfection was achieved by the addition of ortho-nitrophenyl- -galactosidase andmonitoring of absorbance at 420 nm at t = 0 min and t = 5 min. -Arrestin-2 Recruitment Assay at the GLP-1R

[0148] HEK293T cells were seeded in a 6-well plate at a cell density of 250,000 cells per well. After a 24-hour incubation, each well was transfected with 100 ng of GLP-1R-Rluc8 plus 3000ng of GFP2- -Arrestin-2 (R393E, R395E) cDNAs, using a 1:4 cDNA / lipofectamine ratio inOpti-MEM. After 6 hours of incubation, each well was diluted 2-fold with pre-warmed to 37°C - 47 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 DMEM solution, supplemented with 20% FBS and 2% L-glutamine. Following a further 12- hour incubation, the cells were gently washed with HBSS and were then trypsinized and plated in 96-well plates at a density of 10,000 cells per well. After 24 hours, the medium was aspirated and replaced with DPBS, followed by further incubation for 1 hour. Serially diluted peptide was then added for another 20-minute incubation. At this point, Ranilla luciferase substrate coelenterazine 400a (Gold Biotechnology, Cat# C-320-10) was further added to a concentration of 5 μM per well, and incubation was continued for another 10 minutes in the dark. Emission at 395 nm and 515 nm were measured. Bioluminescence resonance energy transfer (BRET) as an index of agonist-induced -arrestin recruitment to the GLP-1R was expressed as the ratio I510 nm / I395 nm. BRET values were normalized to the maximum and minimum activity observed in the absence of (cells treated with vehicle) or presence of a maximally effective concentration of the endogenous reference agonist GLP-1 (defined as 0% and 100% respectively). DPP4 incubation and LCMS analysis Luciferase Reporter Assay

[0149] Briefly, peptides (0.1 mM) were incubated at 37 °C with and without DPP4 (4 nM) within reaction buffer (20 mM TRIS, 100 mM NaCl, 1 mM EDTA, pH 8.0). After 18 hours, the reactions were centrifuged and either analyzed directly by LC-MS (Agilent, C18 Zorbax, 2.1 × 50 mm, 1.8 micron; solvent A, 0.1% formic acid in water, solvent B, 0.1% formic acid in acetonitrile; 25-55%B gradient; vaporizer / sheath gas temp, 347 °C; capillary voltage, 3.5 kV; base peak chromatogram scaled total ion chromatogram) or serially diluted with serum- free DMEM and subjected to the previously discussed luciferase reporter assay. DPP4 kinetic degradation of Gly-Pro-pNA

[0150] The methods developed for this experiment were previously described. In brief, pre- warmed vehicle, peptide, or inhibitor reagents at 37 °C were administered to 96-well plates containing DPP4 (4 nM). This mixture was incubated for 30 minutes before the addition of the chromogenic substrate, Gly-Pro-pNA (Carbosynth). The total concentration of DMSO in each reaction was below 1%. The assay was initiated by the addition of substrate in reaction buffer (20 mM TRIS, 100 mM NaCl, 1 mM EDTA, pH 8.0) at final reaction concentrations of 30 μM to 1000 μM. The DPP4 catalyzed reaction was monitored by the production of para- nitroaniline at an absorbance of 405 nm at 3 second intervals over 200 minutes with a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA). Prism 10 was used to fit the initial velocity of each reaction, spanning no longer than 10 minutes. Michaelis- Menten kinetics experiments were used to calculate initial velocity (V0) of the reactions that - 48 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 were plotted vs. initial substrate concentration ([S]0) to compute Km and Vmax according to the following equation: V0 = Vmax[S]0 / (Km+[S]0). Generation of cells stably transfected with Glp-1R and CRE6x-luciferase

[0151] Cells stably expressing human GLP-1R and the CRE6x-luciferase reporter gene were generated in a two-step sequential procedure. First, HEK293 QB1 cells were transiently transfected with GLP-1R cDNA in the pcDNA 3.1+ expression vector, which also encodes Zeocin resistance. Surviving clones were selected in 96-well plates after seeding an average of two cells / well and growing in the presence of Zeocin. Surviving clones were tested for GLP- 1R agonist responsiveness after additional transient co-transfection of a CRE6x-luciferase reporter gene, where corresponding cDNA was inserted in the pGL4.22 expression vector (Promega). This vector, which includes destabilization motifs to confer high turnover reporter gene expression, enabled high signal to background stimulation of GLP-1R expressing cells (determined by luciferase reporter gene assay). In a second step, functionally verified clones that showed high potency / high efficacy GLP-1R mediated signaling were again transiently transfected with CRE6x-luciferase cDNA in pGL4.22 as above. Additional encoding of Puromycin resistance by the pGL4.22 cDNA construct enabled further selection of GLP-1R + CRE6x-luciferase expressing, double stably transfected cells by clonal selection in the presence of Zeocin+Puromycin. Again, this was achieved by selection of surviving cell clones followed by functional assessment of GLP-1 responsiveness by luciferase reporter gene assay. The latter procedure paralleled the initial identification of stably GLP-1R expressing cells as above, but did not require additional transient transfection of CRE6x-luciferase cDNA (which in this case was already stably co-transfected in positive clones). Phenotypic stability of doubly transfected clones was verified by functional assessment over several passages of cell culture, where cells were maintained in the presence of Zeocin + Puromycin. A stably transfected clonal cell line (C34L) that consistently showed GLP-1 induced receptor stimulation with high potency and efficacy was chosen for assessment of peptide agonists. - 49 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0152] Scheme S1. Reagents and conditions for Fmoc-aza-amino acid synthesis and SPPS protocols.

[0153] Compound 1: Boc-hydrazine was converted to 1-((9H-Fluoren-9-yl)methyl) 2-tert- butyl pyrazolidine-1,2-dicarboxylate (CAS No.: 222854-34-4) through steps 1-3 using literature procedures (Vertesaljai and coworkers), which was further elaborated to compound 1 following Proulx, C., Picard, É., Boeglin, D., Pohankova, P., Chemtob, S., Ong, H., Lubell, W. D. J. Med. Chem. 2012, 55, 6502−6511.1H NMR (500 MHz, CDCl3): 7.76 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 7.0 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 6.9 Hz, 2H), 4.51 (m, 1H), 4.37 (m, 1H), 3.96, (m, 1H), 3.88 (m, 1H), 3.31 (m, 1H), 3.2 (m, 1H), 2.04 (m, 2H), 1.49 (s, 9H). Compound 2: compound 2 was synthesized following literature protocol outlined by Proulx and colleagues.7(1H NMR 500MHz, DMSO-d6): 7.91 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.0 - 50 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 Hz, 2H), 7.44 (t, J = 7.0 Hz, 2H), 7.35 (t, J= 7.0 Hz, 2H), 4.47 (br s, 2H), 4.36 (t, J = 7.1 Hz, 2H), 3.59 (t, J = 6.7 Hz, 2H), 3.45 (br s, 2H), 2.21 (dt, J = 6.9, 6.9 Hz, 2H).13C NMR (125 MHz, DMSO-d6): 153.24, 143.24, 140.69, 127.79, 127.20, 125.28, 120.13, 68.19, 46.28, 46.11, 45.85, 24.22. Compound 5: synthesis of compound 5 followed literature protocols with a 40% yield that had identical1H NMR and13C NMR in D2O.

[0154] Compound 6: to 3.29 g (20.59 mmol, 1 eq) of compound 5 was added 25 mL of water containing 3.46 g (41.19 mmol, 2.5 eq) of NaHCO3and 25 mL of THF. To this solution with rapid stirring was added dropwise a solution of 5.86 g (22.66 mmol, 1.1 eq) of Fmoc-Cl dissolved in 25 mL THF. Full appearance of 6 was typically observed after 35 minutes on TLC (Rf = 0.2 in 20%EtOAc / Hex). This mixture was diluted with 50 mL diethyl ether and was washed three times with brine, then dried with magnesium sulfate, filtered, and concentrated in-vacuo to generate a yellow-tinged oil. This crude oil was purified on silica with a 10-50% gradient of ethyl acetate in hexane mobile phase to yield 4.6 g of neutral oil. To this was added diethyl ether followed by a minor addition of 4M HCl in dioxane to yield 6 as a white solid (29% yield).1H NMR (500 MHz, MeOD): 7.82 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4Hz, 2H), 4.79 (br s, 2H), 4.32 (t, J = 4.7 Hz, 1H), 3.39 (br s, 2H), 2.12 (br s, 2H), 1.46 (s, 9H).13C NMR (125 MHz, MeOD): 172.44, 155.56, 144.74, 142.74, 129.02, 128.35, 125.71, 121.10, 82.70, 69.64, 46.95, 46.06, 34.20, 28.33.

[0155] Compound 7: synthesis of compound 7 step 1-3 followed literature protocols set forth in Kisseljova, K., Kuznetsov, A., Baudy-Floc’h, A., Järv, J. Bioorg. Chem. 2010, 38, 229–23. Step 1 (91% yield) with similar proton and carbon NMR to literature. Step 2, full conversion was assumed, and crude was brought directly to step 3 for precipitation of neutral product 7 (77% yield). 1H NMR (500MHz, DMSO-d6): 7.88 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 4.45 (br s, 2H), 4.23 (br s, 1H), 2.64 (s, 3H). 13C NMR (125MHz, DMSO-d6): 156.76, 143.83, 140.74, 127.65, 127.08, 125.22, 120.13, 65.44, 46.72, 38.27.

[0156] A8AzaA GLP-1 7-36 amide: in parallel to the activation of compound 7 with triphosgene, a 100 mg portion of rink amide resin (0.064 mmol, 1 eq) containing GLP-1 (9-36) was swelled in CH2Cl2 for 45 minutes in a 10 mL peptide synthesis vessel. The preparation of this peptide on resin followed Simon, M. D.; Heider, P. L.; Adamo, A.; Vinogradov, A. A.; Mong, S. K.; Li, X.; Berger, T.; Policarpo, R. L.; Zhang, C.; Zou, Y.; Liao, X.; Spokoyny, A. M.; Jensen, K. F.; Pentelute, B. L. ChemBioChem 2014, 15, 713-720. To an anhydrous 10 mL round bottom flask under nitrogen was added 51.3 mg (0.191mmol, 3 eq) of 7, and 422 μL of - 51 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 dry CH2Cl2. This mixture was placed on an ice bath along with a separate solution containing 19 mg (0.064 mmol, 1 eq) of triphosgene dissolved in 400 μL of dry CH2Cl2. Dropwise, the triphosgene solution was added to the solution of 7 at 0 °C. This solution was continued to stir for an additional 5 minutes, then was brought to room temperature for another 25 minutes of stirring. To this solution a 67 μL volume of DIPEA (0.4 mmol, 6 eq) was added and stirred for a final 5 minutes before the solution was transferring to CH2Cl2 -drained swollen resin for overnight shaking at room temperature. This resin was washed three times with CH2Cl2 and an additional three times with methanol. The resin was dried in-vacuo overnight and stored in the freezer. The following reactions performed on pre-swollen resin within DMF consisted of the deprotection step followed washing the resin three times with DMF, and subjugation to an activated solution Fmoc-His(Trt)-OH. After 3 hours this solution was drained, and the resin was washed three times with DMF and subjected to Fmoc deprotection conditions previously described. After deprotection, the resin was washed with DMF 3×, CH2Cl23×, and MeOH 3× followed by 1 hour drying in vacuo and then full cleavage and purification.

[0157] E9AzaE GLP-17-36 amide: in parallel to the activation of compound 6, a 88 mg portion of rink amide resin (0.060 mmol, 1 eq) containing GLP-1 (9-36) was swelled in DMF for 45 minutes in a 10 mL peptide synthesis vessel. To a 0.85 mL solution of dry DMF in a nitrogen gas atmosphere was added 91 mg (0.238 mmol, 4 eq) of 6 and an equal equivalent of DIPEA to neutralize. This solution was brought to –10 °C and to it was added 43 mg (0.214 mmol, 3.6 eq) of 4-nitrophenyl chloroformate in one portion. Complete consumption of 6 was typically observed after 5 minutes. A 82.8 μL (0.475 mmol, 8 eq) volume of DIPEA was then added and stirred for 1 minute followed by the prompt transfer of the activated compound to GLP-110- 36 peptide on resin for overnight shaking. Full turnover of the peptide on resin starting material was observed through mini-cleaved resin subjected to ESI-MS. Further peptide elongation was achieved by overnight coupling of Fmoc-Ala-OH to the hydrazine moiety with standard coupling equivalents, followed by histidine coupling, deprotection, drying, and full cleavage.- 52 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425

[0158] A8AzaG GLP-17-36 amide: in parallel to the activation of Fmoc-hydrazine, a 80 mg portion of rink amide resin (0.056 mmol, 1 eq) containing GLP-1 (9-36) was swelled in 700 μL of anhydrous DMF for 45 minutes in a 10 mL peptide synthesis vessel. To solution cooled to 0 °C of 44.2 mg (0.174 mmol, 3.1 eq) Fmoc-hydrazine in 1.74 mL of dry DMF under anhydrous N2was added 43.1 mg (0.168 mmol, 3 eq) of DSC. This solution was stirred for 15 minutes, then brought to room temperature and reacted for an additional hour. Following this reaction, the contents of this mixture were transferred to the swollen resin and to the resulting solution was added 58.6 μL (0.337 mmol, 6 eq) of DIPEA followed by capping of the vessel and light shaking for 18 hours. The following day the resin was washed three times with DMF and Fmoc deprotection as previously described. The coupling of Fmoc-His(Trt)-OH was performed followed by deprotection, drying, and purification protocols. Molecular Simulation Methods Partial charges derivation

[0159] Partial charges for Aib, AzaA, AzaG and AzaP were derived using the R.E.D. webserver. Two conformations were prepared: For Aib, one near the αR-helix and the other close to the extended conformation; for AzaA, AzaG and AzaP, one in proximity to the β-turn - 53 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 (with φ, ψ values being –90° and 0°, respectively)and the other near the extended conformation. In both conformations, the residue was capped by an acetyl group at the N-terminus and an N- methyl group at the C-terminus. Atomic partial charges were generated in accordance with the Amber99SB force field. The “RESP-A1” charge model at the HF / 6-31G* theory level was used to compute the charge set. The surface options in molecular electrostatic potential calculation were set to IOp(6 / 33=2,6 / 41=4,6 / 42=6). During charge fitting, the charges of atoms belonging to the cap residues were restrained to the values in the Amber99SB force field. For Aib, AzaA, and AzaG, the partial charges for N, H, C, and O were restrained to –0.4157 e, 0.2719 e, 0.5973 e, and –0.5679 e, respectively. The scaling factor for 1,4 electrostatic energy was set to 0.8333, and the scaling factor for 1,4 van der Waals energy was set to 0.5000. The cap residues were removed from the molecule to create the designated molecular fragment. The derived partial charges for Aib, AzaA, AzaG and AzaP are shown in Figure 7. Additional bonded parameters

[0160] Bonded parameters absent in the Amber99SB force field library were adopted from the GAFF2 force field. The parameters for the improper dihedral “CT-N-N-C” were not located in GAFF2 either, leading us to utilize the most commonly used parameters for this particular improper dihedral. Table S1 shows the additional parameters we used in the simulations. Bias-exchange metadynamics (BE-META) simulations

[0161] Ac-Xaa-NMe (Xaa being one amino acid from the set [Ala, Aib, AzaA, AzaG, AzaP], Ac denoting the acetyl group, and NMe denoting the N-methyl group) and His-Xaa-Glu-NMe, (N-terminus of His carries a positive charge as –NH3+; His and Glu are employed in accordance with the sequence of GLP-1) were subjected to simulation using the bias-exchange metadynamics (BE-META) method within the Amber99SB force field along with the TIP3P water model. The BE-META simulations were conducted using GROMACS 2018.6 patched with the PLUMED 2.5.1 plugin.

[0162] Two independent simulations, initiated with distinct initial structures (S1 and S2) for each peptide, were executed to assess the convergence of simulation outcomes. After constructing the two initial conformations, each structure underwent initial energy minimization in a vacuum using the steepest descent algorithm. Subsequently, solvation was achieved by placing the structure in a pre-equilibrated box of water molecules, maintaining a minimum distance of 1.0 nm between the peptide and the box walls. The energy minimization of each solvated system followed, employing the steepest descent algorithm and a four-step equilibration process. The initial two equilibration steps involved a 50 ps isothermal-isochoric - 54 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 (NVT) simulation, followed by a 50 ps isothermal-isobaric (NPT) simulation to allow solvent molecules to equilibrate. During both simulations, the heavy atoms of the peptide were restrained by a harmonic potential with a force constant of 1000 kJ⋅mol–1⋅nm–2. Two subsequent steps without restraints, comprising a 100 ps NVT simulation and a 100 ps NPT simulation, were conducted to equilibrate the entire system. The temperature was maintained at 300 K in all NVT and NPT simulations, employing the v-rescale thermostat on both the peptide and solvent coupling groups with a time coupling constant of 0.1 ps. A pressure of 1 bar was applied in all NPT simulations, utilizing the Parrinello-Rahman barostat with a time coupling constant of 2.0 ps and an isothermal compressibility of 4.5 × 10−5bar−1. During equilibrations, the LINCS constraint algorithm was implemented for all bonds. Periodic boundary conditions were enforced in all directions of the unit cell. A cutoff of 1.0 nm was set for both Lennard-Jones and electrostatic interactions. Electrostatic interaction beyond the cutoff distance was treated using the particle mesh Ewald approach, with a Fourier spacing of 0.12 nm and cubic interpolation. To account for the 1.0 nm cutoff of Lennard-Jones interactions, a long-range dispersion correction for energy and pressure was applied. The leapfrog algorithm was employed for integrating dynamic equations, with a time step of 2 fs.

[0163] BE-META production simulations were conducted within the NPT ensemble. The MD parameters employed for production simulations were the same as those outlined in NPT equilibrations, with the distinction that the LINCS constraint algorithm was applied to bonds involving hydrogen atoms. For each dipeptide, a single biased replica was employed biasingthe 2D collective variable (CV) ( , ) of the amino acid. In the case of His-Xaa-Glu-NMe, fourbiased replicas were employed biasing the 2D CVs ( 2, 2), ( 3, 3), ( 1, 2), and ( 2, 3).Gaussian hills, characterized by a height of 0.1 kJ mol−1and a width of 0.31416 rad, were introduced every 4 ps. Additionally, five neutral replicas (i.e., replicas with no bias) were incorporated to acquire an unbiased structural ensemble for subsequent analysis. Exchange attempts between different replicas were made every 5 ps. During the simulation of the His- AzaP-Glu-NMe, we noted the formation of cis peptide bonds between His and AzaP. Consequently, conformations containing cis peptide bonds were excluded from the subsequent analysis. Principal component analysis

[0164] To evaluate the convergence of the two distinct sets of simulations originating from initial structures S1 and S2, dihedral principal component analysis (dPCA) was initiallyconducted on the backbone ( , ) dihedrals. In the case of dipeptides, the ( , ) of the amino- 55 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425acid were utilized, while for His-Xaa-Glu-NMe, ( 1, 2, 2, 3, 3) were employed. The last50 ns trajectories from the neutral replicas were extracted for analysis. dPCA involved frames from both S1 and S2 simulations. After obtaining common principal components (PCs), frames from S1 and S2 simulations were individually projected into the 3D space defined by the top three PCs. Subsequently, a 3D density distribution was calculated for each simulation (S1 and S2). To assess convergence, specifically the similarity between the S1 and S2 density distributions, the normalized integrated product (NIP) was computed. The NIP value ranges from 0 (indicating no overlap) to 1.0 (indicating perfect similarity), with simulations having NIP values ≥0.9 considered as converged. Upon confirming convergence, additional structural analysis was undertaken, utilizing trajectories from the last 50 ns of the neutral replicas in the S1 simulation for each peptide. Conventional MD simulations

[0165] The conformation of GLP-1 and its receptor was extracted from chain P and R of the PDB entry 6X18. We employed the Chimera25+Modeller software to generate the conformation of non-terminal loops with missing 3D coordinates in chain R. The missing loop, comprising the initial 28 amino acids in the N-terminal region of chain R, was considered flexible and had minimal impact on the region of interest. Thus, it was excluded from the simulation. A total of 5 models were created, and the best-scoring model served as the initial structure for MD simulations. The conformations of variants GLP-1A8Aib, GLP-1A8AzaA, GLP- 1A8AzaG,receptor were prepared using the Maestro software of the Schrödinger suites based on the wild-type conformation.

[0166] Five systems, namely GLP-1, GLP-1A8Aib, GLP-1A8AzaA, GLP-1A8AzaG, and GLP-1A8AzaPbound to the receptor, underwent simulation in the Amber99sb force field with the TIP3P water model using GROMACS 2018.6. During the simulation, only the region within 8 Å of residue 8 of GLP-1 was relaxed, while the heavy atoms of the protein outside this region were restrained by a harmonic potential with a force constant of 1,000 kJ mol–1nm–2. The initial structure underwent energy minimization in a vacuum and was then placed in the center of a cubic box containing pre-equilibrated water molecules. The minimum distance between the atoms of the complex and the box walls was 5 Å. The solvated system underwent further energy minimization to eliminate any unfavorable contacts. Subsequently, a 100-ps NVT simulation at 300 K was conducted. From this step onward, 5 parallel runs were initiated for each system, each starting with different initial velocities. Following the NVT simulation, a subsequent 300- ns NPT simulation at 300 K and 1 bar was performed in each run. The v-rescale thermostat - 56 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 separately regulated the temperature of the protein and solvent, employing coupling time constants of 0.1 ps. Pressure control was achieved using the Parrinello-Rahman barostat, with a coupling time constant of 2.0 ps and an isothermal compressibility of 4.5 × 10−5bar-1. Dynamics evolution utilized the leapfrog algorithm with a time step of 2 fs. Hydrogen-involved bonds were constrained through the LINCS algorithm. Electrostatic and van der Waals interactions were truncated at 1.0 nm, and electrostatics were treated using the particle mesh Ewald summation with a Fourier spacing of 0.12 nm and an order of 4. To accommodate the 1.0-nm cutoff of Lennard-Jones interactions, a long-range dispersion correction was applied to correct energy and pressure. Trajectories between 100 ns and 300 ns were employed for subsequent analysis. INCORPORATION BY REFERENCE

[0167] All U.S. and PCT patent publications and U.S. patents mentioned herein are hereby incorporated by reference in their entirety as if each individual patent publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. OTHER EMBODIMENTS

[0168] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. - 57 - FH13045379.3

Claims

ATTORNEY DOCKET NO.: TUV-19425 We claim:

1. A peptide comprising an aza-amino acid residue (Aza); wherein the peptide is selected from the group consisting of Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP- 2), Glucose-dependent insulinotropic polypeptide (GIP), Glucagon (GCG), Growth hormone releasing hormone (GHRH), FGF2, FGF21, Tyr-melanostatin, Endomorphin-2, Enterostatin, - casmorphin, Trypsinogen pro-peptide, Corticotropin-like intermediate lobe peptide, Gastrin- release peptide, Aprotinin, GCP-2, MDC, MCP-2, Eotaxin, IP-10, Insulin-like growth factor-1,Interleukin-2, Interleukin-1 , -Microglobulin, PHM, GRH-(1-29), GRH-(1-44),Oxyntomodulin, Secretin (SCT), Vasoactive Intestinal Peptide (VIP), Neuropeptide Y (NPY), Met-enkephalin, Pancreatic Polypeptide (PP), Peptide YY (PYY 1-36 and 3-36 form), Exenatide, Substance P, Parathyroid hormone (hPTH 1-34), BNP, Liraglutide, Tirzepatide, Semaglutide, Retratrutide, Taspoglutide, Lixisenatide, Albiglutide, Dulaglutide, Stromal cell-derived factor 1( and ), BI-456906 MAR423, CCL5, CCL11, Pituitary Adenylate Cyclase-ActivatingPolypeptide, NNC0090-2746, and Ecnoglutide peptides having at least 85% sequence identity to any one of them; and an amino acid residue of the peptide is replaced with the aza-amino acid residue (Aza).- 58 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 59 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 60 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 61 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 62 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 63 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 64 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 65 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 66 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 67 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 68 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 69 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 70 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425- 71 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 3. The peptide of claim 1, wherein the aza-amino acid residue (Aza) is selected from aza- glycine (AzaG), aza-alanine (AzaA), aza-proline (AzaP) and aza-glutamic acid (AzaE).

4. The peptide of any one of claims 1-3, wherein a 2-aminoisobutyric acid (X), -methyl-L- Leucine (meL), alanine (A), glutamic acid (E), glycine (G), leucine (L), lysine (K), phenylalanine (F), isoleucine (I), aspartic acid (D), arginine (R), or proline (P) is replaced with the aza-amino acid residue (Aza).

5. The peptide of any one of claims 1-4, wherein the peptide is selected from the group consisting of: H*A*E*G*TFTSDVSSYL*EG*QAAK*EF*I*AWL*VK*G*R-NH2, H*A*E*G*TFTSDVSSYL*EG*QAAK*EF*I*AWL*VK*G*RG-OH, H*A*D*G*SFSDEMNTI*L*DNL*AARDF*INWL*IQTK*ITD, Y*A*E*G*TFISDYSI*AMDK*IHQQDFVNWL*L*AQK*G*K*K*NDWK*HNI*TQ, HS*Q*G*TFTSDYSK*YL*DSR*R*AQDFVQWL*MNT, YA*DAI*FTNSYR*K*VL*G*QL*SAR*K*L*L*QDIMSR*QQG*ESNQER*G*AR*AR*L, HS*Q*G*TFTSDYSK*YL*DSR*R*AQDFVQWL*MNTK*R*NR*NNI*A, HS*D*G*TFTSEL*SR*L*R*DSAR*L*NR*L*L*NGL*V, HD*A*VFTDNYTR*L*R*K*NMAVK*K*YL*NSI*L*N, YP*SK*P*DNP*G*EDAP*AEDL*AR*YYSAL*R*HYI*NL*I*TR*QR*Y, Y*G*G*F*M, AP*L*EP*VYP*G*DNATP*EQMAQYAADL*R*R*YI*NML*TR*P*R*Y, YP*IKP*EAP*G*EDASPEEL*NR*YYASL*R*HYL*NL*VTR*QR*Y–NH2, H*G*E*G*TFTSDLSK*QME*E*E*AVR*LF*IEW*L*K*NG*G*P*SSG*AP*P*P*S–NH2, R*P*K*P*QQFFG*L*M, YP*SK*P*DNP*G*EDAP*AEDMAR*YYSAL*R*HYI*NL*I*TR*QR*Y, HA*E*G*TFTSDVSSYL*EG*QAA(X)*EF*I*AWL*VR*G*R*G wherein X attached to the - amine of lysine is: - 72 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425, YAib*E*G*TFTSDYSI*Aib*L*DKI*AQ(X)AFVQWL*I*A G*G*P*SSG*AP*P*P*S–NH2where X attached to the -amine of lysine is:HAib*E*G*TFTSDVSSYL*EG*QAA(X)EFI*AWL*VR*G*R*G wherein X attached to the - amine of lysine is:, YAib*Q*G*TFTSDYSImeL*LDK(X)AQAib*AFIEYL*L*G*G*P*SSG*AP*P*P*S–NH2where X attached to the -amine of lysine is:HAib*E*G*T*FTSDVSSYL*EGQAAK*EFIAWL*VK*Aib*R–NH2, HG*E*G*TFTSDL*SK*QMEEEAVR*L*FI*EWL*K*NGGP*SSGAPPSK*K*K*K*K*K*– NH2, - 73 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 H*S*Q*G*TFTSDLSK*Y*L*E*E*E*AVR*E*F*IAW*LKNGGPSR*HY*LNLVTR*QR*Y*- NH2, H*Aib*H*G*TFTSDY*SIY*LE(X)Y*AAib*EF*VQW*LLE*GGPSSGAPPPS-NH2where X attached to the -amine of lysine is:H*Aib*E*G*TFTSDV*SSY*LEG*QALR*HY*INW*LTR*QR*Y*-NH2, and H*Aib*Q*G*TFTSD(X)SK*Y*LD*E*RAA*Q*DF*VQW*LLD*G*G*P*SSG*AP*P*P*S– NH2where X attached to the -amine of lysine is:or a peptide having at least 85% sequence identity to any one of them; wherein at least one amino acid residue marked with * is replaced with the aza-amino acid.

6. The peptide of any one of claims 1-4, wherein the peptide has at least 90% sequence identity to GLP-1, GIP or semaglutide.

7. The peptide of claim 6, wherein the peptide has at least 95% sequence identity to GLP-1, GIP or semaglutide.

8. The peptide of claim 7, wherein the peptide is GLP-1, GIP or semaglutide.

9. The peptide of any one of claims 1-4, wherein the peptide is selected from A8Aza GLP- - 74 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 1, E9Aza GLP-1 and G10Aza GLP-1.

10. The peptide of any one of claims 1-4, wherein the peptide is selected from A8AzaA GLP- 1, A8AzaG GLP-1, A8AzaP GLP-1, E9AzaE GLP-1 and G10AzaG GLP-1.

11. The peptide of any one of claims 1-4, wherein the peptide is A2Aza GIP.

12. The peptide of claim 11, wherein the peptide is A2AzaA GIP.

13. The peptide of any one of claims 1-4, wherein the peptide is X2Aza semaglutide.

14. The peptide of claim 13, wherein the peptide is X2AzaA semaglutide.

15. The peptide of any one of claims 1-4, wherein the peptide is X2Aza NNC0090-2746.

16. The peptide of claim 15, wherein the peptide is X2AzaA NNC0090-2746.

17. A method of: a) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non- insulin dependent diabetes, and / or obesity; b) reducing body weight and / or food intake, and / or inducing satiety; c) treating or preventing Alzheimer's disease, nonalcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated liver disease (MASLD), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; d) treating chronic kidney disease, traumatic brain injury, alcohol addiction, and / or substance addiction; e) treating or preventing emesis; f) treating or preventing effects of aging; g) support of islet transplant survival in Type 1 diabetes; and h) treating or preventing arthritis and related diseases, including but not limited to: Osteoarthritis, Rheumatoid arthritis, Gout, Ankylosing spondylitis, Psoriatic arthritis, Juvenile arthritis, - 75 - FH13045379.3ATTORNEY DOCKET NO.: TUV-19425 Fibromyalgia, Infectious arthritis, Spondyloarthritis, Sjogren's syndrome, Scleroderma, and Polymyalgia rheumatica. comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

18. A method of treating cardiovascular disease and / or hypertension, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

19. A method of treating a neurodegenerative disease (e.g., Alzheimer’s, Parkinsons, other forms of dementia, traumatic brain injury), comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

20. A method of treating alcohol use disorder, substance use disorder or smoking addiction, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

21. A method of treating inflammation, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

22. A method of treating diabetes, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

23. A method of treating obesity, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-16.

24. The method of any one of claims 17-23, wherein the subject was not responsive to or did not adequately tolerate a previous course of therapy with an incretin mono- or dual- or tri- agonist, wherein the azapeptide replaces non-tolerated or ineffective incretin-based mono- or dual agonist therapy. - 76 - FH13045379.3

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