Long-acting glucagon-like polypeptide-1 (GLP-1) receptor agonists and methods of use

Abstract

The present invention relates to isolated polypeptides that are long-acting analogs of human GLP-1. The disclosed GLP-1 receptor agonist polypeptides have a longer (i.e., "long-acting") elimination half-life (t) compared to endogenous GLP-1 and known synthetic GLP-1 receptor agonist polypeptides. 1 / 2 ), as well as beneficial physicochemical properties such as improved solubility and thermal stability. The present invention also relates to methods of using and making the presently disclosed GLP-1 receptor agonist polypeptides in a variety of therapeutic indications. The disclosed GLP-1 receptor agonist polypeptides are particularly useful in methods of treating metabolic diseases or disorders such as type 2 diabetes, methods of treating obesity, and methods of effecting weight loss, as well as in methods of treating non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 126,736, filed December 17, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to compounds that are glucagon-like polypeptide-1 (GLP-1) receptor agonists and methods for making the same. The invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various disorders. [Background technology]

[0003] Endogenous GLP-1 is released from the intestine in response to nutrient ingestion. Following food ingestion and digestion, carbohydrates and fats appear in the intestinal lumen, stimulating the so-called incretin effect, i.e., the release of incretins such as GLP-1 from intestinal L-cells. Once released, GLP-1 targets the pancreas, where it promotes insulin secretion in a "glucose-dependent" manner. In other words, this GLP-1-mediated effect on insulin persists when blood glucose levels are high, but safely dissipates when blood glucose levels fall. Thus, GLP-1 activity self-regulates, reducing the risk of hypoglycemia (a condition in which blood glucose levels drop to dangerous levels). The elimination half-life (t 1 / 2 ) is short, less than 5 minutes, making this endogenous peptide unsuitably short-lived for use as a therapeutic agent.

[0004] Synthetic analogs of GLP-1 have been designed to have longer half-lives and to similarly enhance insulin secretion in a glucose-dependent manner, like endogenous GLP-1, for use in treating type 2 diabetes and for weight loss.

[0005] Certain GLP-1 receptor agonists, including Bydureon® (exenatide), marketed by AstraZeneca of Cambridge, UK; Trulicity® (dulaglutide), marketed by Eli Lilly and Co., of Indianapolis, IN, USA; and Victoza® (liraglutide), Ozempic® (injectable semaglutide), and Rybelsus® (oral semaglutide), marketed by Novo Nordisk A / S of Bagsvaerd, Denmark, have each been approved by numerous regulatory authorities, including the United States Food and Drug Administration (USFDA) and the European Medicines Agency (EMA), for the treatment of patients with type 2 diabetes. These commercially available GLP-1 receptor agonists were developed and formulated for injection and / or oral administration to patients. However, patient adherence to injectable and oral treatments for type 2 diabetes is notoriously poor, preventing many patients from realizing the full and sustained therapeutic potential of GLP-1 receptor agonists. Many patients skip or discontinue regular self-administration of prescribed injectable and oral GLP-1 receptor agonists, and therefore fail to adequately treat and control their type 2 diabetes condition.

[0006] Thus, there remains a need for improved GLP-1 receptor agonist polypeptides that have high potency at the GLP-1 receptor, have excellent physicochemical properties, and are tailored for delivery to patients via more reliable modes of administration than injection and oral administration therapies. Summary of the Invention

[0007] It has now been found that compounds of the present invention, and pharmaceutically acceptable compositions thereof, are effective as GLP-1 receptor agonists. Such compounds have the following general formula: Amino acid sequence of SEQ ID NO: 200: HX2X3GTX6X7X8X9X10 SX 12 X 13 X 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH2) (SEQ ID NO: 200); where each variable is as defined and explained herein.

[0008] The compounds of the present invention have a long elimination half-life (t 1 / 2 ), and are therefore described herein as "long-acting" GLP-1 receptor agonists.

[0009] The compounds of the present invention and their pharmaceutically acceptable compositions are useful for treating various diseases, disorders, or conditions associated with the GLP-1 receptor. Such diseases, disorders, or conditions include metabolic diseases or disorders such as type 2 diabetes, obesity, and the need to achieve weight loss. In certain embodiments, the present invention also relates to methods for treating nonalcoholic fatty liver disease (NAFLD) and / or nonalcoholic steatohepatitis (NASH). [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows long-term weight loss (day 27) in Long-Evans (LE) diet-induced obese (DIO) rats treated with exemplary long-acting GLP-1 analog polypeptides. [Figure 2A] Figure 1 shows a comparison of in vivo weight loss efficacy in DIO rats for compound A120 vs. semaglutide (% of baseline vs. vehicle over 28 days, n=6 / dose). Ozempic® (semaglutide, Novo Nordisk A / S, Bagsvaerd, Denmark) is approved by the US FDA for the treatment of type 2 diabetes. [Figure 2B] Figure 1 shows a comparison of the in vivo type 2 diabetes efficacy (i.e., HbA1c reduction) of compound A120 versus semaglutide in DIO rats (Δ-HbA1c versus vehicle over 28 days, n=9 / dose). Antidiabetic efficacy was compared in the ZDF rat model of type 2 diabetes. [Figure 3] FIG. 1 shows a cross-sectional view of a representative osmotic minipump for drug delivery. [Figure 4] 1 shows the potency shift of a conjugated GLP-1 receptor agonist upon human serum albumin (HSA) binding. [Figure 5] 1 shows the changes in plasma concentrations of compound A120 in rats and monkeys after intravenous and subcutaneous administration. [Figure 6] Dose and concentration response of weight loss in male Long-Evans (LE) diet-induced obese (DIO) rats treated with Compound A120 for 27 days is shown. 18-week-old male LE DIO rats (14 weeks on a high-fat diet) received six doses (SC; every other day) of Compound A120 ranging from 1 to 300 mcg / kg / day (n=6 animals / dose). Shaded and open circles represent repeat doses of semaglutide (3, 30, 300 mcg / kg / day) from two separate studies. [Figure 7] Figure 1 shows semaglutide and A120-induced weight loss in male LEDIO rats over 27 days. [Figure 8] Figure 1 shows semaglutide- and A120-induced changes in body composition in male LE DIO rats, highlighting the fact that the changes in body composition were primarily due to a loss of fat mass (body composition was measured using quantitative magnetic resonance on day 26). [Figure 9] Figure 1 shows the mean daily food intake inhibition and cumulative food intake of mice receiving various doses of compound A120 or semaglutide. [Figure 10] Figure 1 shows weight loss in male Zucker Diabetic Fatty (ZDF) rats administered compound A120 or semaglutide (SC injection; every other day). [Figure 11]Figure 1 shows semaglutide- and A120-induced changes in body composition in male ZDF rats, highlighting the fact that the changes in body composition were primarily due to a loss of fat mass (body composition was measured on day 25). DETAILED DESCRIPTION OF THE INVENTION

[0011] General Description of Certain Embodiments of the Invention The compounds of the present invention and pharmaceutical compositions thereof are useful as agonists of the GLP-1 receptor, particularly as agonists of the human GLP-1 receptor. The present invention also relates to methods of making and using such compounds, i.e., GLP-1 receptor agonist polypeptides. The compounds of the present invention are long-acting GLP-1 receptor agonists. These GLP-1 receptor agonist polypeptides are particularly useful in methods of treating metabolic diseases or disorders, such as type 2 diabetes and obesity, and in methods of weight loss. In certain embodiments, the present invention also relates to methods of treating nonalcoholic fatty liver disease (NAFLD) and / or nonalcoholic steatohepatitis (NASH).

[0012] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 200: HX2X3GTX6X7X8X9X 10 SX 12 X 13 X 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 SGAPPPS-(OH / NH) (SEQ ID NO: 200): or a pharmaceutically acceptable salt thereof, wherein: X2 is A, 2-aminoisobutyric acid (Aib), or G; X3 is E or N-methylGlu; X6 is F or Y; X7 is S or T; X8 is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X9 is D or E; X 10 is I, L, N-methylLeu, or V; X 12 is E, K, Q, or S; X 13 is Aib, E, K, Q, S, W, or Y; X 14 is E, R, I, K, L, M, Q, or Y; X 16 is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn); X 17 is E, K, or Q; X 18 is A, K, S, or Y; X 19 is A, K, or V; X 20 is E, K, or R; X 21 is Aib, E, H, K, L, Q, or Y; X 24 is A, Aib, E, K, Q, S, or Y; X 28 is D, E, K, N, Q, S, or Y; and X 32 is Dap, H, K, R, or S; where X 16 is Dab, Dap, K, k, or Orn, it is covalently linked, optionally via a spacer, to a lipophilic substituent; where X 16 If E, then X 14 , X 17 , X 18 , X 19 , X 20 , or X 21at least one of is selected to be lysine, wherein the lysine residue is covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the isolated polypeptide optionally further comprises two amino acid residues having side chains covalently linked to form a cross-linking moiety).

[0013] In some embodiments, the bridging moiety is a lactam bridging moiety. For example, in some embodiments, the isolated polypeptide optionally further comprises two amino acid residues, one of which is lysine and the other of which is glutamic acid, wherein the amino-containing side chain of the lysine and the carboxy-containing side chain of the glutamic acid are covalently linked by loss of water to form a lactam bridging moiety. In some embodiments, the lactam bridging moiety is each X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 21 and X 28 in place; X respectively 28 and X 21 in place; X respectively 20 and X 24 in place; X respectively 24 and X 20 or X respectively 12 and X 16 It is formed via a covalent bond between a lysine side chain and a glutamic acid side chain at position 1.

[0014] In one embodiment, the isolated polypeptide comprises a K covalently linked to a lipophilic substituent, optionally via a spacer. 12 or K 27 and not an analog of exenatide, namely HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 300).

[0015] In some embodiments, X2 is Aib.

[0016] In some embodiments, X 24 If S, then X 10 is V.

[0017] In some embodiments, X 14 is Y.

[0018] In some embodiments, X 14 is M and X 16 is K covalently attached, optionally via a spacer, to a lipophilic substituent.

[0019] In some embodiments, X 16 is a lysine covalently linked to a lipophilic substituent, optionally via a spacer.

[0020] In some embodiments, X 16 If E, then X 21 is K or E. In some embodiments, X 16 If E, then X 21 is K. In some embodiments, X 16 If E, then X 21 is E.

[0021] In some embodiments, the peptide comprises X 20 and X 24 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0022] definition It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "solvent" includes a combination of two or more such solvents, reference to a "peptide" includes one or more peptides or mixtures of peptides, reference to a "drug" includes one or more drugs, reference to an "osmotic delivery device" includes one or more osmotic delivery devices, etc. Unless specifically stated or apparent from the context, the term "or," as used herein, is understood to be inclusive and encompasses both "or" and "and."

[0023] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within normal tolerances in the art, for example, within two standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are intended to be modified by the term "about."

[0024] Unless specifically stated or clear from the context, the term "substantially," as used herein, is understood to mean within a narrow range of variation or otherwise within normal tolerances in the art. Substantially can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

[0026] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0027] The terms "drug," "therapeutic agent," and "beneficial agent" are used interchangeably and refer to any therapeutically active substance that is delivered to a subject to produce a desired beneficial effect. In one embodiment of the invention, the drug is a polypeptide. In another embodiment of the invention, the drug is a small molecule, e.g., a hormone such as an androgen or estrogen. The devices and methods of the present invention are well suited for the delivery of proteins, small molecules, and combinations thereof.

[0028] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and typically refer to molecules comprising a chain of two or more amino acids (e.g., most typically L-amino acids, but also including, e.g., D-amino acids, modified amino acids, amino acid analogs, and amino acid mimetics).

[0029] In some embodiments, naturally occurring L-amino acids are represented by the conventional three letter or single capital letter amino acid symbols in Table 1. In other embodiments, both naturally occurring L-amino acids and D-amino acids are represented by the conventional three letter or single capital letter amino acid symbols in Table 1. In still other embodiments, D-amino acids correspond to the lower case single letter amino acid symbols corresponding to the single letter symbols in Table 2, i.e., a, l, m, f, w, k, q, e, s, p, v, i, c, y, h, r, n, d, and t. [Table 1] [Table 2]

[0030] Peptides may be naturally occurring, synthetically produced, or recombinantly expressed. Peptides may also contain additional groups that modify the amino acid chain, such as functional groups added by post-translational modifications. Examples of post-translational modifications include, but are not limited to, acetylation, alkylation (including methylation), biotinylation, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, phosphopantetheinylation, phosphorylation, selenation, and C-terminal amidation. The term peptide also includes peptides containing modifications of the amino and / or carboxy termini. Modifications of the terminal amino group include, but are not limited to, desamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., where lower alkyl is a C1-C4 alkyl). The term peptide also includes modifications of amino acids between the amino and carboxy termini, such as, but not limited to, those described above. In one embodiment, the peptide may be modified by the addition of a small molecule drug.

[0031] The terminal amino acid at one end of a peptide chain typically has a free amino group (i.e., the amino terminus). The terminal amino acid at the other end of the chain typically has a free carboxyl group (i.e., the carboxy terminus). Typically, the amino acids that make up a peptide are numbered sequentially, starting with the amino terminus and increasing toward the carboxy terminus of the peptide.

[0032] As used herein, the phrase "amino acid residue" refers to an amino acid that is incorporated into a peptide by an amide bond or an amide bond mimetic.

[0033] As used herein, the term "insulinotropic" typically refers to the ability of a compound, e.g., a peptide, to stimulate or affect the production and / or activity of insulin (e.g., insulinotropic hormones). Such compounds typically stimulate or otherwise affect the secretion or biosynthesis of insulin in a subject. Thus, an "insulinotropic peptide" is an amino acid-containing molecule that can stimulate or otherwise affect the secretion or biosynthesis of insulin.

[0034] The term "insulinotropic peptide" as used herein includes, but is not limited to, glucagon-like peptide 1 (GLP-1), and derivatives and analogs thereof, GLP-1 receptor agonists, such as exenatide, having the amino acid sequence of SEQ ID NO: 300, and derivatives and analogs thereof.

[0035] The term "acylated," as used herein, with respect to the disclosed polypeptides, means that the disclosed polypeptides are substituted with one or more lipophilic substituents, each optionally via a spacer, where "lipophilic substituent" and "spacer" are defined herein. Certain lipophilic substituents, each optionally via a spacer, can bind to albumin, conferring affinity for albumin to the resulting acylated polypeptide. The degree to which lipophilic substituents, each optionally via a spacer, confer affinity for albumin to the resulting acylated polypeptide varies and depends on many factors, including the identity of the lipophilic substituent, the optional spacer, the polypeptide, and the covalent attachment site to the polypeptide.

[0036] The terms "linear" or "linear polypeptide" refer to "non-acylated" polypeptides, in other words, the disclosed GLP-1 receptor agonist polypeptides that do not have a lipophilic substituent, each optionally mediated by a spacer, where "lipophilic substituent" and "spacer" are defined herein.

[0037] As used herein, the term "conjugate" or conjugated polypeptide refers to an "acylated" polypeptide, in other words, the disclosed GLP-1 receptor agonist polypeptides bearing one or more lipophilic substituents, each optionally via a spacer.

[0038] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed polypeptides in which the parent polypeptide has been modified by converting any acidic or basic moieties present therein into their salt forms. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), the entire contents of which are incorporated herein by reference.

[0039] The term "vehicle" as used herein refers to a medium used to carry a compound, such as a drug or a particle containing a drug. The vehicle of the present invention typically includes components such as a polymer and a solvent. The suspension vehicle of the present invention typically includes a solvent and a polymer used to prepare a suspension formulation further comprising a drug particle formulation.

[0040] As used herein, the phrase "phase separation" refers to the formation of multiple phases (e.g., a liquid phase and a gel phase) in a suspension vehicle, such as when the suspension vehicle contacts an aqueous environment. In some embodiments of the present invention, the suspension vehicle is formulated to exhibit phase separation upon contact with an aqueous environment containing less than approximately 10% water.

[0041] As used herein, the phrase "single-phase" refers to a homogeneous system of solid, semi-solid, or liquid that is physically and chemically uniform throughout.

[0042] As used herein, the term "dispersion" refers to dissolving, dispersing, suspending, or otherwise distributing a compound, eg, a drug particle formulation, in a suspension vehicle.

[0043] As used herein, the phrase "chemically stable" refers to the formation of an acceptable percentage of degradation products in the formulation over a defined period of time generated by chemical pathways such as deamidation (usually by hydrolysis), aggregation, or oxidation.

[0044] As used herein, the phrase "physically stable" refers to a formulation in which an acceptable percentage of aggregates (e.g., dimers and other high molecular weight products) are formed. Furthermore, a physically stable formulation does not change its physical state, for example, from liquid to solid or from amorphous to crystalline form.

[0045] The term "viscosity" as used herein typically refers to a value determined from the ratio of shear stress to shear rate, essentially as follows (see, e.g., Considine, D. M. & Considine, G. D., Encyclopedia of Chemistry, 4th Edition, Van Nostrand, Reinhold, NY, 1984): F / A=μ*V / L (Equation 1) Where F / A = shear stress (force per unit area), μ = a proportionality constant (viscosity), and V / L = velocity per layer thickness (shear rate).

[0046] From this relationship, the ratio of shear stress to shear rate defines viscosity. Measurements of shear stress and shear rate are typically determined using parallel plate rheometry, performed under selected conditions (e.g., a temperature of about 37°C). Other methods for determining viscosity include measuring kinematic viscosity using a viscometer, such as a Cannon-Fenske viscometer, a Cannon-Fenske Ubbelohde viscometer for opaque solutions, or an Ostwald viscometer. Generally, the suspension vehicle of the present invention has a viscosity that prevents the particle formulation suspended therein from settling during storage and is sufficient for use in delivery methods, such as implantable drug delivery devices.

[0047] As used herein, the term "non-aqueous" refers to a total water content, e.g., of a suspension formulation, typically about 10% by weight or less, e.g., about 7% by weight or less, about 5% by weight or less, and / or less than about 4% by weight. Also, the particle formulations of the present invention contain less than about 10% by weight, e.g., less than about 5% by weight, of residual water.

[0048] The term "subject" as used herein refers to any member of the subphylum Chordata, including, but not limited to, humans and other primates, including non-human primates such as rhesus monkeys and other monkey species, and chimpanzees and other ape species; livestock, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs; and birds, including domestic, wild, and game birds, such as chickens, turkeys, and other poultry, ducks, and geese. The term does not denote a particular age or sex. Thus, it is intended to encompass both adults and newborns.

[0049] As used herein, the terms "treat," "treatment," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered even in the absence of symptoms. For example, treatment may be administered to susceptible individuals (e.g., in light of a history of symptoms and / or genetic or other susceptibility factors) prior to the onset of symptoms. Treatment may be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0050] The term "osmotic delivery device" as used herein typically refers to a device used to deliver a drug (e.g., a disclosed GLP-1 receptor agonist polypeptide) to a subject. The device includes, for example, a reservoir (e.g., made from a titanium alloy) having a lumen containing a suspension formulation including a drug (e.g., a disclosed GLP-1 receptor agonist polypeptide) and an osmotic agent formulation. A piston assembly located within the lumen separates the suspension formulation from the osmotic agent formulation. A semipermeable membrane is located at a first distal end of the reservoir adjacent to the osmotic agent formulation, and a diffusion moderator (defining a delivery orifice through which the suspension formulation exits the device) is located at a second distal end of the reservoir adjacent to the suspension formulation. Typically, the osmotic delivery device is implanted within a subject, for example, subdermally or subcutaneously (e.g., in the medial, lateral, or posterior upper arm and abdominal region). An exemplary osmotic delivery device is the DUROS® (ALZA Corporation, Mountain View, Calif.) delivery device. Examples of terms synonymous with "osmotic delivery device" include, but are not limited to, "osmotic drug delivery device," "osmotic drug delivery system," "osmotic device," "osmotic delivery device," "osmotic delivery system," "osmotic pump," "implantable drug delivery device," "drug delivery system," "drug delivery device," "implantable osmotic pump," "implantable drug delivery system," and "implantable delivery system." Other terms for "osmotic delivery device" are known in the art.

[0051] As used herein, the term "continuous delivery" typically refers to the substantially continuous release of a drug from an osmotic delivery device into tissues near the implantation site, e.g., subdermal and subcutaneous tissues. For example, osmotic delivery devices release drugs at an essentially predetermined rate based on the principles of osmosis. Extracellular fluid passes through a semipermeable membrane directly into the osmotic engine of the osmotic delivery device, which expands to drive a piston at a slow and constant rate of movement. The piston movement forces the drug formulation through the orifice of the diffusion moderator. Thus, the release of the drug from the osmotic delivery device occurs at a slow, controlled, and constant rate.

[0052] As used herein, the term "substantially steady-state delivery" typically refers to delivery of a drug at or near a target concentration over a defined period of time, and the amount of drug delivered from an osmotic delivery device is substantially zero-order delivery. Substantially zero-order delivery of an active agent (e.g., a disclosed GLP-1 receptor agonist polypeptide) means that the rate of drug delivered is constant and independent of the drug available in the delivery system; for example, for zero-order delivery, as determined by standard methods (e.g., linear regression), when the rate of drug delivered is graphed against time and a line is fit to the data, the line has a slope of approximately zero.

[0053] As used herein, the phrase "drug half-life" refers to the time it takes for a drug to be eliminated from plasma to half its concentration. Drug half-life is usually measured by monitoring how the drug breaks down when injected or administered intravenously. Drugs are usually detected using, for example, radioimmunoassay (RIA), chromatographic methods, electrochemiluminescence (ECL) assays, enzyme-linked immunosorbent assays (ELISA), or immunoenzyme sandwich assays (IEMA).

[0054] The terms "μg" and "mcg" and "ug" are understood to mean "micrograms." Similarly, the terms "μl" and "uL" are understood to mean "microliters," and the terms "μM" and "uM" are understood to mean "micromoles."

[0055] The term "serum" is intended to mean a blood product in which a substance can be detected. Thus, the term serum includes at least whole blood, serum, and plasma. For example, "the amount of [substance] in the serum of a subject" would encompass "the amount of [substance] in the plasma of a subject."

[0056] Baseline is defined as the last assessment on or the day before the first placement of the osmotic delivery device (containing drug or placebo).

[0057] Endogenous GLP-1, GLP-1 receptors, and certain GLP-1 analogs The phrase "incretin mimetic" as used herein includes, but is not limited to, GLP-1 peptide, GLP-1(7-36), GLP-1 receptor agonists, peptide derivatives of GLP-1, peptide analogs of GLP-1, exenatide, exenatide having the amino acid sequence of exendin-4 (the natural form of exenatide, exenatide-LAR, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, and taspoglutide. Incretin mimetics are also referred to herein as "insulinotropic peptides." Incretin mimetics that target the GLP-1 receptor are also known in the literature as "GLP-1 receptor agonists" or "GLP-1 agonists," and both terms are used interchangeably herein.

[0058] The term "GLP-1" refers to a polypeptide, glucagon-like peptide-1(7-36)amide, a 30-residue peptide hormone released from intestinal L-cells after nutrient consumption. GLP-1 has the amino acid sequence of (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2), SEQ ID NO: 301. GLP-1 is a regulatory peptide that binds to the extracellular domain of the GLP-1 receptor (GLP-1R), a G protein-coupled receptor on β cells, and stimulates the insulin response to nutrients absorbed from the intestine via adenyl cyclase activity and cAMP production [Baggio 2007, "Biology of incretins: GLP-1 and GIP," Gastroenterology, vol. 132(6):2131-57; Holst 2008, "The incretin system and its role in type 2 diabetes mellitus," Mol Cell Endocrinology, vol. 297(1-2):127-36]. The agonistic effects of GLP-1R are diverse.GLP-1 maintains glucose homeostasis by enhancing endogenous glucose-dependent insulin secretion, increasing beta-cell glucose receptivity and sensitivity to GLP-1, suppressing glucagon release, restoring first- and second-phase insulin secretion, slowing gastric emptying, reducing food intake, and increasing satiety [Holst 2008 Mol. Cell Endocrinology; Kjems 2003 “The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects,” Diabetes, vol. 52(2): 380-86; Holst 2013 “Incretin hormones and the satiation signal,” Int J Obes (Lond), vol. 37(9):1161-69; Seufert 2014, “The extra-pancreatic effects of GLP-1 receptor agonists: a focus on the cardiovascular, “Gastrointestinal and central nervous systems,” Diabetes Obes Metab, vol. 16(8): 673-88]. Given the mechanism of action of GLP-1, the risk of hypoglycemia is minimal.

[0059] Glucagon-like peptide-1 (7-36) amide (GLP-1) is a 30-residue peptide hormone released from intestinal L-cells after nutrient consumption. It enhances glucose-induced insulin secretion from pancreatic beta cells, increases insulin expression, inhibits beta cell apoptosis, promotes beta cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety, and increases peripheral glucose disposal. These multiple effects have generated significant interest in discovering long-lasting agonists of the GLP-1 receptor (GLP-1R) for the treatment of type 2 diabetes. The term "exenatide" as used herein includes exenatide, (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2), exenatide having the amino acid sequence of SEQ ID NO: 300, native exendin-4, exenatide peptide, exenatide peptide analogs, and exenatide peptide derivatives.

[0060] Certain disclosed GLP-1 receptor agonist polypeptides, including those in Table 4 below, exhibit one or more of superior solubility, stability, biological activity and specificity, and longer half-life than endogenous GLP-1 and known GLP-1 receptor agonists. Certain disclosed GLP-1 receptor agonist polypeptides have been developed to accommodate less frequent administration than required for currently marketed GLP-1 receptor agonists. Certain disclosed GLP-1 receptor agonist polypeptides have been developed for administration by weekly or monthly injection. Certain disclosed GLP-1 receptor agonist polypeptides have been developed for administration via implantation of a delivery device containing the GLP-1 receptor agonist polypeptide, the delivery device containing a dose of the GLP-1 receptor agonist polypeptide for up to 3 months, 6 months, 9 months, 1 year, 18 months, or 2 years.

[0061] Description of exemplary embodiments In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 210: HX2X3GTX6X7X8X9X 10 SX 12 X13 X 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 SGAPPPS-(OH / NH) (SEQ ID NO: 210): or a pharmaceutically acceptable salt thereof, wherein: X2 is A, 2-aminoisobutyric acid (Aib), or G; X3 is E or N-methylGlu; X6 is F or Y; X7 is S or T; X8 is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X9 is D or E; X 10 is I, L, N-methylLeu, or V; X 12 is E, K, Q, or S; X 13 is Aib, E, K, Q, S, W, or Y; X 14 is E, R, I, K, L, M, Q, or Y; X 16 is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn); X 17 is E, K, or Q; X 18 is A, K, S, or Y; X 19 is A, K, or V; X 20 is E, K, or R; X 21 is Aib, E, H, K, L, Q, or Y; X 24 is A, Aib, E, K, Q, S, or Y; X 28is D, E, K, N, Q, S, or Y; and X 32 is Dap, H, K, R, or S; However, the isolated polypeptide is not exenatide).

[0062] In some embodiments, X2 is Aib.

[0063] In some embodiments, X 24 If S, then X 10 is V.

[0064] In some embodiments, X 14 is Y.

[0065] In some embodiments, X 14 is M and X 16 is K.

[0066] In some embodiments, X 16 is K.

[0067] In some embodiments, X 16 If E, then X 21 is K or E. In some embodiments, X 16 If E, then X 21 is K. In some embodiments, X 16 If E, then X 21 is E.

[0068] In some embodiments, X2 is A. In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0069] As used herein, Aib alternatively refers to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine or 2-methylalanine.

[0070] In some embodiments, X3 is E. In some embodiments, X3 is N-methylGlu.

[0071] In some embodiments, X6 is F. In some embodiments, X6 is Y.

[0072] In some embodiments, X7 is S. In some embodiments, X7 is T.

[0073] In some embodiments, X8 is Dap. In some embodiments, X8 is E. In some embodiments, X8 is K. In some embodiments, X8 is N. In some embodiments, X8 is N-methyl Ser. In some embodiments, X8 is Q. In some embodiments, X8 is S. In some embodiments, X8 is s. In some embodiments, X8 is Y.

[0074] As used herein, Dap refers to diaminopimelic acid.

[0075] As used herein, s refers to D-serine.

[0076] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0077] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is N-methylLeu. In some embodiments, X 10 is V.

[0078] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q. In some embodiments, X 12 is S.

[0079] In some embodiments, X 13In some embodiments, X is Aib. 13 is E. In some embodiments, X 13 is K. In some embodiments, X 13 is Q. In some embodiments, X 13 is S. In some embodiments, X 13 is W. In some embodiments, X 13 is Y.

[0080] In some embodiments, X 14 is E. In some embodiments, X 14 is R. In some embodiments, X 14 is I. In some embodiments, X 14 is K. In some embodiments, X 14 is L. X 14 is M. In some embodiments, X 14 is Q. In some embodiments, X 14 is Y.

[0081] In some embodiments, X 16 In some embodiments, X is Dab. 16 In some embodiments, X is Dab. 16 is E. In some embodiments, X 16 is K. In some embodiments, X 16 is k. In some embodiments, X 16 is Orn.

[0082] As used herein, Dab refers alternatively to 2,4-diaminobutanoic acid, 2,4-diaminobutyric acid, or α,γ-diaminobutyric acid.

[0083] As used herein, k refers to D-lysine.

[0084] As used herein, Orn refers to ornithine.

[0085] In some embodiments, X17 is E. In some embodiments, X 17 is K. In some embodiments, X 17 is Q.

[0086] In some embodiments, X 18 is A. In some embodiments, X 18 is K. In some embodiments, X 18 is S. In some embodiments, X 18 is Y.

[0087] In some embodiments, X 19 is A. In some embodiments, X 19 is K. In some embodiments, X 19 is V.

[0088] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0089] In some embodiments, X 21 In some embodiments, X is Aib. 21 is E. In some embodiments, X 21 is H. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q. In some embodiments, X 21 is Y.

[0090] In some embodiments, X 24 is A. In some embodiments, X 24 In some embodiments, X is Aib. 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is Q. In some embodiments, X24 is S. In some embodiments, X 24 is Y.

[0091] In some embodiments, X 28 is D. In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X 28 is N. In some embodiments, X 28 is Q. In some embodiments, X 28 is S. In some embodiments, X 28 is Y.

[0092] In some embodiments, X 32 In some embodiments, X is Dap. 32 is H. In some embodiments, X 32 is K. In some embodiments, X 32 is R. In some embodiments, X 32 is S.

[0093] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0094] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include:

[0095] In some embodiments, X2 is Aib and X 10 is V. In some embodiments, X2 is Aib and X 14 is Y. In some embodiments, X2 is Aib and X 16 is K. In some embodiments, X2 is Aib and X 19is V. In some embodiments, X2 is Aib and X 19 is A. In some embodiments, X2 is Aib and X 20 is R. In some embodiments, X2 is Aib and X 20 is K. In some embodiments, X2 is Aib and X 21 is K. In some embodiments, X2 is Aib and X 21 is Q. In some embodiments, X2 is Aib and X 24 is S. In some embodiments, X2 is Aib and X 24 is E.

[0096] In some embodiments, X 10 is V and X 14 is Y. In some embodiments, X 10 is V and X 16 is K. In some embodiments, X 10 is V and X 19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21 is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X 10 is V and X 24 is E.

[0097] In some embodiments, X 14 is Y and X 16 is K. In some embodiments, X 14 is Y and X 19is V. In some embodiments, X 14 is Y and X 19 is A. In some embodiments, X 14 is Y and X 20 is R. In some embodiments, X 14 is Y and X 20 is K. In some embodiments, X 14 is Y and X 21 is K. In some embodiments, X 14 is Y and X 21 is Q. In some embodiments, X 14 is Y and X 24 is S. In some embodiments, X 14 is Y and X 24 is E.

[0098] In some embodiments, X 16 is K and X 19 is V. In some embodiments, X 16 is K and X 19 is A. In some embodiments, X 16 is K and X 20 is R. In some embodiments, X 16 is K and X 20 is K. In some embodiments, X 16 is K and X 21 is K. In some embodiments, X 16 is K and X 21 is Q. In some embodiments, X 16 is K and X 24 is S. In some embodiments, X 16 is K and X 24 is E.

[0099] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0100] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0101] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20 is R and X 21 is Q. In some embodiments, X 20 is R and X 24 is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21 is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E In some embodiments, X 21 is K and X 24 is S. In some embodiments, X 21 is K and X 24 is E.

[0102] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0103] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include:

[0104] In some embodiments, X2 is Aib and X 10 is V and X 14 is Y. In some embodiments, X2 is Aib and X 10 is V and X 16 is K. In some embodiments, X2 is Aib and X 10 is V and X 19 is V. In some embodiments, X2 is Aib and X 10 is V and X 19 is A. In some embodiments, X2 is Aib and X 10 is V and X 20 is R. In some embodiments, X2 is Aib and X 10 is V and X 20 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is Q. In some embodiments, X2 is Aib and X 10 is V and X 24 is S. In some embodiments, X2 is Aib and X 10 is V and X 24 is E.

[0105] In some embodiments, X2 is Aib and X 14 is Y and X 16 is K. In some embodiments, X2 is Aib and X 14 is Y and X 19 is V. In some embodiments, X2 is Aib and X 14 is Y and X 19 is A. In some embodiments, X2 is Aib and X 14 is Y and X 20 is R. In some embodiments, X2 is Aib and X 14 is Y and X 20 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is Q. In some embodiments, X2 is Aib and X 14 is Y and X 24 is S. In some embodiments, X2 is Aib and X 14 is Y and X 24 is E.

[0106] In some embodiments, X 10 is V and X 14 is Y and X 16 is K. In some embodiments, X 10 is V and X 14 is Y and X 19 is V. In some embodiments, X 10 is V and X 14 is Y and X 19 is A. In some embodiments, X 10 is V and X 14 is Y and X 20 is R. In some embodiments, X 10 is V and X 14 is Y and X 20is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is Q. In some embodiments, X 10 is V and X 14 is Y and X 24 is S. In some embodiments, X 10 is V and X 14 is Y and X 24 is E.

[0107] In some embodiments, X 14 is Y and X 16 is K and X 19 is V. In some embodiments, X 14 is Y and X 16 is K and X 19 is A. In some embodiments, X 14 is Y and X 16 is K and X 20 is R. In some embodiments, X 14 is Y and X 16 is K and X 20 is K. In some embodiments, X 14 is Y and X 16 is K and X 21 is K. In some embodiments, X 14 is Y and X 16 is K and X 21 is Q. In some embodiments, X 14 is Y and X 16 is K and X 24 is S. In some embodiments, X 14 is Y and X 16 is K and X 24 is E.

[0108] In some embodiments, X 14 is Y and X 19 is V and X21 is K. In some embodiments, X 14 is Y and X 19 is A and X 21 is K. In some embodiments, X 14 is Y and X 20 is R and X 21 is K. In some embodiments, X 14 is Y and X 20 is K and X 21 is K. In some embodiments, X 14 is Y and X 21 is K and X 24 is S. In some embodiments, X 14 is Y and X 21 is K and X 24 is E.

[0109] In some embodiments, X 16 is K and X 19 is A and X 20 is R. In some embodiments, X 16 is K and X 19 is A and X 20 is K. In some embodiments, X 16 is K and X 19 is A and X 21 is K. In some embodiments, X 16 is K and X 19 is A and X 21 is Q. In some embodiments, X 16 is K and X 19 is A and X 24 is S. In some embodiments, X 16 is K and X 19 is A and X 24 is E.

[0110] In some embodiments, X 19 is V and X 20 is R and X 21 is K. In some embodiments, X 19 is V and X20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X 19 is V and X 20 is R and X 24 is E.

[0111] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0112] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0113] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E.

[0114] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 200: HX2X3GTX6X7X8X9X 10 SX 12X 13 X 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 200), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is A, 2-aminoisobutyric acid (Aib), or G; X3 is E or N-methylGlu; X6 is F or Y; X7 is S or T; X8 is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X9 is D or E; X 10 is I, L, N-methylLeu, or V; X 12 is E, K, Q, or S; X 13 is Aib, E, K, Q, S, W, or Y; X 14 is E, R, I, K, L, M, Q, or Y; X 16 is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn); X 17 is E, K, or Q; X 18 is A, K, S, or Y; X 19 is A, K, or V; X 20 is E, K, or R; X 21 is Aib, E, H, K, L, Q, or Y; X 24 is A, Aib, E, K, Q, S, or Y; X 28is D, E, K, N, Q, S, or Y; and X 32 is Dap, H, K, R, or S; where X 16 is covalently linked, optionally via a spacer, to a lipophilic substituent when is Dab, Dap, K, k, or Orn; where X 16 If E, then X 14 , X 17 , X 18 , X 19 , X 20 , or X 21 at least one of is selected to be lysine, wherein the lysine residue is covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the isolated polypeptide optionally further comprises two amino acid residues having side chains covalently linked to form a cross-linking moiety).

[0115] In one embodiment, the isolated polypeptide comprises a K covalently linked to a lipophilic substituent, optionally via a spacer. 12 or K 27 and not an analog of exenatide, namely HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 300).

[0116] In some embodiments, X2 is Aib.

[0117] In some embodiments, X 24 If S, then X 10 is V.

[0118] In some embodiments, X 14 is Y.

[0119] In some embodiments, X 14 is M and X 16 is K covalently attached, optionally via a spacer, to a lipophilic substituent.

[0120] In some embodiments, X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer.

[0121] In some embodiments, X 16 If E, then X 21 is K or E. In some embodiments, X 16 If E, then X 21 is K. In some embodiments, X 16 If E, then X 21 is E.

[0122] In some embodiments, the peptide is: 20 and X 24 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0123] In some embodiments, X2 is A. In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0124] In some embodiments, X3 is E. In some embodiments, X3 is N-methylGlu.

[0125] In some embodiments, X6 is F. In some embodiments, X6 is Y.

[0126] In some embodiments, X7 is S. In some embodiments, X7 is T.

[0127] In some embodiments, X8 is Dap. In some embodiments, X8 is E. In some embodiments, X8 is K. In some embodiments, X8 is N. In some embodiments, X8 is N-methyl Ser. In some embodiments, X8 is Q. In some embodiments, X8 is S. In some embodiments, X8 is s. In some embodiments, X8 is Y.

[0128] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0129] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is N-methylLeu. In some embodiments, X 10 is V.

[0130] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q. In some embodiments, X 12 is S.

[0131] In some embodiments, X 13 In some embodiments, X is Aib. 13 is E. In some embodiments, X 13 is K. In some embodiments, X 13 is Q. In some embodiments, X 13 is S. In some embodiments, X 13 is W. In some embodiments, X 13 is Y.

[0132] In some embodiments, X 14 is E. In some embodiments, X 14 is R. In some embodiments, X 14 is I. In some embodiments, X 14 is K. In some embodiments, X 14 is L. X 14 is M. In some embodiments, X 14 is Q. In some embodiments, X 14 is Y.

[0133] In some embodiments, X 17 is E. In some embodiments, X 17 is K. In some embodiments, X 17 is Q.

[0134] In some embodiments, X 18 is A. In some embodiments, X 18 is K. In some embodiments, X 18 is S. In some embodiments, X 18 is Y.

[0135] In some embodiments, X 19 is A. In some embodiments, X 19 is K. In some embodiments, X 19 is V.

[0136] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0137] In some embodiments, X 21 In some embodiments, X is Aib. 21 is E. In some embodiments, X 21 is H. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q. In some embodiments, X 21 is Y.

[0138] In some embodiments, X 24 is A. In some embodiments, X 24 In some embodiments, X is Aib. 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24is Q. In some embodiments, X 24 is S. In some embodiments, X 24 is Y.

[0139] In some embodiments, X 28 is D. In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X 28 is N. In some embodiments, X 28 is Q. In some embodiments, X 28 is S. In some embodiments, X 28 is Y.

[0140] In some embodiments, X 32 In some embodiments, X is Dap. 32 is H. In some embodiments, X 32 is K. In some embodiments, X 32 is R. In some embodiments, X 32 is S.

[0141] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0142] In some embodiments, X 16 is Dab, Dap, K, or Orn, optionally covalently linked to a lipophilic substituent via a spacer. In some embodiments, X 16 is a Dab covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 is Dap covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 is k covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 is Orn covalently linked to a lipophilic substituent, optionally via a spacer.

[0143] In some embodiments, X 16 is E and X 14 , X 17 , X 18 , X 19 , X 20 , or X 21 In some embodiments, at least one of X is selected to be lysine, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. 16 If E, then X 14 is a lysine residue, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 17 is a lysine residue, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 18 is a lysine residue, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 19 is a lysine residue, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 20 is a lysine residue, where the lysine residue is covalently attached to a lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 21 is a lysine residue, where the lysine residue is covalently attached, optionally via a spacer, to a lipophilic substituent.

[0144] In some embodiments, the isolated polypeptide further comprises two amino acid residues having side chains that are covalently linked to form a bridging moiety. In some embodiments, the isolated polypeptide optionally further comprises two amino acid residues, one of which is lysine (K) and the other of which is glutamic acid (E), wherein the amino-containing side chain of the lysine and the carboxy-containing side chain of the glutamic acid are covalently linked by loss of water to form a lactam bridging moiety. In some embodiments, the lactam bridging moieties are each represented by X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 21 and X 28 in place; X respectively 28 and X 21 in place; X respectively 20 and X 24 in place; X respectively 24 and X 20 or X respectively 12 and X 16 In some embodiments, the lactam bridging moiety is formed through a covalent bond between the side chain of a lysine (K) residue and the side chain of glutamic acid (E) at position X. 17 and X 21 In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 21 and X 17 In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 21 and X 28 In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 28 and X 21 In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 20 and X 24In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 24 and X 20 In some embodiments, the lactam bridging moiety is formed between the side chains of a lysine (K) residue and a glutamic acid (E) residue at positions X and X, respectively. 12 and X 16 The nucleotides are formed by covalent bonds between the side chains of lysine (K) and glutamic acid (E) residues at positions 1 and 2, respectively.

[0145] In some embodiments, K is X, X 12 , X 13 , X 14 , X 17 , X 18 , X 19 , X 20 , X 21 , X 24 , X 28 , or X 32 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0146] In some embodiments, K is X 14 , X 17 , X 18 , X 19 , X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0147] In some embodiments, X 16 can be covalently attached to the lipophilic substituent, optionally via a spacer.

[0148] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 201: HX2X3GTX6X7X8X9X 10 SX 12 X 13 YEX 16 X 17 X18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 201), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is A, 2-aminoisobutyric acid (Aib), or G; X3 is E or N-methylGlu; X6 is F or Y; X7 is S or T; X8 is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X9 is D or E; X 10 is I, L, N-methylLeu, or V; X 12 is E, K, Q, or S; X 13 is Aib, E, K, Q, S, W, or Y; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 17 is E, K, or Q; X 18 is A, K, S, or Y; X 19 is A, K, or V; X 20 is E, K, or R; X 21 is Aib, E, H, K, L, Q, or Y; X 24 is A, Aib, E, K, Q, S, or Y; X 28 is D, E, K, N, Q, S, or Y; and X 32 is Dap, H, K, R, or S; where the peptides are X 17 and X21 in place; X respectively 21 and X 17 in place; X respectively 21 and X 28 in place; X respectively 28 and X 21 in place; X respectively 20 and X 24 or X respectively 24 and X 20 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0149] In some embodiments, X2 is Aib.

[0150] In some embodiments, X 24 If S, then X 10 is V.

[0151] In some embodiments, the peptide comprises X 20 and X 24 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0152] In some embodiments, X2 is A. In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0153] In some embodiments, X3 is E. In some embodiments, X3 is N-methylGlu.

[0154] In some embodiments, X6 is F. In some embodiments, X6 is Y.

[0155] In some embodiments, X7 is S. In some embodiments, X7 is T.

[0156] In some embodiments, X8 is Dap. In some embodiments, X8 is E. In some embodiments, X8 is K. In some embodiments, X8 is N. In some embodiments, X8 is N-methyl Ser. In some embodiments, X8 is Q. In some embodiments, X8 is S. In some embodiments, X8 is s. In some embodiments, X8 is Y.

[0157] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0158] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is N-methylLeu. In some embodiments, X 10 is V.

[0159] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q. In some embodiments, X 12 is S.

[0160] In some embodiments, X 13 In some embodiments, X is Aib. 13 is E. In some embodiments, X 13 is K. In some embodiments, X 13 is Q. In some embodiments, X 13 is S. In some embodiments, X 13 is W. In some embodiments, X 13 is Y.

[0161] In some embodiments, X 17 is E. In some embodiments, X 17 is K. In some embodiments, X 17is Q.

[0162] In some embodiments, X 18 is A. In some embodiments, X 18 is K. In some embodiments, X 18 is S. In some embodiments, X 18 is Y.

[0163] In some embodiments, X 19 is A. In some embodiments, X 19 is K. In some embodiments, X 19 is V.

[0164] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0165] In some embodiments, X 21 In some embodiments, X is Aib. 21 is E. In some embodiments, X 21 is H. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q. In some embodiments, X 21 is Y.

[0166] In some embodiments, X 24 is A. In some embodiments, X 24 In some embodiments, X is Aib. 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is Q. In some embodiments, X 24 is S. In some embodiments, X 24 is Y.

[0167] In some embodiments, X 28 is D. In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X 28 is N. In some embodiments, X 28 is Q. In some embodiments, X 28 is S. In some embodiments, X 28 is Y.

[0168] In some embodiments, X 32 In some embodiments, X is Dap. 32 is H. In some embodiments, X 32 is K. In some embodiments, X 32 is R. In some embodiments, X 32 is S.

[0169] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0170] In some embodiments, the isolated peptide comprises X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 21 and X 28 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 28 and X 21In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0171] In some embodiments, K is X, X 12 , X 13 , X 17 , X 18 , X 19 , X 20 , X 21 , X 24 , X 28 , or X 32 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0172] In some embodiments, K is X 17 , X 18 , X 19 , X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0173] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0174] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:200 or SEQ ID NO:201 include:

[0175] In some embodiments, X2 is Aib and X 10is V. In some embodiments, X2 is Aib and X 14 is Y. In some embodiments, X2 is Aib and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 19 is V. In some embodiments, X2 is Aib and X 19 is A. In some embodiments, X2 is Aib and X 20 is R. In some embodiments, X2 is Aib and X 20 is K. In some embodiments, X2 is Aib and X 21 is K. In some embodiments, X2 is Aib and X 21 is Q. In some embodiments, X2 is Aib and X 24 is S. In some embodiments, X2 is Aib and X 24 is E.

[0176] In some embodiments, X 10 is V and X 14 is Y. In some embodiments, X 10 is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X 19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21 is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X10 is V and X 24 is E.

[0177] In some embodiments, X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 14 is Y and X 19 is V. In some embodiments, X 14 is Y and X 19 is A. In some embodiments, X 14 is Y and X 20 is R. In some embodiments, X 14 is Y and X 20 is K. In some embodiments, X 14 is Y and X 21 is K. In some embodiments, X 14 is Y and X 21 is Q. In some embodiments, X 14 is Y and X 24 is S. In some embodiments, X 14 is Y and X 24 is E.

[0178] In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is V. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is A. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X21 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is E.

[0179] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X 21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0180] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0181] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20 is R and X 21 is Q. In some embodiments, X 20 is R and X 24 is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21 is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E. In some embodiments, X 20 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0182] In some embodiments, X 21 is K and X 24 is S. In some embodiments, X 21 is K and X 24 is E.

[0183] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0184] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:200 or SEQ ID NO:201 include:

[0185] In some embodiments, X2 is Aib and X 10 is V and X 14 is Y. In some embodiments, X2 is Aib and X 10 is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 10 is V and X 19 is V. In some embodiments, X2 is Aib and X 10 is V and X 19 is A. In some embodiments, X2 is Aib and X 10 is V and X 20 is R. In some embodiments, X2 is Aib and X 10 is V and X 20 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is Q. In some embodiments, X2 is Aib and X 10 is V and X 24 is S. In some embodiments, X2 is Aib and X 10 is V and X 24 is E.

[0186] In some embodiments, X2 is Aib and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 14 is Y and X 19 is V. In some embodiments, X2 is Aib and X 14 is Y and X 19 is A. In some embodiments, X2 is Aib and X 14 is Y and X 20 is R. In some embodiments, X2 is Aib and X 14is Y and X 20 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is Q. In some embodiments, X2 is Aib and X 14 is Y and X 24 is S. In some embodiments, X2 is Aib and X 14 is Y and X 24 is E.

[0187] In some embodiments, X 10 is V and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X 14 is Y and X 19 is V. In some embodiments, X 10 is V and X 14 is Y and X 19 is A. In some embodiments, X 10 is V and X 14 is Y and X 20 is R. In some embodiments, X 10 is V and X 14 is Y and X 20 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is Q. In some embodiments, X 10 is V and X 14 is Y and X 24 is S. In some embodiments, X 10 is V and X 14 is Y and X 24 is E.

[0188] In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is V. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is R. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is Q. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is S. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is E.

[0189] In some embodiments, X 14 is Y and X 19 is V and X 21 is K. In some embodiments, X 14 is Y and X 19 is A and X 21is K. In some embodiments, X 14 is Y and X 20 is R and X 21 is K. In some embodiments, X 14 is Y and X 20 is K and X 21 is K. In some embodiments, X 14 is Y and X 21 is K and X 24 is S. In some embodiments, X 14 is Y and X 21 is K and X 24 is E.

[0190] In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is E.

[0191] In some embodiments, X 19is V and X 20 is R and X 21 is K. In some embodiments, X 19 is V and X 20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X 19 is V and X 20 is R and X 24 is E.

[0192] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0193] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0194] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E. In some embodiments, X 20 is K and X 21is K and X 24 is E and the peptide is X 20の Side chain and X 24 The lactam bridge is formed via an amide bond between the side chains of

[0195] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 202: HX2EGTFTX8X9X 10 SX 12 QX 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 202), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X9 is D or E; X 10 is I, L, or V; X 12 is E or K; X 14 is M or Y; X 16 is Dap, E, or K; X 17 is E or K; X 18 is A or Y; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is E, K, N, or Q; and X 32is H or S; where X 16 is covalently linked to a lipophilic substituent, optionally via a spacer, when is Dap or K; where X 16 If E, then X 17 , X 20 , or X 21 at least one of is selected to be lysine, wherein the lysine residue is covalently linked, optionally via a spacer, to a lipophilic substituent; where the peptides are X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 21 and X 28 in place; X respectively 28 and X 21 in place; X respectively 20 and X 24 in place; X respectively 24 and X 20 or X respectively 12 and X 16 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0196] In some embodiments, X2 is Aib.

[0197] In some embodiments, X 24 If S, then X 10 is V.

[0198] In some embodiments, X 14 is Y.

[0199] In some embodiments, X 14 is M and X 16 is K covalently attached, optionally via a spacer, to a lipophilic substituent.

[0200] In some embodiments, X 16is K covalently attached to the lipophilic substituent, optionally via a spacer.

[0201] In some embodiments, X 16 If E, then X 21 is K or E. In some embodiments, X 16 If E, then X 21 is K. In some embodiments, X 16 If E, then X 21 is E.

[0202] In some embodiments, the peptide comprises X 20 and X 24 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0203] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0204] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0205] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0206] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0207] In some embodiments, X 12 is E. In some embodiments, X 12 is K.

[0208] In some embodiments, X 14 is M. In some embodiments, X 14 is Y.

[0209] In some embodiments, X 16 is Dap covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 If E, then X 17 , X 20 , or X 21 At least one of is selected to be lysine, and the lysine residue is covalently linked, optionally via a spacer, to a lipophilic substituent.

[0210] In some embodiments, X 17 is E. In some embodiments, X 17 is K.

[0211] In some embodiments, X 18 is A. In some embodiments, X 18 is Y.

[0212] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0213] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0214] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0215] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X24 is S.

[0216] In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0217] In some embodiments, X 32 is H. In some embodiments, X 32 is S.

[0218] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0219] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 21 and X 28 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 28 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 12 and X 16 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 16 and X 12 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0220] In some embodiments, K is X 12 , X 17 , X 20 , X 21 , X 24 , or X 28 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0221] In some embodiments, K is X 17 , X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0222] In some embodiments, X 16 can be covalently attached to the lipophilic substituent, optionally via a spacer.

[0223] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 203: HX2EGTFTX8X9X 10 SX 12 QYEX 16 X 17 X 18 X 19X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 203), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X9 is D or E; X 10 is I, L, or V; X 12 is E or K; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 17 is E or K; X 18 is A or Y; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is E, K, N, or Q; and X 32 is H or S; each K independently represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; where the peptides are X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 21 and X 28 in place; X respectively 28 and X 21 in place; X respectively 20 and X 24 or X respectively 24 and X 20and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0224] In some embodiments, X2 is Aib.

[0225] In some embodiments, X 24 If S, then X 10 is V.

[0226] In some embodiments, the peptide comprises X 20 and X 24 The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0227] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0228] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0229] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0230] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0231] In some embodiments, X 12 is E. In some embodiments, X 12 is K.

[0232] In some embodiments, X 17 is E. In some embodiments, X 17 is K.

[0233] In some embodiments, X 18is A. In some embodiments, X 18 is Y.

[0234] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0235] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0236] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0237] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0238] In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0239] In some embodiments, X 32 is H. In some embodiments, X 32 is S.

[0240] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S39 -(OH).

[0241] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 21 and X 28 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 28 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0242] In some embodiments, K is X 12 , X 17 , X 20 , X 21 , X 24 , or X 28 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0243] In some embodiments, K is X 17 , X 20 , or X 21When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0244] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0245] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:202 or SEQ ID NO:203 include:

[0246] In some embodiments, X2 is Aib and X 10 is V. In some embodiments, X2 is Aib and X 14 is Y. In some embodiments, X2 is Aib and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 19 is V. In some embodiments, X2 is Aib and X 19 is A. In some embodiments, X2 is Aib and X 20 is R. In some embodiments, X2 is Aib and X 20 is K. In some embodiments, X2 is Aib and X 21 is K. In some embodiments, X2 is Aib and X 21 is Q. In some embodiments, X2 is Aib and X 24 is S. In some embodiments, X2 is Aib and X 24 is E.

[0247] In some embodiments, X 10 is V and X 14 is Y. In some embodiments, X 10 is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21 is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X 10 is V and X 24 is E.

[0248] In some embodiments, X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 14 is Y and X 19 is V. In some embodiments, X 14 is Y and X 19 is A. In some embodiments, X 14 is Y and X 20 is R. In some embodiments, X 14 is Y and X 20 is K. In some embodiments, X 14 is Y and X 21 is K. In some embodiments, X 14 is Y and X 21 is Q. In some embodiments, X 14 is Y and X 24 is S. In some embodiments, X 14 is Y and X 24 is E.

[0249] In some embodiments, X 16is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is V. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is A. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 21 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is E.

[0250] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X 21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0251] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0252] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20 is R and X 21 is Q. In some embodiments, X 20 is R and X 24 is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21 is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E. In some embodiments, X 20 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0253] In some embodiments, X 21 is K and X 24is S. In some embodiments, X 21 is K and X 24 is E.

[0254] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0255] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:202 or SEQ ID NO:203 include:

[0256] In some embodiments, X2 is Aib and X 10 is V and X 14 is Y. In some embodiments, X2 is Aib and X 10 is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 10 is V and X 19 is V. In some embodiments, X2 is Aib and X 10 is V and X 19 is A. In some embodiments, X2 is Aib and X 10 is V and X 20 is R. In some embodiments, X2 is Aib and X 10 is V and X 20 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is Q. In some embodiments, X2 is Aib and X 10 is V and X 24 is S. In some embodiments, X2 is Aib and X 10 is V and X 24 is E.

[0257] In some embodiments, X2 is Aib and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 14 is Y and X 19 is V. In some embodiments, X2 is Aib and X 14 is Y and X 19 is A. In some embodiments, X2 is Aib and X 14 is Y and X 20 is R. In some embodiments, X2 is Aib and X 14 is Y and X 20 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is Q. In some embodiments, X2 is Aib and X 14 is Y and X 24 is S. In some embodiments, X2 is Aib and X 14 is Y and X 24 is E.

[0258] In some embodiments, X 10 is V and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X 14 is Y and X 19 is V. In some embodiments, X 10 is V and X 14 is Y and X 19 is A. In some embodiments, X 10 is V and X 14 is Y and X 20 is R. In some embodiments, X10 is V and X 14 is Y and X 20 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is Q. In some embodiments, X 10 is V and X 14 is Y and X 24 is S. In some embodiments, X 10 is V and X 14 is Y and X 24 is E.

[0259] In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is V. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is R. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is Q. In some embodiments, X14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is S. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is E.

[0260] In some embodiments, X 14 is Y and X 19 is V and X 21 is K. In some embodiments, X 14 is Y and X 19 is A and X 21 is K. In some embodiments, X 14 is Y and X 20 is R and X 21 is K. In some embodiments, X 14 is Y and X 20 is K and X 21 is K. In some embodiments, X 14 is Y and X 21 is K and X 24 is S. In some embodiments, X 14 is Y and X 21 is K and X 24 is E.

[0261] In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is E.

[0262] In some embodiments, X 19 is V and X 20 is R and X 21 is K. In some embodiments, X 19 is V and X 20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X 19 is V and X 20 is R and X 24 is E.

[0263] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0264] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0265] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E. In some embodiments, X 20 is K and X 21 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0266] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 204: HX2EGTFTX8DX 10 SX 12 QX 14 EX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 204), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X 10 is I, L, or V; X 12 is E, K, or Q; X 14 is M or Y; X 16 is (diaminopimelic acid) Dap covalently attached to a lipophilic substituent, optionally via a spacer, or K covalently attached to a lipophilic substituent, optionally via a spacer; X 17 is E or K; X 18 is A or Y; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is N or Q; X 32 is H or S; where the peptides are X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 20 and X 24 in place; X respectively 24 and X 20 position, optionally including a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain).

[0267] In some embodiments, X2 is Aib.

[0268] In some embodiments, X 24 If S, then X 10 is V.

[0269] In some embodiments, X 14 is Y.

[0270] In some embodiments, X 14 is M and X 16 is K covalently attached, optionally via a spacer, to a lipophilic substituent.

[0271] In some embodiments, X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer.

[0272] In some embodiments, the peptide comprises X 20 and X 24 The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0273] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0274] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0275] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0276] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q.

[0277] In some embodiments, X 14 is M. In some embodiments, X 14 is Y.

[0278] In some embodiments, X 16 is Dap covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer.

[0279] In some embodiments, X 17 is E. In some embodiments, X 17is K.

[0280] In some embodiments, X 18 is A. In some embodiments, X 18 is Y.

[0281] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0282] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0283] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0284] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0285] In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0286] In some embodiments, X 32 is H. In some embodiments, X 32 is S.

[0287] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39-S 39 -(OH).

[0288] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0289] In some embodiments, K is X 12 , X 17 , X 20 , X 21 , or X 24 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0290] In some embodiments, K is X 17 , X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0291] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0292] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 205: HX2EGTFTX8DX 10 SX 12 QYEX 16 X 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 An isolated polypeptide comprising SGAPPPS-(OH / NH) (SEQ ID NO: 205), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X 10 is I, L, or V; X 12 is E, K, or Q; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 17 is E or K; X 18 is A or Y; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is N or Q; X 32 is H or S; where the peptides are X 17 and X 21 in place; X respectively 21 and X 17 in place; X respectively 20 and X 24 or X respectively 24 and X 20 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0293] In some embodiments, X2 is Aib.

[0294] In some embodiments, X 24 If S, then X 10 is V.

[0295] In some embodiments, the peptide comprises X 20 and X 24 The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0296] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0297] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0298] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0299] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q.

[0300] In some embodiments, X 17 is E. In some embodiments, X 17 is K.

[0301] In some embodiments, X 18 is A. In some embodiments, X 18 is Y.

[0302] In some embodiments, X 19 is A. In some embodiments, X19 is V.

[0303] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0304] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0305] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0306] In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0307] In some embodiments, X 32 is H. In some embodiments, X 32 is S.

[0308] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0309] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0310] In some embodiments, K is X 12 , X 17 , X 20 , X 21 , or X 24 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0311] In some embodiments, K is X 17 , X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0312] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0313] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:204 or SEQ ID NO:205 include:

[0314] In some embodiments, X2 is Aib and X 10 is V. In some embodiments, X2 is Aib and X 14 is Y. In some embodiments, X2 is Aib and X 16is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 19 is V. In some embodiments, X2 is Aib and X 19 is A. In some embodiments, X2 is Aib and X 20 is R. In some embodiments, X2 is Aib and X 20 is K. In some embodiments, X2 is Aib and X 21 is K. In some embodiments, X2 is Aib and X 21 is Q. In some embodiments, X2 is Aib and X 24 is S. In some embodiments, X2 is Aib and X 24 is E.

[0315] In some embodiments, X 10 is V and X 14 is Y. In some embodiments, X 10 is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X 19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21 is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X 10 is V and X 24 is E.

[0316] In some embodiments, X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 14 is Y and X 19 is V. In some embodiments, X 14 is Y and X 19 is A. In some embodiments, X 14 is Y and X 20 is R. In some embodiments, X 14 is Y and X 20 is K. In some embodiments, X 14 is Y and X 21 is K. In some embodiments, X 14 is Y and X 21 is Q. In some embodiments, X 14 is Y and X 24 is S. In some embodiments, X 14 is Y and X 24 is E.

[0317] In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is V. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 19 is A. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 21 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer, and X 24 is E.

[0318] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X 21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0319] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0320] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20is R and X 21 is Q. In some embodiments, X 20 is R and X 24 is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21 is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E. In some embodiments, X 20 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0321] In some embodiments, X 21 is K and X 24 is S. In some embodiments, X 21 is K and X 24 is E.

[0322] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0323] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:204 or SEQ ID NO:205 include:

[0324] In some embodiments, X2 is Aib and X 10 is V and X 14 is Y. In some embodiments, X2 is Aib and X 10is V and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 10 is V and X 19 is V. In some embodiments, X2 is Aib and X 10 is V and X 19 is A. In some embodiments, X2 is Aib and X 10 is V and X 20 is R. In some embodiments, X2 is Aib and X 10 is V and X 20 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is Q. In some embodiments, X2 is Aib and X 10 is V and X 24 is S. In some embodiments, X2 is Aib and X 10 is V and X 24 is E.

[0325] In some embodiments, X2 is Aib and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X2 is Aib and X 14 is Y and X 19 is V. In some embodiments, X2 is Aib and X 14 is Y and X 19 is A. In some embodiments, X2 is Aib and X 14 is Y and X 20 is R. In some embodiments, X2 is Aib and X 14 is Y and X 20 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is Q. In some embodiments, X2 is Aib and X 14 is Y and X 24 is S. In some embodiments, X2 is Aib and X 14 is Y and X 24 is E.

[0326] In some embodiments, X 10 is V and X 14 is Y and X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer. In some embodiments, X 10 is V and X 14 is Y and X 19 is V. In some embodiments, X 10 is V and X 14 is Y and X 19 is A. In some embodiments, X 10 is V and X 14 is Y and X 20 is R. In some embodiments, X 10 is V and X 14 is Y and X 20 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is Q. In some embodiments, X 10 is V and X 14 is Y and X 24 is S. In some embodiments, X 10 is V and X 14 is Y and X 24 is E.

[0327] In some embodiments, X 14 is Y;X 16is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is V. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is R. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 20 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is K. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 21 is Q. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is S. In some embodiments, X 14 is Y;X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 24 is E.

[0328] In some embodiments, X 14 is Y and X 19 is V and X 21 is K. In some embodiments, X 14 is Y and X 19 is A and X 21 is K. In some embodiments, X 14 is Y and X 20 is R and X 21is K. In some embodiments, X 14 is Y and X 20 is K and X 21 is K. In some embodiments, X 14 is Y and X 21 is K and X 24 is S. In some embodiments, X 14 is Y and X 21 is K and X 24 is E.

[0329] In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is R. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 20 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21 is K. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 21 is Q. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is S. In some embodiments, X 16 is K covalently linked to a lipophilic substituent, optionally via a spacer; X 19 is A;X 24 is E.

[0330] In some embodiments, X 19 is V and X 20 is R and X 21 is K. In some embodiments, X 19is V and X 20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X 19 is V and X 20 is R and X 24 is E.

[0331] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0332] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0333] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E. In some embodiments, X 20 is K and X 21 is K and X 24 is E and the peptide is X 20 and the side chain of X 24The lactam bridge is formed via an amide bond between the side chains of

[0334] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 206: HX2EGTFTX8DX 10 SKQX 14 EX 16 EAX 19 X 20 X 21 FIX 24 WLKX 28 An isolated polypeptide comprising GGPSSGAPPPS-(OH / NH) (SEQ ID NO: 206), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X 10 is I, L, or V; X 14 is M or Y; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is N or Q; where the peptides are X 21 and X 17 in place; X respectively 20 and X 24 or X respectively 24 and X 20 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0335] In some embodiments, X2 is Aib.

[0336] In some embodiments, X 24 If S, then X 10 is V.

[0337] In some embodiments, X 14 is Y.

[0338] In some embodiments, the peptide comprises X 20 and X 24 The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0339] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0340] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0341] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0342] In some embodiments, X 14 is M. In some embodiments, X 14 is Y.

[0343] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0344] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0345] In some embodiments, X 21 is E. In some embodiments, X21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0346] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0347] In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0348] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0349] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0350] In some embodiments, K is X20 , X 21 , or X 24 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0351] In some embodiments, K is X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0352] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0353] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 207: HX2EGTFTX8DX 10 SKQYEX 16 EAX 19 X 20 X 21 FIX 24 WLKX 28 An isolated polypeptide comprising GGPSSGAPPPS-(OH / NH) (SEQ ID NO: 207), or a pharmaceutically acceptable salt thereof, is provided, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X 10 is I, L, or V; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is N or Q; where the peptides are X 21 and X 17 in place; X respectively 20 and X 24 or X respectively 24 and X 20 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0354] In some embodiments, X2 is Aib.

[0355] In some embodiments, X 24 If S, then X 10 is V.

[0356] In some embodiments, the peptide comprises X 20 and X 24 The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0357] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0358] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0359] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0360] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0361] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0362] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0363] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0364] In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0365] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0366] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 17 and X 21 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0367] In some embodiments, K is X 20 , X 21 , or X 24 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0368] In some embodiments, K is X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0369] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0370] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:206 or SEQ ID NO:207 include:

[0371] In some embodiments, X2 is Aib and X 10 is V. In some embodiments, X2 is Aib and X 14 is Y. In some embodiments, X2 is Aib and X 19 is V. In some embodiments, X2 is Aib and X 19 is A. In some embodiments, X2 is Aib and X 20 is R. In some embodiments, X2 is Aib and X 20 is K. In some embodiments, X2 is Aib and X 21 is K. In some embodiments, X2 is Aib and X 21 is Q. In some embodiments, X2 is Aib and X 24 is S. In some embodiments, X2 is Aib and X24 is E.

[0372] In some embodiments, X 10 is V and X 14 is Y. In some embodiments, X 10 is V and X 19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21 is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X 10 is V and X 24 is E.

[0373] In some embodiments, X 14 is Y and X 19 is V. In some embodiments, X 14 is Y and X 19 is A. In some embodiments, X 14 is Y and X 20 is R. In some embodiments, X 14 is Y and X 20 is K. In some embodiments, X 14 is Y and X 21 is K. In some embodiments, X 14 is Y and X 21 is Q. In some embodiments, X 14 is Y and X 24 is S. In some embodiments, X 14 is Y and X 24 is E.

[0374] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X 21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0375] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0376] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20 is R and X 21 is Q. In some embodiments, X 20 is R and X 24 is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E. In some embodiments, X 20 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0377] In some embodiments, X 21 is K and X 24 is S. In some embodiments, X 21 is K and X 24 is E.

[0378] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0379] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:206 or SEQ ID NO:207 include:

[0380] In some embodiments, X2 is Aib and X 10 is V and X 14 is Y. In some embodiments, X2 is Aib and X 10 is V and X 19 is V. In some embodiments, X2 is Aib and X 10 is V and X 19 is A. In some embodiments, X2 is Aib and X 10 is V and X 20 is R. In some embodiments, X2 is Aib and X 10is V and X 20 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is K. In some embodiments, X2 is Aib and X 10 is V and X 21 is Q. In some embodiments, X2 is Aib and X 10 is V and X 24 is S. In some embodiments, X2 is Aib and X 10 is V and X 24 is E.

[0381] In some embodiments, X2 is Aib and X 14 is Y and X 19 is V. In some embodiments, X2 is Aib and X 14 is Y and X 19 is A. In some embodiments, X2 is Aib and X 14 is Y and X 20 is R. In some embodiments, X2 is Aib and X 14 is Y and X 20 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is K. In some embodiments, X2 is Aib and X 14 is Y and X 21 is Q. In some embodiments, X2 is Aib and X 14 is Y and X 24 is S. In some embodiments, X2 is Aib and X 14 is Y and X 24 is E.

[0382] In some embodiments, X 10 is V and X 14 is Y and X 19 is V. In some embodiments, X 10 is V and X 14 is Y and X 19is A. In some embodiments, X 10 is V and X 14 is Y and X 20 is R. In some embodiments, X 10 is V and X 14 is Y and X 20 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is K. In some embodiments, X 10 is V and X 14 is Y and X 21 is Q. In some embodiments, X 10 is V and X 14 is Y and X 24 is S. In some embodiments, X 10 is V and X 14 is Y and X 24 is E.

[0383] In some embodiments, X 14 is Y and X 19 is V and X 21 is K. In some embodiments, X 14 is Y and X 19 is A and X 21 is K. In some embodiments, X 14 is Y and X 20 is R and X 21 is K. In some embodiments, X 14 is Y and X 20 is K and X 21 is K. In some embodiments, X 14 is Y and X 21 is K and X 24 is S. In some embodiments, X 14 is Y and X 21 is K and X 24 is E.

[0384] In some embodiments, X 19 is V and X 20 is R and X21 is K. In some embodiments, X 19 is V and X 20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X 19 is V and X 20 is R and X 24 is E.

[0385] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0386] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0387] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E. In some embodiments, X 20 is K and X 21 is K and X 24is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0388] In certain embodiments, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 208: HAibEGTFTSDX 10 SKQYEX 16 EAX 19 X 20 X 21 FIX 24 An isolated polypeptide comprising WLKNGGPSSGAPPPS-(OH / NH) (SEQ ID NO: 208), or a pharmaceutically acceptable salt thereof, is provided, wherein: X 10 is L or V; X 16 is K covalently attached to the lipophilic substituent, optionally via a spacer; X 19 is A or V; X 20 is E, K, or R; X 21 is K or Q; X 24 is E, K, or S; where the peptides are X 21 and X 17 in place; X respectively 20 and X 24 or X respectively 24 and X 20 and optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain at position (

[0389] In some embodiments, X 24 If S, then X 10 is V.

[0390] In some embodiments, the peptide comprises X 20 and X 24The hydroxylase comprises a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions 111 and 112, respectively.

[0391] In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0392] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0393] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0394] In some embodiments, X 21 is K. In some embodiments, X 21 is Q.

[0395] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0396] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0397] In some embodiments, the isolated peptide comprises X 21 and X 17 In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 20 and X 24In some embodiments, the isolated peptide further comprises a lactam bridge formed through an amide bond between the lysine and glutamic acid side chains at positions X, X 24 and X 20 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0398] In some embodiments, K is X 20 , X 21 , or X 24 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0399] In some embodiments, K is X 20 , or X 21 When selected at position 1, it represents a lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent.

[0400] In some embodiments, X 16 is covalently attached to the lipophilic substituent, optionally via a spacer.

[0401] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 208 include:

[0402] In some embodiments, X 10 is V and X 19 is V. In some embodiments, X 10 is V and X 19 is A. In some embodiments, X 10 is V and X 20 is R. In some embodiments, X 10 is V and X 20 is K. In some embodiments, X 10 is V and X 21 is K. In some embodiments, X 10 is V and X 21is Q. In some embodiments, X 10 is V and X 24 is S. In some embodiments, X 10 is V and X 24 is E.

[0403] In some embodiments, X 19 is V and X 20 is R. In some embodiments, X 19 is V and X 20 is K. In some embodiments, X 19 is V and X 21 is K. In some embodiments, X 19 is V and X 21 is Q. In some embodiments, X 19 is V and X 24 is S. In some embodiments, X 19 is V and X 24 is E.

[0404] In some embodiments, X 19 is A and X 20 is R. In some embodiments, X 19 is A and X 20 is K. In some embodiments, X 19 is A and X 21 is K. In some embodiments, X 19 is A and X 21 is Q. In some embodiments, X 19 is A and X 24 is S. In some embodiments, X 19 is A and X 24 is E.

[0405] In some embodiments, X 20 is R and X 21 is K. In some embodiments, X 20 is R and X 21 is Q. In some embodiments, X 20 is R and X 24is S. In some embodiments, X 20 is R and X 24 is E In some embodiments, X 20 is K and X 21 is K. In some embodiments, X 20 is K and X 21 is Q. In some embodiments, X 20 is K and X 24 is S. In some embodiments, X 20 is K and X 24 is E. In some embodiments, X 20 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0406] In some embodiments, X 21 is K and X 24 is S. In some embodiments, X 21 is K and X 24 is E.

[0407] In some embodiments, X 21 is Q and X 24 is S. In some embodiments, X 21 is Q and X 24 is E.

[0408] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 208 include:

[0409] In some embodiments, X 10 is V and X 19 is V and X 20 is R. In some embodiments, X 10 is V and X 19 is V and X 20 is K. In some embodiments, X 10 is V and X19 is V and X 21 is K. In some embodiments, X 10 is V and X 19 is V and X 21 is Q. In some embodiments, X 10 is V and X 19 is V and X 24 is S. In some embodiments, X 10 is V and X 19 is V and X 24 is E.

[0410] In some embodiments, X 10 is V and X 19 is V and X 24 is S. In some embodiments, X 10 is V and X 19 is A and X 24 is S. In some embodiments, X 10 is V and X 20 is R and X 24 is S. In some embodiments, X 10 is V and X 20 is K and X 24 is S. In some embodiments, X 10 is V and X 21 is K and X 24 is S. In some embodiments, X 10 is V and X 21 is Q and X 24 is S.

[0411] In some embodiments, X 19 is V and X 20 is R and X 21 is K. In some embodiments, X 19 is V and X 20 is R and X 21 is Q. In some embodiments, X 19 is V and X 20 is R and X 24 is S. In some embodiments, X19 is V and X 20 is R and X 24 is E.

[0412] In some embodiments, X 19 is A and X 20 is R and X 21 is K. In some embodiments, X 19 is A and X 20 is R and X 21 is Q. In some embodiments, X 19 is A and X 20 is R and X 24 is S. In some embodiments, X 19 is A and X 20 is R and X 24 is E.

[0413] In some embodiments, X 20 is R and X 21 is K and X 24 is S. In some embodiments, X 20 is R and X 21 is K and X 24 is E.

[0414] In some embodiments, X 20 is K and X 21 is K and X 24 is S. In some embodiments, X 20 is K and X 21 is K and X 24 is E. In some embodiments, X 20 is K and X 21 is K and X 24 is E and the peptide is X 20 and the side chain of X 24 The lactam bridge is formed via an amide bond between the side chains of

[0415] Conjugation of a lipophilic substituent to any of the peptides, optionally via a spacer In some embodiments, any of the disclosed polypeptides is optionally substituted with one or more lipophilic substituents, each optionally via a spacer, where "lipophilic substituent" and "spacer" are defined herein. In some embodiments, any of the disclosed polypeptides comprising an amino acid sequence selected from the group consisting of the amino acid sequences represented by any of the consensus sequences SEQ ID NO:1 through SEQ ID NO:162 and SEQ ID NO:1000 through SEQ ID NO:1162, each optionally via a spacer, can comprise or be modified with one or more lipophilic substituents, or can be further modified by the covalent attachment of one or more lipophilic substituents, each optionally via a spacer. In some embodiments, a lipophilic substituent can be attached to an amino group of the polypeptide (e.g., the ε-amino group of a lysine residue) via a carboxyl group of the lipophilic substituent or, optionally, an amino group of the spacer, where the carboxyl group of the spacer forms an amide bond with the ε-amino group of the lysine residue.

[0416] lipophilic substituent Conjugation of one or more "lipophilic substituents," each optionally via a "spacer," to any of the disclosed polypeptides of the present invention is intended to promote binding to serum albumin and thereby prolong the action of the polypeptide by delaying renal clearance of the conjugated polypeptide. As used herein, a "lipophilic substituent" includes substituents containing 4 to 40 carbon atoms, 8 to 25 carbon atoms, 12 to 22 carbon atoms, or 6 to 20 carbon atoms. The lipophilic substituent can be attached to an amino group of the polypeptide (e.g., the ε-amino group of a lysine residue) through the carboxyl group of the lipophilic substituent, or can be attached to an amino group of a spacer, which further forms an amide bond with the amino group of the amino acid (e.g., lysine) residue to which it is attached. In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with or without an optional spacer, as defined in more detail below.

[0417] In some embodiments, the lipophilic substituent comprises a straight-chain or branched-chain alkyl group. In some embodiments, the lipophilic substituent is an acyl group of a straight-chain or branched-chain fatty acid. In some embodiments, the lipophilic substituent is an acyl group of a straight-chain or branched-chain fatty acid further substituted with one or more carboxylic acid and / or hydroxamic acid groups.

[0418] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each without an optional spacer. In some embodiments, the lipophilic substituent is —CO(CH) 16 In some embodiments, the lipophilic substituent is —CO(CH) 18 In some embodiments, the lipophilic substituent is —CO(CH) 20 CO2H.

[0419] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each without an optional spacer. In some embodiments, the lipophilic substituent is represented by Formula I: -CO-(CH2) m -Z Formula I is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; The lipophilic substituent forms an amide bond between an amino group of the disclosed polypeptide (eg, the ε-amino group of lysine) and the CO-group of the lipophilic substituent.

[0420] In some embodiments, Z is -COH. In some embodiments, m is 14 to 20. In some embodiments, the lipophilic substituent is covalently attached to the isolated polypeptide via a spacer. In some embodiments, the lipophilic substituent -CO-(CH) m-Z is linked to the amino group of the isolated polypeptide via a spacer, which forms a bridge between the amino group of the isolated polypeptide and the CO-group of the lipophilic substituent.

[0421] In some embodiments, m is selected from the group consisting of 4-20, 8-20, 12-20, 14-20, 16-20, 14, 16, 18, and 20.

[0422] In some embodiments, Z is —COH and the lipophilic substituent is of the formula —CO—(CH) m In some embodiments, the compound has -CO-(CH) m -Z is -CO-(CH2)4-CO2H, -CO-(CH2)5-CO2H, -CO-(CH2)6-CO2H, -CO-(CH2)7-CO2H, -CO-(CH2)8-CO2H, -CO-(CH2)9-CO2H, -CO-(CH2) 10 -CO2H, -CO-(CH2) 11 -CO2H, -CO-(CH2) 12 -CO2H, -CO-(CH2) 13 -CO2H, -CO-(CH2) 14 -CO2H, -CO-(CH2) 15 -CO2H, -CO-(CH2) 16 -CO2H, -CO-(CH2) 17 -CO2H, -CO-(CH2) 18 -CO2H, -CO-(CH2) 19 -CO2H, -CO-(CH2) 20 -CO2H.

[0423] In some embodiments, the lipophilic substituent is —CO—(CH) 14 In some embodiments, the lipophilic substituent is —CO—(CH) 16 In some embodiments, the lipophilic substituent is —CO—(CH) 18 In some embodiments, the lipophilic substituent is —CO—(CH) 20 -CO2H.

[0424] In some embodiments, Z is -CH3 and the lipophilic substituent is of the formula -CO-(CH2) m In some embodiments, the compound has -CO-(CH) m -Z is -CO-(CH2)4-CH3, -CO-(CH2)5-CH3, -CO-(CH2)6-CH3, -CO-(CH2)7-CH3, -CO-(CH2)8-CH3, -CO-(CH2)9-CH3, -CO-(CH2) 10 -CH3, -CO-(CH2) 11 -CH3, -CO-(CH2) 12 -CH3, -CO-(CH2) 13 -CH3, -CO-(CH2) 14 -CH3, -CO-(CH2) 15 -CH3, -CO-(CH2) 16 -CH3, -CO-(CH2) 17 -CH3, -CO-(CH2) 18 -CH3, -CO-(CH2) 19 -CH3 and -CO-(CH2) 20 -CH3.

[0425] Lipophilic Substituents and Spacers In some embodiments, the lipophilic substituent is attached to the parent peptide by a "spacer." In some embodiments, provided herein is any of the disclosed polypeptides comprising an amino acid sequence selected from the group consisting of the amino acid sequences represented by any of the consensus sequences SEQ ID NO: 1 through SEQ ID NO: 143, comprising a lipophilic substituent, wherein the lipophilic substituent is linked to the ε-amino group of a lysine via a spacer, which forms a bridge between the amino group of the disclosed polypeptide and the CO- group of the lipophilic substituent.

[0426] In some embodiments, the spacer comprises one or more amino acids, e.g., a single amino acid such as Glu, Asp, Gly, or Lys, a dipeptide such as 2(Glu), Glu-Gly, or a polypeptide such as 3(Glu), 4(Glu), 2(Glu)-Gly, etc. In some embodiments, when the spacer comprises one or more amino acids, e.g., Glu, Asp, Gly, or Lys, one carboxyl group of the spacer can form an amide bond with an amino group of a disclosed polypeptide, and an amino group of the spacer can form an amide bond with a carboxyl group of a lipophilic substituent.

[0427] In some embodiments, when the spacer comprises Glu or Asp, which further comprises a carboxylic acid terminal side chain, the terminal carboxyl group of the side chain of the Glu or Asp-containing spacer can form an amide bond with an amino group of a disclosed polypeptide, and the amino group of the Glu or Asp-containing spacer, i.e., γGlu or βAsp, can form an amide bond with the carboxyl group of the lipophilic substituent.

[0428] In some embodiments, the spacer is -γGlu-γGlu-dpeg-. In some embodiments, the spacer is -γGlu-γGlu-dpeg-γGlu-γGlu-. In some embodiments, the spacer is -[COCH2(OCH2CH2)2NH]2-γGlu-.

[0429] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some non-limiting embodiments, the lipophilic substituent and the spacer form a monovalent group selected from the group consisting of those listed in Table 3: [Table 3]

[0430] In some embodiments, the lipophilic substituent and spacer have Formula II: -(Y) n -CO-(CH2) m -Z Formula II Form a monovalent group of (In the formula, Y is selected from the group consisting of γGlu, Asp, Lys, and Gly; Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0431] In some embodiments, Z is -CO2H. In some embodiments, m is 14-20. In some embodiments, Y is γGlu. In some embodiments, n is 1-5.

[0432] In some embodiments, Y is selected from the group consisting of γGlu and GIy. In some embodiments, Y is γGlu. In some embodiments, Y is GIy.

[0433] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula III: -(V) r -(Y) n -CO-(CH2) m -Z Formula III is a monovalent group of (In the formula, V is -[COCH2(OCH2CH2) t NH]-; Y is selected from the group consisting of γGlu, Asp, and Gly; Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10; r is 1 to 6; t is 1 to 6).

[0434] In some embodiments, Z is —CO 2 H. In some embodiments, Z is —CH 3 .

[0435] In some embodiments, Y is γGlu. In some embodiments, Y is Asp. In some embodiments, Y is GIy.

[0436] In some embodiments, m is selected from the group consisting of 4 to 20, 8 to 20, 12 to 20, 14 to 20, 16 to 20, 14, 16, 18, and 20. In some embodiments, m is 14 to 20.

[0437] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1-5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0438] In some embodiments, r is 1 to 3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5.

[0439] In some embodiments, t is 1 to 3. In some embodiments, t is selected from the group consisting of 1, 2, 3, 4, 5, and 6.

[0440] In some embodiments, Y is γGlu; ​​Z is —CO 2 H; m is 16; n is 1; r is 2; and t is 2.

[0441] In one embodiment, -(V) r -(Y) n - is -[COCH2(OCH2CH2)2NH]2-γGlu-.

[0442] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula IV: -(Y1) n1 -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z formula IV It is of During the ceremony, Z is -CH3 or -CO2H; m is 4 to 24; Y1 is selected from the group consisting of γGlu, Asp, and Gly; Y2 is selected from the group consisting of γGlu, Asp, and Gly; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; r is 1 to 8; n1 is 0 to 10; n2 is a number between 0 and 10.

[0443] In some embodiments, Z is —CO 2 H. In some embodiments, Z is —CH 3 .

[0444] In some embodiments, m is selected from the group consisting of 4 to 20, 8 to 20, 12 to 20, 14 to 20, 16 to 20, 14, 16, 18, and 20. In some embodiments, m is 14 to 20.

[0445] In some embodiments, Y is γGlu. In some embodiments, Y is Asp. In some embodiments, Y is GIy.

[0446] In some embodiments, Y2 is γGlu. In some embodiments, Y2 is Asp. In some embodiments, Y2 is GIy.

[0447] In some embodiments, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n1 is 0 to 3. In some embodiments, n1 is 0. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5.

[0448] In some embodiments, n2 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n2 is 0-3. In some embodiments, n2 is 0. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, n2 is 3. In some embodiments, n2 is 4. In some embodiments, n2 is 5.

[0449] In some embodiments, r is 1-3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, r is 6. In some embodiments, r is 7. In some embodiments, r is 8.

[0450] In some embodiments, r is 1, n1 is 2, and n2 is 0.

[0451] In some embodiments, r is 1, n1 is 2, and n2 is 2.

[0452] In some embodiments, Y1 is γGlu and Y2 is γGlu.

[0453] In some embodiments, Y1 is γGlu and n2 is 0.

[0454] In some embodiments, Y1 is γGlu, r is 1, n1 is 2, and n2 is 0.

[0455] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - is selected from the group consisting of -γGlu-γGlu-dpeg-, -γGlu-γGlu-dpeg-γGlu-γGlu-, -γGlu-γGlu-dpeg-γGlu-, -γGlu-γGlu-dpeg-dpeg-, -γGlu-γGlu-dpeg-dpeg-γGlu-, -dpeg-dpeg-γGlu-, -γGlu-γGlu-γGlu-dpeg-, and -γGlu-dpeg-.

[0456] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula V: -(γGlu) n -CO-(CH2) m -Z Formula V is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0457] In some embodiments, Z is —CH 3 . In some embodiments, Z is —CO 2 H.

[0458] In some embodiments, m is 14-20.

[0459] In some embodiments, m is selected from the group consisting of 4-20, 8-20, 12-20, 14-20, 16-20, 14, 16, 18, and 20.

[0460] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0461] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula VI: -(γGlu) n -(Gly)-CO-(CH2) m -Z Equation VI is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0462] In some embodiments, (γGlu) n is selected from the group consisting of γGlu; ​​2(γGlu); 3(γGlu); 4(γGlu); and 5(γGlu). In some embodiments, -(γGlu) n -(Gly)- is selected from the group consisting of 2(γGlu),Gly; and 3(γGlu),Gly.

[0463] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula VII: -(Gly)-(γGlu) n -(CO-(CH2) m -Z Formula VII is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0464] In some embodiments, certain variables represented in certain preceding formulas include:

[0465] In some embodiments, Z is —CH 3 . In some embodiments, Z is —CO 2 H.

[0466] In some embodiments, m is selected from the group consisting of 4-20, 8-20, 12-20, 14-20, 16-20, 14, 16, 18, and 20.

[0467] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0468] In some embodiments, n is 1 and Z is -CO2H. In some embodiments, n is 1 and Z is -CH3. In some embodiments, n is 2 and Z is -CO2H. In some embodiments, n is 2 and Z is -CH3. In some embodiments, n is 3 and Z is -CO2H. In some embodiments, n is 3 and Z is -CH3. In some embodiments, n is 4 and Z is -CO2H. In some embodiments, n is 4 and Z is -CH3. In some embodiments, n is 5 and Z is -CO2H. In some embodiments, n is 5 and Z is -CH3.

[0469] In some embodiments, n is 1, Z is -CO2H, and m is 14 to 20. In some embodiments, n is 1, Z is -CO2H, and m is 14. In some embodiments, n is 1, Z is -CO2H, and m is 16. In some embodiments, n is 1, Z is -CO2H, and m is 18.

[0470] In some embodiments, n is 1, Z is -CH3, and m is 14 to 20. In some embodiments, n is 1, Z is -CH3, and m is 14. In some embodiments, n is 1, Z is -CH3, and m is 16. In some embodiments, n is 1, Z is -CH3, and m is 18.

[0471] In some embodiments, n is 2, Z is -CO2H, and m is 14 to 20. In some embodiments, n is 2, Z is -CO2H, and m is 14. In some embodiments, n is 2, Z is -CO2H, and m is 16. In some embodiments, n is 2, Z is -CO2H, and m is 18.

[0472] In some embodiments, n is 2, Z is -CH3, and m is 14 to 20. In some embodiments, n is 2, Z is -CH3, and m is 14. In some embodiments, n is 2, Z is -CH3, and m is 16. In some embodiments, n is 2, Z is -CH3, and m is 18.

[0473] In some embodiments, n is 3, Z is -CO2H, and m is 14 to 20. In some embodiments, n is 3, Z is -CO2H, and m is 14. In some embodiments, n is 3, Z is -CO2H, and m is 16. In some embodiments, n is 3, Z is -CO2H, and m is 18.

[0474] In some embodiments, n is 3, Z is -CH3, and m is 14 to 20. In some embodiments, n is 3, Z is -CH3, and m is 14. In some embodiments, n is 3, Z is -CH3, and m is 16. In some embodiments, n is 3, Z is -CH3, and m is 18.

[0475] In some embodiments, n is 4, Z is -CO2H, and m is 14 to 20. In some embodiments, n is 4, Z is -CO2H, and m is 14. In some embodiments, n is 4, Z is -CO2H, and m is 16. In some embodiments, n is 4, Z is -CO2H, and m is 18.

[0476] In some embodiments, n is 4, Z is -CH3, and m is 14 to 20. In some embodiments, n is 4, Z is -CH3, and m is 14. In some embodiments, n is 4, Z is -CH3, and m is 16. In some embodiments, n is 4, Z is -CH3, and m is 18.

[0477] In some embodiments, n is 5, Z is -CO2H, and m is 14 to 20. In some embodiments, n is 5, Z is -CO2H, and m is 14. In some embodiments, n is 5, Z is -CO2H, and m is 16. In some embodiments, n is 5, Z is -CO2H, and m is 18.

[0478] In some embodiments, n is 5, Z is -CH3, and m is 14 to 20. In some embodiments, n is 5, Z is -CH3, and m is 14. In some embodiments, n is 5, Z is -CH3, and m is 16. In some embodiments, n is 5, Z is -CH3, and m is 18.

[0479] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula VIII: -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2) m -Z Formula VIII is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; Y1 is selected from the group consisting of γGlu, Asp, and Gly; Y2 is selected from the group consisting of γGlu, Asp, and Gly; V is -[COCH2(OCH2CH2) t NH]-; r is 1 to 6; n1 is 0 to 10; n2 is 0 to 10; t is 1 to 6).

[0480] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula IX: -(Y) n -(V) r -CO-(CH2) m -Z Formula IX is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; Y is selected from the group consisting of γGlu, Asp, and Gly; V is -[COCH2(OCH2CH2) t NH]-; r is 1 to 6; n is 1 to 10; t is 1 to 6).

[0481] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each accompanied by a spacer. In some embodiments, the lipophilic substituent and the spacer are represented by the formula X: -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z formula is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; Y2 is selected from the group consisting of γGlu, Asp, and Gly; r is 1 to 8; n2 is 0 to 10).

[0482] In some embodiments, -(dpeg) r -(Y2) n2 - is selected from the group consisting of dpeg,γGlu; ​​and dpeg,dpeg,γGlu.

[0483] In one embodiment, -(dpeg) r -(Y2) n2 - is -dpeg-dpeg-γGlu-.

[0484] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with an optional spacer. In some embodiments, the lipophilic substituent and the spacer are represented by Formula XI: -(Y1) n1 -(dpeg) r -CO-(CH2) m -Z Formula XI is a monovalent group of (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; Y1 is selected from the group consisting of γGlu, Asp, and Gly; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; r is 1 to 8; n1 is 0 to 10).

[0485] In some embodiments, Z is —CO 2 H.

[0486] In some embodiments, m is selected from the group consisting of 4 to 20, 8 to 20, 12 to 20, 14 to 20, 16 to 20, 14, 16, 18, and 20. In some embodiments, m is 14 to 20.

[0487] In some embodiments, Y is γGlu. In some embodiments, Y is Asp. In some embodiments, Y is GIy.

[0488] In some embodiments, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n1 is 0 to 3. In some embodiments, n1 is 0. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5.

[0489] In some embodiments, r is 1-3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, r is 6. In some embodiments, r is 7. In some embodiments, r is 8.

[0490] In some embodiments, r is 1 and n1 is 2.

[0491] In some embodiments, Y1 is γGlu, r is 1, and n1 is 2.

[0492] In some embodiments, -(Y1) n1 -(dpeg) r - is selected from the group consisting of -γGlu-γGlu-dpeg-, -γGlu-γGlu-dpeg-dpeg-, -γGlu-γGlu-γGlu-dpeg-, and -γGlu-dpeg-.

[0493] Further Exemplary Spacers In some embodiments, the spacer is represented by Formula XII: -N(R1)(CHR2) p CO-[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r Formula XII containing a divalent group of (In the formula, each R1 and R3 is hydrogen or C1-C4 alkyl; each R2 is H or CO2H; p is 1, 2, 3, 4, 5, or 6; q is 1, 2, or 3; r is 0 or 1, This spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent).

[0494] In some embodiments, the spacer is represented by Formula XIII: [-N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r Formula XIII containing a divalent group of (In the formula, each R3 is hydrogen or C1-C4 alkyl; q is 1, 2, or 3; r is 0 or 1, This spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent).

[0495] In some embodiments, certain variables represented in certain formulas include:

[0496] In some embodiments, R1 is hydrogen. In some embodiments, R3 is hydrogen. In some embodiments, each R1 and each R3 is hydrogen.

[0497] In some embodiments, at least one R2 is CO2H. In some embodiments, one R2 is CO2H.

[0498] In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0499] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[0500] In some embodiments, r is 0. In some embodiments, r is 1.

[0501] In some embodiments, the spacer is γ-glutamyl, i.e., —NH(CHCOH)(CH)CO—. In some embodiments, the spacer is γ-aminobutanoyl, i.e., —NH(CH)CO—. In some embodiments, the spacer is β-asparagyl, i.e., —NH(CHCOH)(CH)CO—. In some embodiments, the spacer is —NH(CH)CO—. In some embodiments, the spacer is glycyl. In some embodiments, the spacer is β-alanyl.

[0502] In some embodiments, the spacer is -NHCH(COH)(CH)CO--[N(R)((CH)O(CH)O) q (CH2)CO-] r In some embodiments, the spacer is -NH(CH2)3CO--[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] rIn some embodiments, the spacer is -NHCH(COH)(CH)CO-NH((CH)O(CH)O)(CH)CO-. In some embodiments, the spacer is -NH(CH)CO-NH((CH)O(CH)O)(CH)CO-. In some embodiments, the spacer is -NHCH(COH)CHCO--[N(R)((CH)O(CH)O) q (CH2)CO-]r. In some embodiments, the spacer is -NH(CH2)2CO--[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r is.

[0503] In some embodiments, the spacer is represented by Formula XIV: -(Y) n - Formula XIV containing a divalent group of (In the formula, Y is selected from the group consisting of γGlu, Asp, Lys, and Gly; n is 1 to 10).

[0504] In some embodiments, Y is selected from the group consisting of γGlu and GIy. In some embodiments, Y is γGlu. In some embodiments, Y is GIy.

[0505] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0506] In some embodiments, the spacer is represented by Formula XV: -(γGlu) n - Formula XV containing a divalent group of (In the formula, n is 1 to 10).

[0507] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0508] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0509] In some embodiments, the spacer is represented by Formula XVI: -(γGlu) n -(Gly)- Formula XVI containing a divalent group of (In the formula, n is 1 to 10).

[0510] In some embodiments, (γGlu) n is selected from the group consisting of γGlu; ​​2(γGlu); 3(γGlu); 4(γGlu); and 5(γGlu). In some embodiments, -(γGlu) n -(Gly)- is selected from the group consisting of 2(γGlu),Gly; and 3(γGlu),Gly.

[0511] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0512] In some embodiments, the spacer is represented by Formula XVII: -(Gly)-(γGlu) n - Formula XVII containing a divalent group of (In the formula, n is 1 to 10).

[0513] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0514] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0515] In some embodiments, the spacer is represented by Formula XVIII: -(V) r -(Y) n - Formula XVIII containing a divalent group of (In the formula, Y is selected from the group consisting of γGlu, Asp, and Gly; V is -[COCH2(OCH2CH2) t NH]-; r is 1 to 6; n is 1 to 10; t is 1 to 6).

[0516] In some embodiments, Y is γGlu. In some embodiments, Y is Asp. In some embodiments, Y is GIy.

[0517] In some embodiments, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is 1-5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0518] In some embodiments, r is 1 to 3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5.

[0519] In some embodiments, t is 1 to 3. In some embodiments, t is selected from the group consisting of 1, 2, 3, 4, 5, and 6.

[0520] In one embodiment, -(V) r -(Y) n - is -[COCH2(OCH2CH2)2NH]2-γGlu-.

[0521] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0522] In some embodiments, the spacer is represented by Formula XIX: -(Y1) n1 -(dpeg) r -(Y2) n2 - Formula XIX containing a divalent group of (In the formula, Y1 is selected from the group consisting of γGlu, Asp, and Gly; Y2 is selected from the group consisting of γGlu, Asp, and Gly; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; r is 1 to 8; n1 is 0 to 10; n2 is 0 to 10).

[0523] In some embodiments, Y is γGlu. In some embodiments, Y is Asp. In some embodiments, Y is GIy.

[0524] In some embodiments, Y2 is γGlu. In some embodiments, Y2 is Asp. In some embodiments, Y2 is GIy.

[0525] In some embodiments, n1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n1 is 0 to 3. In some embodiments, n1 is 0. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5.

[0526] In some embodiments, n2 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n2 is 0-3. In some embodiments, n2 is 0. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, n2 is 3. In some embodiments, n2 is 4. In some embodiments, n2 is 5.

[0527] In some embodiments, r is 1-3. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, r is 6. In some embodiments, r is 7. In some embodiments, r is 8.

[0528] In some embodiments, r is 1, n1 is 2, and n2 is 0.

[0529] In some embodiments, r is 1, n1 is 2, and n2 is 2.

[0530] In some embodiments, Y1 is γGlu and Y2 is γGlu.

[0531] In some embodiments, Y1 is γGlu and n2 is 0.

[0532] In some embodiments, Y1 is γGlu, r is 1, n1 is 2, and n2 is 0.

[0533] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - is selected from the group consisting of -γGlu-γGlu-dpeg-, -γGlu-γGlu-dpeg-γGlu-γGlu-, -γGlu-γGlu-dpeg-γGlu-, -γGlu-γGlu-dpeg-dpeg-, -γGlu-γGlu-dpeg-dpeg-γGlu-, -dpeg-dpeg-γGlu-, -γGlu-γGlu-γGlu-dpeg-, and -γGlu-dpeg-.

[0534] In some embodiments, the spacer forms a bridge between an amino group of a disclosed polypeptide and a CO- group of a lipophilic substituent, hi some embodiments, one end of the spacer forms a covalent bond with an amino group of a disclosed polypeptide, and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0535] Thus, in some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by consensus sequences selected from the group consisting of SEQ ID NO: 1000 to SEQ ID NO: 1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and optionally a spacer.

[0536] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by consensus sequences selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent of Formula I.

[0537] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by consensus sequences selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent of Formula I and a spacer selected from the group consisting of those described by Formulas XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX.

[0538] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by a consensus sequence selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and a spacer selected from the group consisting of those described by Formulas I, II, III, IV, V, VI, VII, VIII, IX, X, and XI.

[0539] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by a consensus sequence selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and a spacer selected from the group consisting of those described by Formula I.

[0540] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by a consensus sequence selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and a spacer selected from the group consisting of those described by Formula II.

[0541] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by a consensus sequence selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and a spacer selected from the group consisting of those described by Formula III.

[0542] In some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by a consensus sequence selected from the group consisting of SEQ ID NO:1000 to SEQ ID NO:1162, or a pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and a spacer selected from the group consisting of those described by Formula IV.

[0543] As used herein, (γGlu) and 2(γGlu) both mean -(γGlu)-(γGlu)- or -CO(CH)CH(COH)NH-CO(CH)CH(COH)NH-; (γGlu) and 3(γGlu) both mean -(γGlu)-(γGlu)-(γGlu)- or -CO(CH)CH(COH)NH-CO(CH)CH(COH)NH-CO(CH)CH(COH)NH-; and so forth; when a variable occurs more than once in a given formula, each occurrence of that variable is determined independently. For example, for the -(Y)- group, where Y can be γGlu, Asp, Lys, or Gly, each Y is independently selected to be one of four amino acids. Thus, by non-limiting example, -(Y)3- can be -(γGlu)-(γGlu)-(γGlu)-, -(γGlu)-(Asp)-(γGlu)-, -(Gly)-(Asp)-(γGlu)-, or -(Gly)-(γGlu)-(γGlu)-.

[0544] bridging part In some embodiments, any of the disclosed polypeptides are optionally substituted with one or more bridging moieties. As used herein, the term "bridging moiety" refers to a covalent bond or any bivalent linker or moiety that connects two side chains of two separate amino acid residues. In some embodiments, any of the disclosed polypeptides are optionally substituted with one or more lactam bridging moieties. As used herein, the term "lactam bridging moiety" refers to a lactam bridge or lactam bond that connects the amino-containing side chain and the carboxy-containing side chain of two separate amino acid residues.

[0545] Exemplary Compound: GLP-1 Receptor Agonist Polypeptide In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Table 4: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [ka] As used herein, dpeg represents -COCH2O(CH2)2O(CH2)2NH-; and dpeg-dpeg represents -COCH2O(CH2)2O(CH2)2NH-COCH2O(CH2)2O(CH2)2NH; Carboxy-terminal amino acid, i.e., -S 39 -S shown as (NH2) 39 is represented as -NH-CH(CHOH)-CONH.

[0546] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-162, or a pharmaceutically acceptable salt thereof.

[0547] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 45, 46, 54, 55, 62, 68, 70, 71, 74, 75, 77, 78, 85, 86, 87, 88, 89, 92, 94, 103, 107, 108, 111, 112, 114, 115, 120, 132, 134, 139, 147, and 149.

[0548] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, 54, 55, 62, 68, 70, 74, 75, 77, 78, 85, 86, 87, 88, 89, 92, 94, 103, 107, 108, 111, 112, 114, 115, 120, 132, 134, 139, 147, and 149.

[0549] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 45, 46, 54, 55, 77, 78, 108, 111, 112, 114, 115, 120, 132, 134, and 139, or a pharmaceutically acceptable salt thereof.

[0550] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, 54, 55, 77, 78, 108, 111, 112, 114, 115, 120, 132, 134, and 139.

[0551] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 115, 120, and 132, or a pharmaceutically acceptable salt thereof.

[0552] In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0553] In some embodiments, the present invention provides a compound set forth in Table 4 above, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-162, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is an acetate salt. In some embodiments, the pharmaceutically acceptable salt is a trifluoroacetic acid (TFA) salt. In some embodiments, the pharmaceutically acceptable salt is a hydrochloric acid (HCl) salt.

[0554] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 55 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 55.

[0555] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 115. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 115. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 115.

[0556] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In one embodiment, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 120.

[0557] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 132 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 132. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0558] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1000-1162, or a pharmaceutically acceptable salt thereof.

[0559] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1006, 1045, 1046, 1054, 1055, 1062, 1068, 1070, 1071, 1074, 1075, 1077, 1078, 1085, 1086, 1087, 1088, 1089, 1092, 1094, 1103, 1107, 1108, 1111, 1112, 1114, 1115, 1120, 1132, 1134, 1139, 1147, and 1149.

[0560] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1006, 1045, 1046, 1054, 1055, 1077, 1078, 1108, 1111, 1112, 1114, 1115, 1120, 1132, 1134, and 1139, or a pharmaceutically acceptable salt thereof.

[0561] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1055, 1115, 1120, and 1132, or a pharmaceutically acceptable salt thereof.

[0562] In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1055, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1115, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1120, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1132, or a pharmaceutically acceptable salt thereof.

[0563] In some embodiments, the present invention provides a compound set forth in Table 6 above, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1000-1162, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is an acetate salt. In some embodiments, the pharmaceutically acceptable salt is a trifluoroacetic acid (TFA) salt. In some embodiments, the pharmaceutically acceptable salt is a hydrochloric acid (HCl) salt.

[0564] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1055 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1055. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1055. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1055.

[0565] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1115 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1115. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1115. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1115.

[0566] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1120 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1120. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1120. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1120.

[0567] In one embodiment, the compound is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1132 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is an acetate salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1132. In some embodiments, the compound is a TFA salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1132. In some embodiments, the compound is an HCl salt of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1132. [Table 7]

[0568] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2117, 2118, 2119, 2120, 2121, 2135, 2136, 2137, 2138, 2140, and 2141, or a pharmaceutically acceptable salt thereof.

[0569] In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2117, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2118, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2119, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2120, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2121, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2135, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2136, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2137, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:2138, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 2140, or a pharmaceutically acceptable salt thereof. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 2141, or a pharmaceutically acceptable salt thereof.

[0570] In some embodiments, the pharmaceutically acceptable salt is an acetate salt. In some embodiments, the pharmaceutically acceptable salt is a trifluoroacetic acid (TFA) salt. In some embodiments, the pharmaceutically acceptable salt is a hydrochloric acid (HCl) salt.

[0571] Polypeptide Intermediates and General Methods for Providing the Compounds The compounds of the invention may generally be prepared or isolated by synthetic and / or semi-synthetic methods known to those skilled in the art for similar compounds, and by methods detailed in the Examples herein.

[0572] In the following schemes, where a particular protecting group ("PG"), leaving group ("LG"), or transformation condition is shown, one skilled in the art will recognize that other protecting groups, leaving groups, and transformation conditions are suitable and contemplated. Such groups and transformations are described in detail in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, M.B. Smith and J. March, 5 th Edition, John Wiley & Sons, 2001, Comprehensive Organic Transformations, RC Larock, 2 nd Edition, John Wiley & Sons, 1999, and Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999, each of which is incorporated herein by reference in its entirety.

[0573] As used herein, the phrase "leaving group" (LG) includes, but is not limited to, halogen (e.g., fluoride, chloride, bromide, iodide), sulfonate (e.g., mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium, and the like.

[0574] As used herein, the phrase "oxygen protecting group" includes, for example, carbonyl protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known in the art and are described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rdedition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters (e.g., acetyl, benzyl), allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, benzyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formates, benzoyl formates, chloroacetates, trifluoroacetates, methoxyacetates, triphenylmethoxyacetates, p-chlorophenoxyacetates, 3-phenylpropionates, 4-oxopentanoates, 4,4-(ethylenedithio)pentanoates, pivaloates (trimethylacetyl), crotonates, 4-methoxycrotonates, benzoates, p-benzylbenzoates, 2,4,6-trimethylbenzoates, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl ether, triethylsilyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, triisopropylsilyl ether, and other trialkylsilyl ethers. Alkyl ethers include methyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trityl ether, t-butyl ether, allyl ether, and allyloxycarbonyl ether or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl ether, methylthiomethyl ether, (2-methoxyethoxy)methyl ether, benzyloxymethyl ether, beta-(trimethylsilyl)ethoxymethyl ether, and tetrahydropyranyl ether.Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2- and 4-picolyl.

[0575] Amino protecting groups are well known in the art and are described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Suitable amino-protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allylamines, amides, and the like. Examples of such groups include t-butyloxycarbonyl (Boc), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (Cbz), allyl, phthalimido, benzyl (Bn), dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dmb), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like.

[0576] In certain embodiments, the present invention also relates to synthetic peptide intermediates of the disclosed GLP-1 receptor agonists. In some embodiments, the polypeptide intermediates of the present disclosure have the amino acid sequence of SEQ ID NO: 400: HX2X3GTX6X7X8X9X 10 SX 12 X 13 X 14 EX 16 X 17 X 18 X 19 X20 X 21 FIX 24 WLKX 28 GGPX 32 SGAPPPS-(OH / NH) (SEQ ID NO: 400), or a pharmaceutically acceptable salt thereof, wherein: X2 is A, 2-aminoisobutyric acid (Aib), or G; X3 is E or N-methylGlu; X6 is F or Y; X7 is S or T; X8 is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X9 is D or E; X 10 is I, L, N-methylLeu, or V; X 12 is E, K, Q, or S; X 13 is Aib, E, K, Q, S, W, or Y; X 14 is E, R, I, K, L, M, Q, or Y; X 16 is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn); X 17 is E, K, or Q; X 18 is A, K, S, or Y; X 19 is A, K, or V; X 20 is E, K, or R; X 21 is Aib, E, H, K, L, Q, or Y; X 24 is A, Aib, E, K, Q, S, or Y; X 28 is D, E, K, N, Q, S, or Y; and X 32 is Dap, H, K, R, or S; wherein each Dab, Dap, K or Orn independently represents a Dab, Dap, K or Orn optionally covalently linked to a protecting group, or a spacer optionally linked to a protecting group).

[0577] In some embodiments, the spacer is not attached to a protecting group.

[0578] In one embodiment, the synthetic peptide intermediate is a K covalently bonded to a protecting group. 12 Or K 27 or the precursor of exenatide, i.e., HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 300), optionally with a spacer attached to a protecting group.

[0579] In some embodiments, X2 is Aib.

[0580] In some embodiments, X 24 If S, then X 10 is V.

[0581] In some embodiments, X 14 is Y.

[0582] In some embodiments, X 14 is M and X 16 is K.

[0583] In some embodiments, X 16 is K.

[0584] In some embodiments, X 16 If E, then X 21 is K or E. In some embodiments, X 16 If E, then X 21 is K. In some embodiments, X 16 If E, then X 21 is E.

[0585] In some embodiments, X2 is A. In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0586] In some embodiments, X3 is E. In some embodiments, X3 is N-methylGlu.

[0587] In some embodiments, X6 is F. In some embodiments, X6 is Y.

[0588] In some embodiments, X7 is S. In some embodiments, X7 is T.

[0589] In some embodiments, X8 is Dap. In some embodiments, X8 is E. In some embodiments, X8 is K. In some embodiments, X8 is N. In some embodiments, X8 is N-methyl Ser. In some embodiments, X8 is Q. In some embodiments, X8 is S. In some embodiments, X8 is s. In some embodiments, X8 is Y.

[0590] In some embodiments, X9 is D. In some embodiments, X9 is E.

[0591] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is N-methylLeu. In some embodiments, X 10 is V.

[0592] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q. In some embodiments, X 12 is S.

[0593] In some embodiments, X 13 In some embodiments, X is Aib. 13 is E. In some embodiments, X 13 is K. In some embodiments, X 13 is Q. In some embodiments, X 13 is S. In some embodiments, X 13 is W. In some embodiments, X 13 is Y.

[0594] In some embodiments, X 14 is E. In some embodiments, X 14 is R. In some embodiments, X 14 is I. In some embodiments, X 14 is K. In some embodiments, X 14 is L. X 14 is M. In some embodiments, X 14 is Q. In some embodiments, X 14 is Y.

[0595] In some embodiments, X 16 In some embodiments, X is Dab. 16 In some embodiments, X is Dap. 16 is E. In some embodiments, X 16 is K. In some embodiments, X 16 is k. In some embodiments, X 16 is Orn.

[0596] In some embodiments, X 17 is E. In some embodiments, X 17 is K. In some embodiments, X 17 is Q.

[0597] In some embodiments, X 18 is A. In some embodiments, X 18 is K. In some embodiments, X18 is S. In some embodiments, X 18 is Y.

[0598] In some embodiments, X 19 is A. In some embodiments, X 19 is K. In some embodiments, X 19 is V.

[0599] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0600] In some embodiments, X 21 In some embodiments, X is Aib. 21 is E. In some embodiments, X 21 is H. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q. In some embodiments, X 21 is Y.

[0601] In some embodiments, X 24 is A. In some embodiments, X 24 In some embodiments, X is Aib. 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is Q. In some embodiments, X 24 is S. In some embodiments, X 24 is Y.

[0602] In some embodiments, X 28 is D. In some embodiments, X 28 is E. In some embodiments, X 28 is K. In some embodiments, X28 is N. In some embodiments, X 28 is Q. In some embodiments, X 28 is S. In some embodiments, X 28 is Y.

[0603] In some embodiments, X 32 In some embodiments, X is Dap. 32 is H. In some embodiments, X 32 is K. In some embodiments, X 32 is R. In some embodiments, X 32 is S.

[0604] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0605] In some embodiments, a polypeptide intermediate of the present disclosure has the amino acid sequence of SEQ ID NO: 405: HX2EGTFTX8DX 10 SX 12 QYEKX 17 X 18 X 19 X 20 X 21 FIX 24 WLKX 28 GGPX 32 SGAPPPS-(OH / NH) (SEQ ID NO: 405), or a pharmaceutically acceptable salt thereof, wherein: X2 is 2-aminoisobutyric acid (Aib), or G; X8 is N or S; X 10 is I, L, or V; X 12 is E, K, or Q; X 17 is E or K; X 18 is A or Y; X 19 is A or V; X 20 is E, K, or R; X 21 is E, K, L, or Q; X 24 is E, K, or S; X 28 is N or Q; X 32 is H or S; where each K represents a lysine optionally covalently bound to a protecting group, or a spacer optionally bound to a protecting group).

[0606] In some embodiments, the spacer is not attached to a protecting group.

[0607] In some embodiments, X2 is Aib.

[0608] In some embodiments, the lysine residue at position 16 of SEQ ID NO:405 is covalently linked to a protecting group or to a spacer that optionally links to a protecting group.

[0609] In some embodiments, X 24 If S, then X 10 is V.

[0610] In some embodiments, the peptide comprises X 20 and X 24 The lysine and glutamic acid side chains are preferably linked together at positions 1111-1115 and 1111-1116, respectively.

[0611] In some embodiments, X2 is Aib. In some embodiments, X2 is G.

[0612] In some embodiments, X8 is N. In some embodiments, X8 is S.

[0613] In some embodiments, X 10 is I. In some embodiments, X 10 is L. In some embodiments, X 10 is V.

[0614] In some embodiments, X 12 is E. In some embodiments, X 12 is K. In some embodiments, X 12 is Q.

[0615] In some embodiments, X 17 is E. In some embodiments, X 17 is K.

[0616] In some embodiments, X 18 is A. In some embodiments, X 18 is Y.

[0617] In some embodiments, X 19 is A. In some embodiments, X 19 is V.

[0618] In some embodiments, X 20 is E. In some embodiments, X 20 is K. In some embodiments, X 20 is R.

[0619] In some embodiments, X 21 is E. In some embodiments, X 21 is K. In some embodiments, X 21 is L. In some embodiments, X 21 is Q.

[0620] In some embodiments, X 24 is E. In some embodiments, X 24 is K. In some embodiments, X 24 is S.

[0621] In some embodiments, X 28 is N. In some embodiments, X 28 is Q.

[0622] In some embodiments, X 32 is H. In some embodiments, X 32 is S.

[0623] In some embodiments, the carboxy terminal amino acid, i.e., S 39 -S 39 In some embodiments, the carboxy-terminal amino acid S 39 -S 39 -(OH).

[0624] Exemplary Polypeptide Intermediates In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Tables 8 and 9: [Table 8]

[0625] In some embodiments, the present invention provides a peptide intermediate of compound A120 (SEQ ID NO: 120). In some embodiments, the present invention provides a peptide intermediate described in Table 8 above. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 3120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 4120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 5120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 6120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 7120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 8120. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 9120.

[0626] In some embodiments, the present invention provides a method for converting a polypeptide intermediate, such as compound D3120 (SEQ ID NO: 3120), into an activated acyl group, i.e., (LG)CO(CH), as illustrated in Scheme 1 below. zz

[0013] A method for preparing compounds of the invention, such as compound A120 (SEQ ID NO: 120), is provided, comprising acylation with COH, where ZZ is 14-22, and LG is a leaving group as defined herein:

[0627] [Table 9] [Table 10]

[0628] In some embodiments, the present invention provides a peptide intermediate of compound A115 (SEQ ID NO: 115). In some embodiments, the present invention provides a peptide intermediate described in Table 9 above. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 3115. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 4115. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 5115. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 6115. In some embodiments, the peptide intermediate is a peptide having the amino acid sequence of SEQ ID NO: 7115.

[0629] In some embodiments, the present invention provides a method for converting a polypeptide intermediate, such as compound D3115 (SEQ ID NO: 3115), to the following activated acyl group: (LG)CO(CH) zz

[0013] A method for preparing compounds of the invention, such as compound A115 (SEQ ID NO: 115), is provided, comprising acylating with COH, where ZZ is 14-22, and LG is a leaving group as defined herein:

[0630] [Table 11]

[0631] How to use According to another embodiment, the present invention relates to a method for treating a metabolic disease or disorder in a subject in need thereof, the method comprising providing to the subject an effective amount of a GLP-1 receptor agonist polypeptide of the present disclosure or a pharmaceutical composition thereof. Metabolic diseases or disorders include type 1 diabetes, type 2 diabetes, and obesity. Furthermore, the present invention relates to a method for achieving weight loss in a subject, including a diabetic subject, the method comprising providing to the subject an effective amount of a GLP-1 receptor agonist polypeptide of the present disclosure. In certain embodiments, the present invention also relates to a method for treating non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH).

[0632] In some embodiments, there is provided a method of treating obesity in a human subject, a method of inducing weight loss in a human subject, or a method of suppressing appetite in a human subject, comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides.

[0633] In some embodiments, a method of treating diabetes in a human subject is provided, comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides. In some embodiments, the diabetes is type 1 diabetes. In some embodiments, the diabetes is type 1 diabetes.

[0634] In some embodiments, provided are methods of treating non-alcoholic fatty liver disease (NAFLD) in a human subject, the method comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides.

[0635] In some embodiments, provided are methods of treating non-alcoholic steatohepatitis (NASH) in a human subject, the method comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides.

[0636] The GLP-1 receptor agonist polypeptides of the present disclosure are particularly useful for treating diabetes, and the methods include providing an effective amount of a GLP-1 receptor agonist polypeptide to a diabetic subject. In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used to treat a subject with type 1 or type 2 diabetes to control or reduce the subject's blood glucose levels, where blood glucose levels can be monitored or estimated based on measured blood levels of glycated hemoglobin (hemoglobin A1c, HbA1c).

[0637] (i) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used for the treatment of a subject with type 1 diabetes; (ii) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used for the treatment of a subject with type 2 diabetes; (iii) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used for the treatment of obesity; (iv) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used to induce weight loss in a subject, such as a diabetic subject. (v) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used for the treatment of non-alcoholic fatty liver disease (NAFLD). (vi) In some embodiments, the GLP-1 receptor agonist polypeptides of the present disclosure are used for the treatment of non-alcoholic steatohepatitis (NASH). wherein the GLP-1 receptor agonist polypeptide of use (i), (ii), (iii), (iv), (v) or (vi) comprises any isolated polypeptide of the present disclosure, including those selected from the group consisting of SEQ ID NOs: 6, 45, 46, 54, 55, 77, 78, 108, 111, 112, 114, 115, 120, 132, 134, and 139, or a pharmaceutically acceptable salt thereof.

[0638] In some embodiments, the GLP-1 receptor agonist polypeptide of use (i), (ii), (iii), (iv), (v) or (vi) comprises any isolated polypeptide of the present disclosure, including those selected from the group consisting of SEQ ID NOs: 55, 115, 120 and 132, or a pharmaceutically acceptable salt thereof.

[0639] In some embodiments, the GLP-1 receptor agonist polypeptide of method (i), (ii), (iii), (iv), (v), or (vi) comprises the isolated polypeptide of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide of method (i), (ii), (iii), (iv), (v), or (vi) comprises the isolated polypeptide of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide of method (i), (ii), (iii), (iv), (v), or (vi) comprises the isolated polypeptide of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide of method (i), (ii), (iii), (iv), (v), or (vi) comprises the isolated polypeptide of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0640] In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 1 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 1 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 1 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 1 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132 or a pharmaceutically acceptable salt thereof.

[0641] In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 2 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 2 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 2 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120 or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with type 2 diabetes, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132 or a pharmaceutically acceptable salt thereof.

[0642] In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with obesity, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with obesity, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with obesity, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with obesity, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0643] In some embodiments, the GLP-1 receptor agonist polypeptide is used to effect weight loss in a subject, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide is used to effect weight loss in a subject, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide is used to effect weight loss in a subject, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1 receptor agonist polypeptide is used to effect weight loss in a subject, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0644] In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NAFLD, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NAFLD, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NAFLD, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NAFLD, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0645] In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NASH, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NASH, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NASH, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof. In some embodiments, a GLP-1 receptor agonist polypeptide is used to treat a subject with NASH, and the isolated polypeptide is the isolated polypeptide of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

[0646] In some embodiments, provided are methods of treating a neurological disease or disorder in a human subject, comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides. In some embodiments, the neurological disease or disorder is selected from the group consisting of Parkinson's disease (PD) and Alzheimer's disease (AD).

[0647] In some embodiments, a method of treating Parkinson's disease in a human subject is provided, comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides. In clinical trials, moderately advanced PD patients treated with exenatide for 12 months showed improvements in motor and cognitive symptoms, with the benefits persisting for as long as 12 months after treatment ended (Aviles-Olmos, I., et al., J Clin Invest, 2013. 123(6): 2730-6; Athauda, ​​D. and Foltynie, T., Drug Discov Today. 2016. 21(5): 802-18; Simuni, T. and Brundin, P., J Parkinsons Dis, 2014. 4(3): 345-7).

[0648] In some embodiments, a method for treating Alzheimer's disease in a human subject is provided, comprising administering to the subject any of the polypeptides disclosed herein, a pharmaceutical composition comprising any of the disclosed polypeptides, or an osmotic delivery device comprising any of the disclosed polypeptides. Various preclinical studies on AD have shown that administration of GLP-1 receptor agonists provides neuroprotective effects accompanied by improved cognitive function and positively impacts most neuropathological features in AD (Grieco, M. et al., Front Neurosci. 2019;13:1112; Holscher, C., Acta Physiologica Sinica, 2014, 66(5):497-510).

[0649] The term "patient" or "subject" as used herein refers to a rodent or animal, preferably a mammal, and most preferably a human.

[0650] combination In some embodiments, the GLP-1 receptor agonist polypeptide of the present disclosure is combined with a second agent to form a combination drug. In some embodiments, the GLP-1 receptor agonist polypeptide of the present disclosure is combined with a second agent to form a combination drug, and the second agent is an incretin mimetic. In some embodiments, the GLP-1 receptor agonist polypeptide of the present disclosure is combined with a second agent to form a combination drug, and the second agent is an insulinotropic compound.

[0651] Some embodiments of the present invention include the use of the disclosed GLP-1 receptor agonist polypeptides of the present invention in combination with a second therapeutic agent, such as a second polypeptide, including, but not limited to, insulinotropic peptides, peptide hormones, e.g., glucagon and incretin mimetics, and their peptide analogs and derivatives, glucagon-like polypeptide-2 (GLP-2), PYY (also known as peptide YY, peptide tyrosine), and its peptide analogs and derivatives, e.g., PYY(3-36); oxyntomodulin, and its peptide analogs and derivatives; and gastric inhibitory peptide (GIP), and its peptide analogs and derivatives. In some embodiments, a pharmaceutical composition comprising a GLP-1 receptor agonist polypeptide in combination with a second agent is used to treat type 2 diabetes.

[0652] In some embodiments, a pharmaceutical composition is provided comprising any of the isolated polypeptides disclosed herein. In some embodiments, a pharmaceutical composition is provided comprising any of the isolated polypeptides disclosed herein and further comprising a second polypeptide. In some embodiments, the second polypeptide is a glucagon receptor agonist. In some embodiments, the second polypeptide is a glucagon analog. In some embodiments, the second polypeptide is an amylin analog. In some embodiments, the second polypeptide is a PYY analog.

[0653] GLP-1, including three forms of the peptide, GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36)amide, as well as peptide analogs of GLP-1, have been shown to stimulate insulin secretion (i.e., be insulinotropic), thereby inducing glucose uptake by cells and lowering serum glucose levels (see, e.g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).

[0654] Numerous GLP-1 receptor agonists (e.g., GLP-1 peptide derivatives and peptide analogs) that exhibit insulinotropic activity are known in the art (e.g., U.S. Pat. Nos. 5,118,666; 5,120,712; 5,512,549; 5,545,618; 5,574,008; 5,574,008; 5,614,492; 5,958,909; 6,191,102; 6,268,343; 6,329,336; 6,451,974; 6,458,924; 6,514,500; 6,593,29 Nos. 5; 6,703,359; 6,706,689; 6,720,407; 6,821,949; 6,849,708; 6,849,714; 6,887,470; 6,887,849; 6,903,186; 7,022,674; 7,041,646; 7,084,243; 7,101,843; 7,138,486; 7,141,547; 7,144,863; and 7,199,217), and in clinical trials (e.g., taspoglutide and albiglutide). An example of a GLP-1 receptor agonist in the practice of the present invention is Victoza® (Novo Nordisk A / S, Bagsvaerd DK) (liraglutide; U.S. Patent Nos. 6,268,343, 6,458,924, and 7,235,627). Once-daily injection of Victoza® (liraglutide) is commercially available in the United States, Europe, and Japan. Another example of a GLP-1 receptor agonist is Ozempic® or Rybelsus® (Novo Nordisk A / S, Bagsvaerd DK) (semaglutide, injectable and oral formulations, respectively). For ease of reference, the family of insulinotropically active GLP-1 receptor agonists, GLP-1 peptides, GLP-1 peptide derivatives, and GLP-1 peptide analogs will be collectively referred to herein as "GLP-1."

[0655] Peptide YY (PYY) is a 36-amino acid peptide amide. PYY inhibits intestinal motility and blood flow (Laburthe, M., Trends Endocrinol Metab. 1(3):168-74 (1990)), mediates intestinal secretion (Cox, HM, et al., Br J Pharmacol 101(2):247-52 (1990); Playford, RJ, et al., Lancet 335(8705):1555-7 (1990)), and stimulates net absorption (MacFayden, RJ, et al., Neuropeptides 7(3):219-27 (1986)). Two major in vivo variants, PYY(1-36) and PYY(3-36), have been identified (e.g., Eberlein, GA, et al., Peptides 10(4), 797-803 (1989)). The sequence of PYY, as well as its peptide analogs and derivatives, are known in the art (e.g., U.S. Pat. Nos. 5,574,010 and 5,552,520).

[0656] Oxyntomodulin is a naturally occurring 37-amino acid peptide hormone found in the colon that has been shown to suppress appetite and promote weight loss (Wynne K, et al., Int J Obes (Lond) 30(12):1729-36(2006)). The sequence of oxyntomodulin and its peptide analogs and derivatives are known in the art (e.g., Bataille D, et al., Peptides 2(Suppl 2):41-44 (1981); and U.S. Patent Application Publication Nos. 2005 / 0070469 and 2006 / 0094652).

[0657] Gastric inhibitory peptide (GIP) is an insulinotropic peptide hormone (Efendic, S., et al., Horm Metab Res. 36:742-6 (2004)), secreted by the mucosa of the duodenum and jejunum in response to absorbed fat and carbohydrates, and stimulates the pancreas to secrete insulin. GIP circulates as a biologically active 42-amino acid peptide. GIP is also known as glucose-dependent insulinotropic protein. GIP is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose (Tseng, C., et al., PNAS 90:1992-1996 (1993)). The sequence of GIP, as well as its peptide analogs and derivatives, are known in the art (e.g., Meier J. J., Diabetes Metab Res Rev. 21(2):91-117 (2005) and Efendic S., Horm Metab Res. 36(11-12):742-6 (2004)).

[0658] Glucagon is a peptide hormone produced by alpha cells in the pancreas that increases glucose levels in the bloodstream. Its effect is opposite to that of insulin, which lowers glucose levels. When glucose levels in the bloodstream become too low, the pancreas releases glucagon. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. High blood glucose levels stimulate the release of insulin. Insulin enables glucose uptake and use by insulin-dependent tissues. Thus, glucagon and insulin are part of a feedback system that keeps blood glucose levels stable.

[0659] Human amylin, or islet amyloid polypeptide (IAPP), is a 37-residue polypeptide hormone. Amylin is co-secreted with insulin from pancreatic β cells in a ratio of approximately 100:1 (insulin:amylin). Pro-islet amyloid polypeptide (i.e., pro-IAPP) is produced in pancreatic β cells as a 67-amino acid, 7,404-dalton propeptide, which undergoes post-translational modifications, including protease cleavage, to produce the 37-residue amylin. Loss of β-cell function, which occurs early in patients with type 1 diabetes and can occur later in patients with type 2 diabetes, leads to deficiencies in insulin and amylin secretion.

[0660] Amylin functions as part of the endocrine pancreas, which are cells in the pancreas that synthesize and secrete hormones. Amylin contributes to blood sugar control; it is secreted from pancreatic islets into the circulation and cleared by renal peptidases. Amylin's metabolic function is well characterized as an inhibitor of the appearance of nutrients such as glucose in plasma. Thus, amylin functions as a synergistic partner to insulin, a peptide that regulates blood sugar levels and regulates the distribution and uptake of glucose in the body. The role of insulin in the body is, among other things, to prevent blood sugar levels from becoming too high, especially after meals.

[0661] Amylin is thought to play a role in blood sugar regulation by slowing gastric emptying and promoting satiety (i.e., fullness), thereby preventing a postprandial (i.e., post-meal) spike in blood sugar levels. The overall effect is to slow the rate at which glucose appears in the blood after eating. Amylin also reduces the secretion of glucagon by the pancreas. Glucagon's role in the body is, among other things, to prevent blood sugar levels from getting too low. This is important because, for example, certain type 1 diabetics tend to secrete excessive amounts of glucagon, which raises blood sugar immediately after eating.

[0662] For many reasons, human amylin, with a serum half-life of approximately 13 minutes, is not suitable for use as a therapeutic agent. Instead, pramlintide (Symlin®, developed by Amylin Pharmaceuticals, Inc., San Diego, CA, USA, and sold by AstraZeneca plc, Cambridge, UK) was developed as a synthetic analog of human amylin for the treatment of type 1 or type 2 diabetes patients who use mealtime insulin but are unable to achieve desired glycemic control despite optimal insulin therapy. Pramlintide differs from human amylin in three of its 37 amino acids. These modifications give pramlintide a longer half-life of approximately 48 minutes in humans and a reduced tendency to aggregate, a characteristic of human amylin. Additional analogs of human amylin have been disclosed, such as in U.S. Patent Application No. 16 / 598,915 (corresponding to PCT International Application No. PCT / US2019 / 055696), both filed October 10, 2019.

[0663] Implantable delivery In some embodiments, there is provided an osmotic delivery device as described herein comprising any of the long-acting GLP-1 receptor agonist polypeptides disclosed herein or a pharmaceutical composition comprising any of the long-acting GLP-1 receptor agonist polypeptides.

[0664] In some embodiments, the osmotic delivery device includes an impermeable reservoir having an inner surface and an outer surface and first and second open ends; a semipermeable membrane in sealing relation with the first open end of the reservoir; an osmotic engine within the reservoir and adjacent to the semipermeable membrane; a piston adjacent to the osmotic engine, the piston forming a movable seal with an inner surface of the reservoir, the piston dividing the reservoir into a first chamber and a second chamber, the first chamber comprising the osmotic engine; a suspension formulation, the second chamber comprising the suspension formulation, the suspension formulation being flowable and comprising the isolated polypeptide; and a diffusion moderator inserted into the second open end of the reservoir, the diffusion moderator adjacent to the suspension formulation.

[0665] The implantable osmotic delivery device typically includes a reservoir having at least one orifice through which the suspension formulation is delivered. The suspension formulation can be stored in the reservoir. In a preferred embodiment, the implantable drug delivery device is an osmotic delivery device, and the delivery of the drug is osmotically driven. Several osmotic delivery devices and their components have been described, such as the DUROS® delivery vise or similar devices (e.g., U.S. Pat. Nos. 5,609,885; 5,728,396; 5,985,305; 5,997,527; 6,113,938; 6,132,420; 6,156,331; 6,217,906; 6,261,584; 6,270,787; 6,287,295; 6,375,978; 6,395,292). ; 6,508,808; 6,544,252; 6,635,268; 6,682,522; 6,923,800; 6,939,556; 6,976,981; 6,997,922; 7,014,636; 7,207,982; and 7,112,335; 7,163,688; see U.S. Patent Application Publication Nos. 2005 / 0175701, 2007 / 0281024, 2008 / 0091176, and 2009 / 0202608).

[0666] Osmotic delivery devices typically consist of an osmotic engine, a piston, and a cylindrical reservoir containing a drug formulation. The reservoir is covered at one end by a controlled-rate semipermeable membrane and at the other end by a diffusion moderator, through which a drug-containing suspension formulation is released from the drug reservoir. The piston separates the drug formulation from the osmotic engine and utilizes a seal to prevent water in the osmotic engine section from entering the drug reservoir. The diffusion moderator, in conjunction with the drug formulation, is designed to prevent bodily fluids from entering the drug reservoir through the orifice.

[0667] Osmotic devices release drugs at a predetermined rate based on the principles of osmosis. Extracellular fluid passes through a semipermeable membrane directly into the salt engine of the osmotic delivery device, which expands to drive a piston at a slow and uniform delivery rate. The movement of the piston forces the drug formulation through an orifice or exit port at a predetermined shear rate. In one embodiment of the present invention, the reservoir of the osmotic device is filled with a suspension formulation, and the device can deliver the suspension formulation to a subject at a predetermined, therapeutically effective delivery rate over an extended period of time (e.g., about 1, about 3, about 6, about 9, about 10, and about 12 months).

[0668] The release rate of a drug from an osmotic delivery device typically provides a subject with a predetermined target dose of the drug, e.g., a therapeutically effective daily dose, delivered over the course of a day; i.e., the release rate of the drug from the device provides a substantially steady-state delivery of the drug at a therapeutic concentration to the subject.

[0669] Typically, for osmotic delivery devices, the volume of the beneficial agent chamber containing the beneficial agent formulation is between about 100 μl and about 1000 μl, more preferably between about 120 μl and about 500 μl, and more preferably between about 150 μl and about 200 μl.

[0670] Typically, the osmotic delivery device is implanted, for example, subdermally or subcutaneously, within a subject to provide subcutaneous drug delivery. The device(s) can be implanted subdermally or subcutaneously in one or both arms (e.g., the inside, outside, or back of the upper arm) or in the abdomen. A preferred site in the abdominal region is under the abdominal skin in an area extending below the ribs and above the belt line. To provide multiple sites for implanting one or more osmotic delivery devices within the abdomen, the abdominal wall is divided into four quadrants as follows: the right upper quadrant extending at least 2-3 centimeters below the right ribs, e.g., at least about 5-8 centimeters below the right ribs, and at least 2-3 centimeters to the right of the midline, e.g., at least about 5-8 centimeters to the right of the midline; the right upper quadrant extending at least 2-3 centimeters above the belt line, e.g., at least about 5-8 centimeters above the belt line, and at least 2-3 centimeters to the right of the midline, e.g., at least about 5-8 centimeters to the right of the midline; The right lower quadrant extends approximately 5-8 centimeters; the left upper quadrant extends at least 2-3 centimeters below the left rib cage, e.g., at least 5-8 centimeters below the left rib cage, and at least 2-3 centimeters to the left of the midline, e.g., at least 5-8 centimeters to the left of the midline; and the left lower quadrant extends at least 2-3 centimeters above the belt line, e.g., at least 5-8 centimeters above the belt line, and at least 2-3 centimeters to the left of the midline, e.g., at least 5-8 centimeters to the left of the midline. This provides multiple available sites for implanting one or more devices on one or more occasions. Implantation and removal of osmotic delivery devices are generally performed by a medical professional using local anesthesia (e.g., lidocaine).

[0671] Termination of treatment by removal of the osmotic delivery device from the subject is simple, which provides the important advantage of immediately stopping delivery of drug to the subject.

[0672] Preferably, the osmotic delivery device has a fail-safe mechanism to prevent accidental over- or bolus delivery of drug in theoretical situations such as blockage or clogging of the outlet (diffusion moderator) through which the drug formulation is delivered. To prevent accidental over- or bolus delivery of drug, the osmotic delivery device is designed and constructed so that the pressure required to partially or completely remove or expel the diffusion moderator from the reservoir exceeds the pressure required to partially or completely remove or expel the semipermeable membrane to the extent necessary to depressurize the reservoir. In such a scenario, pressure will build within the device until the device pushes the semipermeable membrane outward at the other end, thereby releasing osmotic pressure. The osmotic delivery device then becomes static and no longer delivers the drug formulation when the piston is in a sealing relationship with the reservoir.

[0673] The dose and delivery rate can be selected to achieve a desired blood concentration of the drug generally within less than about six half-lives of the drug in the subject after implantation of the device. The blood concentration of the drug is selected to provide optimal therapeutic effect of the drug while avoiding undesirable side effects that may be induced by excessive concentrations of the drug, and at the same time avoiding peaks and troughs that may induce side effects associated with peak or trough plasma concentrations of the drug.

[0674] Suspension formulations can also be used in infusion pumps, such as the ALZET® (DURECT Corporation, Cupertino, Calif.) osmotic pump, which is a miniature infusion pump for continuous medication in laboratory animals (e.g., mice and rats).

[0675] Mode of administration In some embodiments, the method comprises providing to a subject in need of treatment a GLP-1 receptor agonist polypeptide of the present disclosure or a pharmaceutical composition thereof via injection. In some embodiments, the method comprises providing to a subject in need of treatment a GLP-1 receptor agonist polypeptide of the present disclosure or a pharmaceutical composition thereof formulated for oral administration.

[0676] In some embodiments, the method includes providing a GLP-1 receptor agonist polypeptide of the present disclosure or a pharmaceutical composition thereof via implantation to a subject in need of treatment. In some embodiments, the method includes providing continuous delivery of the GLP-1 receptor agonist polypeptide from an osmotic delivery device to a subject in need of treatment. The delivery device, such as an osmotic delivery device, contains sufficient GLP-1 receptor agonist polypeptide of the present disclosure for up to 3, 6, 9, 12, 18, or 24 months of continuous administration. Thus, continuous administration of the GLP-1 receptor agonist polypeptide of the present disclosure via an osmotic delivery device eliminates the need for a daily dose or multiple daily doses of a commercially available GLP-1 receptor agonist polypeptide.

[0677] The substantially steady-state delivery of the GLP-1 receptor agonist polypeptide from the osmotic delivery device is continuous over the administration period. In some embodiments, the subject or patient is a human subject or patient.

[0678] In some embodiments of the present invention, the administration period is, for example, at least about 3 months, at least about 3 months to about 1 year, at least about 4 months to about 1 year, at least about 5 months to about 1 year, at least about 6 months to about 1 year, at least about 8 months to about 1 year, at least about 9 months to about 1 year, at least about 10 months to about 1 year, at least about 1 year to about 2 years, or at least about 2 years to about 3 years.

[0679] In a further embodiment, the therapeutic methods of the present invention provide a significant reduction in the subject's fasting plasma glucose concentration after implantation of an osmotic delivery device (compared to the subject's fasting plasma glucose concentration before implantation of the osmotic delivery device), which reduction is achieved within about 7, 6, 5, 4, 3, 2, 1 day or less after implantation of the osmotic delivery device in the subject. A significant reduction in fasting plasma glucose is typically statistically significant, as demonstrated by application of an appropriate statistical test, or is considered significant to the subject by a physician. A significant reduction in fasting plasma glucose compared to the pre-implant baseline is typically maintained over the course of administration.

[0680] In some embodiments, the present invention relates to a method of treating a disease or condition in a subject in need of treatment. The method includes providing continuous delivery of a drug from an osmotic delivery device, wherein substantially steady-state delivery of the drug at therapeutic concentrations is achieved in the subject. The substantially steady-state delivery of the drug from the osmotic delivery device is continuous over an administration period of at least about three months. The drug has a known or determined half-life in a typical subject. Humans are preferred subjects for practicing the present invention. The present invention includes drugs effective in treating a disease or condition, as well as osmotic delivery devices containing the drugs for use in the method of treating a disease or condition in a subject in need of treatment. Advantages of the present invention include reduced peak-related drug toxicity and attenuation of suboptimal drug therapy associated with troughs.

[0681] In some embodiments, substantially steady-state delivery of the drug at therapeutic concentrations is achieved within a period of about 1 month, 7 days, 5 days, 3 days, or 1 day after implantation of the osmotic delivery device in a subject.

[0682] The present invention also provides methods for promoting weight loss in a subject in need thereof, for treating excess weight or obesity in a subject in need thereof, and / or for suppressing appetite in a subject in need thereof, comprising providing delivery of an isolated GLP-1 receptor agonist polypeptide. In some embodiments, the isolated GLP-1 receptor agonist polypeptide is delivered continuously from an implantable osmotic delivery device. In some embodiments, substantially steady-state delivery of the GLP-1 receptor agonist polypeptide from the osmotic delivery device is achieved and is substantially continuous over the administration period. In some embodiments, the subject is a human.

[0683] The present invention includes an osmotic delivery device containing a GLP-1 receptor agonist polypeptide for use in the method in a subject in need of treatment. The subject may have type 2 diabetes. The subject in need of treatment may have a baseline HbA1c% greater than 10.0%, i.e., may be a high baseline (HBL) subject. The subject may not have previously received medication to treat type 2 diabetes.

[0684] In further embodiments, the therapeutic methods of the invention result in a significant decrease in the subject's fasting plasma glucose concentration after implantation of an osmotic delivery device in the subject (compared to the subject's fasting plasma glucose concentration before implantation of the osmotic delivery device), which significant decrease is achieved within about 7 days or less after implantation of the osmotic delivery device in the subject, within about 6 days or less after implantation of the osmotic delivery device in the subject, within about 5 days or less after implantation of the osmotic delivery device in the subject, within about 4 days or less after implantation of the osmotic delivery device in the subject, within about 3 days or less after implantation of the osmotic delivery device in the subject, within about 2 days or less after implantation of the osmotic delivery device, or within about 1 day or less after implantation of the osmotic delivery device in the subject. In preferred embodiments of the invention, the significant decrease in the subject's fasting plasma glucose concentration after implantation of the osmotic delivery device compared to the subject's fasting plasma glucose concentration before implantation is achieved within about 2 days or less, preferably within about 1 day or less, or more preferably within about 1 day after implantation of the osmotic delivery device in the subject. A significant reduction in fasting plasma glucose is typically statistically significant, as demonstrated by application of an appropriate statistical test, or is considered significant to the subject by a physician. A significant reduction in fasting plasma glucose compared to pre-implant baseline is typically maintained over the course of treatment.

[0685] In embodiments of all aspects of the invention relating to methods for treating a disease or condition in a subject, an exemplary osmotic delivery device includes: an impermeable reservoir having an inner surface and an outer surface and a first and second open end; a semipermeable membrane in sealing relation with the first open end of the reservoir; an osmotic engine within the reservoir and adjacent to the semipermeable membrane; a piston adjacent to the osmotic engine, the piston forming a movable seal with an inner surface of the reservoir and dividing the reservoir into a first chamber and a second chamber, the first chamber containing the osmotic engine; a drug formulation or a suspension formulation containing the drug, the second chamber containing the drug formulation or suspension formulation, the drug formulation or suspension formulation being flowable; and a diffusion moderator inserted in the second open end of the reservoir, the diffusion moderator adjacent to the suspension formulation. In a preferred embodiment, the reservoir comprises titanium or a titanium alloy.

[0686] In embodiments of all aspects of the present invention relating to methods for treating a disease or condition in a subject, the drug formulation can include a drug and a vehicle formulation. Alternatively, a suspension formulation can be used in the method, and can include, for example, a particle formulation containing a drug and a vehicle formulation. The vehicle formulation used in forming the suspension formulation of the present invention can include, for example, a solvent and a polymer.

[0687] The reservoir of the osmotic delivery device can comprise, for example, titanium or a titanium alloy.

[0688] In embodiments of all aspects of the present invention, an implanted osmotic delivery device can be used to provide subcutaneous delivery.

[0689] In embodiments of all aspects of the present invention, the continuous delivery can be, for example, zero-order controlled continuous delivery.

[0690] Pharmaceutical Composition According to another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention, i.e., an isolated polypeptide or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0691] In some embodiments, provided are pharmaceutical compositions comprising any of the disclosed polypeptides formulated as a pharmaceutically acceptable salt thereof, such as a trifluoroacetate salt, acetate salt, or hydrochloride salt. In some embodiments, provided are pharmaceutical compositions comprising any of the disclosed polypeptides formulated as a trifluoroacetate salt. In some embodiments, provided are pharmaceutical compositions comprising any of the disclosed polypeptides formulated as an acetate salt. In some embodiments, provided are pharmaceutical compositions comprising any of the disclosed polypeptides formulated as a hydrochloride salt.

[0692] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.

[0693] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, polymers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in this field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0694] Representative pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, protamine sulfate, water, partial glyceride mixtures of salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0695] In some embodiments, pharmaceutical compositions are provided comprising a pharmaceutically acceptable derivative of any of the disclosed polypeptides formulated as a pharmaceutically acceptable salt thereof, such as a trifluoroacetate, acetate, or hydrochloride salt. By "pharmaceutically acceptable derivative" is meant any non-toxic salt, ester, salt of an ester, or other derivative of a compound of the invention which, upon administration to a recipient, is capable of directly or indirectly providing a compound of the invention, or an active metabolite or residue thereof.

[0696] The pharmaceutical composition comprises a drug and can be formulated as a "particle formulation" as described in more detail below. The pharmaceutical composition and / or particle formulation can also comprise a stabilizing component (also referred to herein as an "excipient"). Examples of stabilizing components include, but are not limited to, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, and surfactants.

[0697] The amount of compound in the compositions of the present invention is such that it is effective to measurably activate one or more GLP-1 receptors in a biological sample or in a patient (e.g., human, rat, monkey, etc.). In certain embodiments, the amount of compound in the compositions of the present invention is such that it is effective to measurably agonize human GLP-1 receptors in a biological sample or in a patient in the absence or presence of human serum albumin. In certain embodiments, the compositions of the present invention are formulated for administration to a patient in need of such a composition. In some embodiments, the compositions of the present invention are formulated for injectable administration to a patient. In some embodiments, the compositions of the present invention are formulated for administration to a patient via an implantable delivery device, such as an osmotic delivery device.

[0698] The isolated polypeptides of the present disclosure (also referred to herein as "active compounds"), and their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the isolated polypeptide, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0699] Pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Exemplary routes of administration include parenteral (e.g., intravenous, intradermal, subdermal, subcutaneous), oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, rectal, or a combination thereof. In some embodiments, pharmaceutical compositions or isolated polypeptides of the present disclosure are formulated for administration by topical administration. In some embodiments, pharmaceutical compositions or isolated polypeptides of the present disclosure are formulated for administration by inhalation administration. In some embodiments, pharmaceutical compositions are suitable for subdermal or subcutaneous implantation and are formulated for administration by a device that delivers the pharmaceutical composition subcutaneously or other suitable delivery mechanism. In some embodiments, pharmaceutical compositions are suitable for subdermal or subcutaneous implantation and are formulated for administration by an implanted device that delivers the pharmaceutical composition subcutaneously. In some embodiments, pharmaceutical compositions are suitable for subdermal or subcutaneous implantation and are formulated for administration by an osmotic delivery device that delivers the pharmaceutical composition subcutaneously, e.g., an implantable osmotic delivery device. Solutions or suspensions used for parenteral, intradermal, subdermal, or subcutaneous application, or a combination thereof, can include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting isotonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0700] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The appropriate fluidity can be achieved, for example, by the addition of lecithin. This can be achieved by the use of a coating such as a polyacrylate, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols (mannitol, sorbitol, sodium chloride, and the like) in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0701] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or a combination of the above-listed ingredients as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the above-listed ingredients.For the preparation of sterile powder for sterile injectable solution, the preparation method is vacuum drying and freeze-drying, thereby obtaining a powder containing active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0702] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash; the compound in the fluid carrier is applied orally, swished in the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0703] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0704] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as generally known in the art.

[0705] In one embodiment, the active compound can be prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0706] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unit dose for treating a subject, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the technical field of compounding such active compound for treating individuals.

[0707] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0708] Drug particle formulation The compound for use in carrying out the present invention, i.e., isolated polypeptide or its pharmaceutically acceptable salt, is typically added to a particle formulation, which is used to make polypeptide-containing particles that are uniformly suspended, dissolved or dispersed in a suspension vehicle, thereby forming a suspension formulation.In some embodiments, the GLP-1 receptor agonist polypeptide is formulated into a particle formulation and converted into particles (e.g., spray-dried).In some embodiments, the particles containing the GLP-1 receptor agonist polypeptide are suspended in a vehicle formulation, resulting in a suspension formulation of suspended particles containing the GLP-1 receptor agonist polypeptide.

[0709] Preferably, the particle formulation can be formed into particles using processes such as spray drying, lyophilization, drying, freeze-drying, milling, granulation, ultrasonic droplet generation, crystallization, precipitation, or other techniques available in the art for forming particles from a mixture of components. In one embodiment of the present invention, the particles are spray-dried. The particles are preferably substantially uniform in shape and size.

[0710] In some embodiments, the present invention provides a drug particle formulation for pharmaceutical use. The particle formulation typically contains a drug and one or more stabilizing components (also referred to herein as "excipients"). Examples of stabilizing components include, but are not limited to, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, and surfactants. The amount of stabilizer in the particle formulation can be determined empirically based on the activity of the stabilizer and the desired properties of the formulation, taking into account the teachings herein.

[0711] In any of the embodiments, the particle formulation may comprise about 50% to about 90% by weight of drug, about 50% to about 85% by weight of drug, about 55% to about 90% by weight of drug, about 60% to about 90% by weight of drug, about 65% to about 85% by weight of drug, about 65% to about 90% by weight of drug, about 70% to about 90% by weight of drug, about 70% to about 85% by weight of drug, about 70% to about 80% by weight of drug, or about 70% to about 75% by weight of drug.

[0712] Typically, the amount of carbohydrate in a particle formulation is determined by concerns about aggregation. In general, the amount of carbohydrate should not be too high to avoid excessive carbohydrate not bound to the drug promoting crystal growth in the presence of water.

[0713] Typically, the amount of antioxidant in a particle formulation is determined by oxidation concerns, while the amount of amino acid in the formulation is determined by oxidation concerns and / or the compactibility of the particles during spray drying.

[0714] Typically, the amount of buffer in a particle formulation is determined by pre-processing concerns, stability concerns, and the compactibility of the particles during spray drying. If all stabilizers are solubilized, a buffer may be required to stabilize the drug during processing such as solution preparation and spray drying.

[0715] Examples of carbohydrates that can be included in the particle formulation include, but are not limited to, monosaccharides (e.g., fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (e.g., lactose, sucrose, trehalose, and cellobiose), polysaccharides (e.g., raffinose, melezitose, maltodextrin, dextran, and starch), and alditols (acyclic polyols; e.g., mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosylsorbitol, and myoinositol). Suitable carbohydrates include disaccharides and / or non-reducing sugars, such as sucrose, trehalose, and raffinose.

[0716] Examples of antioxidants that can be included in the particle formulation include, but are not limited to, methionine, ascorbic acid, sodium thiosulfate, catalase, platinum, ethylenediaminetetraacetic acid (EDTA), citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate.In addition, easily oxidized amino acids, such as cysteine, methionine, and tryptophan, can be used as antioxidants.

[0717] Examples of amino acids that can be included in the particle formulation include, but are not limited to, arginine, methionine, glycine, histidine, alanine, leucine, glutamic acid, isoleucine, L-threonine, 2-phenylamine, valine, norvaline, proline, phenylalanine, tryptophan, serine, asparagine, cysteine, tyrosine, lysine, and norleucine.Suitable amino acids include those that are easily oxidized, such as cysteine, methionine, and tryptophan.

[0718] Examples of buffers that may be included in the particle formulation include, but are not limited to, citrate, histidine, succinate, phosphate, maleate, Tris, acetate, carbohydrate, and glycylglycine. Suitable buffers include citrate, histidine, succinate, and Tris.

[0719] Examples of inorganic compounds that may be included in the particle formulation include, but are not limited to, NaCl, Na2SO4, NaHCO3, KCl, KH2PO4, CaCl2, and MgCl2.

[0720] In addition, particle formulations may contain other stabilizers / excipients such as surfactants and salts.Examples of surfactants include, but are not limited to, polysorbate 20, polysorbate 80, PLURONIC® (BASF Corporation, Mount Olive, NJ) F68, and sodium dodecyl sulfate (SDS).Examples of salts include, but are not limited to, sodium chloride, calcium chloride, and magnesium chloride.

[0721] Particles are typically sized for delivery via an implantable osmotic delivery device. Uniform particle shape and size typically contribute to providing a consistent, uniform release rate from such a delivery device; however, particle preparations with non-normal particle size distribution profiles can also be used. For example, in a typical implantable osmotic delivery device having a delivery orifice, the particle size is less than about 30%, more preferably less than about 20%, and more preferably less than about 10% of the diameter of the delivery orifice. In one embodiment of a particle formulation for use with an osmotic delivery system in which the delivery orifice diameter of the implant is about 0.5 mm, the particle size can be, for example, less than about 150 microns to about 50 microns. In one embodiment of a particle formulation for use with an osmotic delivery system in which the delivery orifice diameter of the implant is about 0.1 mm, the particle size can be, for example, less than about 30 microns to about 10 microns. In one embodiment, the orifice is about 0.25 mm (250 microns) and the particle size is about 2 microns to about 5 microns.

[0722] Those skilled in the art will understand that populations of particles follow the principles of particle size distribution. Widely used, art-recognized methods for describing particle size distribution include, for example, mean diameter and D-values, such as D50 values, which are commonly used to represent the average diameter of the range of particle sizes for a given sample.

[0723] The particles of the particle formulation have a diameter of between about 2 microns and about 150 microns, for example, less than 150 microns in diameter, less than 100 microns in diameter, less than 50 microns in diameter, less than 30 microns in diameter, less than 10 microns in diameter, less than 5 microns in diameter, and about 2 microns in diameter. Preferably, the particles have a diameter of between about 2 microns and about 50 microns.

[0724] The particles of the particle formulation comprising the isolated GLP-1 receptor agonist polypeptide have an average diameter of between about 0.3 microns and about 150 microns. The particles of the particle formulation comprising the isolated GLP-1 receptor agonist polypeptide have an average diameter of between about 2 microns and about 150 microns, e.g., an average diameter of less than 150 microns, an average diameter of less than 100 microns, an average diameter of less than 50 microns, an average diameter of less than 30 microns, an average diameter of less than 10 microns, an average diameter of less than 5 microns, and an average diameter of about 2 microns. In some embodiments, the particles have an average diameter of between about 0.3 microns and 50 microns, e.g., between about 2 microns and about 50 microns. In some embodiments, the particles have an average diameter of between 0.3 microns and 50 microns, e.g., between about 2 microns and about 50 microns, with each particle being less than about 50 microns in diameter.

[0725] Typically, the particles of the particle formulation, when incorporated into a suspension vehicle, do not settle at the delivery temperature for less than about 3 months, preferably less than about 6 months, more preferably less than about 12 months, more preferably less than about 24 months, and most preferably less than about 36 months. The suspension vehicle typically has a viscosity between about 5,000 and about 30,000 poise, preferably between about 8,000 and about 25,000 poise, and more preferably between about 10,000 and about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise plus or minus about 3,000 poise. Generally speaking, smaller particles tend to have slower settling rates in viscous suspension vehicles than larger particles. Therefore, micron- to nano-sized particles are typically desirable. In viscous suspension formulations, particles of the present invention between about 2 microns and about 7 microns will not settle out at room temperature for at least 20 years based on simulation modeling studies. One embodiment of a particle formulation of the present invention for use in implantable osmotic delivery devices comprises particles of a size less than about 50 microns, more preferably less than about 10 microns, more preferably in the range of about 2 microns to about 7 microns.

[0726] In summary, the disclosed polypeptide or its pharmaceutically acceptable salt is formulated into a dry powder of solid-state particles, which maintains the maximum chemical and biological stability of the drug. The particles provide long-term storage stability at high temperatures, thus enabling stable and biologically effective drug delivery to subjects over long periods of time. The particles are suspended in a suspension vehicle and administered to patients.

[0727] Particle suspension in a vehicle In one embodiment, the suspension vehicle provides a stable environment in which the drug particle formulation is dispersed. The drug particle formulation is chemically and physically stable in the suspension vehicle (as described above). The suspension vehicle typically contains one or more polymers and one or more solvents that form a solution of sufficient viscosity to uniformly suspend the drug-containing particles. The suspension vehicle may contain additional components, including, but not limited to, surfactants, antioxidants, and / or other compounds that are soluble in the vehicle.

[0728] The viscosity of the suspension vehicle is typically sufficient to prevent settling of the drug particle formulation during storage and is sufficient for use in a delivery method, such as an implantable osmotic delivery device. The suspension vehicle is biodegradable in that it disintegrates or decomposes over a period of time in response to the biological environment, while the drug particles dissolve in the biological environment and the active pharmaceutical ingredient (i.e., drug) in the particles is absorbed.

[0729] In embodiments, the suspension vehicle is a "single-phase" suspension vehicle, which is a homogeneous system of solid, semi-solid, or liquid that is physically and chemically uniform throughout.

[0730] The solvent in which the polymer is dissolved may affect the properties of the suspension formulation, such as the behavior of the drug particle formulation during storage. The solvent can be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation upon contact with an aqueous environment. In some embodiments of the present invention, the solvent can be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation upon contact with an aqueous environment having less than approximately 10% water.

[0731] The solvent can be any acceptable solvent that is immiscible with water. The solvent can also be selected so that the polymer is soluble in the solvent at high concentrations, such as at polymer concentrations greater than about 30%. Examples of solvents useful in the practice of the present invention include, but are not limited to, lauryl alcohol, benzyl benzoate, benzyl alcohol, lauryl lactate, decanol (also known as decyl alcohol), ethylhexyl lactate, and long-chain (C8-C24) aliphatic alcohols, esters, or mixtures thereof. The solvent used in the suspension vehicle can be "dry" in that it has a low water content. Preferred solvents for use in formulating the suspension vehicle include lauryl lactate, lauryl alcohol, benzyl benzoate, and mixtures thereof.

[0732] Examples of polymers for formulating the suspension vehicles of the present invention include, but are not limited to, polyesters (e.g., polylactic acid and polylactic-polyglycolic acid), pyrrolidone-containing polymers (e.g., polyvinylpyrrolidone having a molecular weight ranging from approximately 2,000 to approximately 1,000,000), esters or ethers of unsaturated alcohols (e.g., vinyl acetate), polyoxyethylene-polyoxypropylene block copolymers, or mixtures thereof. Polyvinylpyrrolidone can be characterized by its K value (e.g., K-17), which is a viscosity index. In one embodiment, the polymer is polyvinylpyrrolidone having a molecular weight ranging from 2,000 to 1,000,000. In a preferred embodiment, the polymer is polyvinylpyrrolidone K-17 (typically having an approximate average molecular weight range of about 7,900 to 10,800). The polymer used in the suspension vehicle may comprise one or more different polymers, or may comprise different grades of a single polymer. The polymer used in the suspension vehicle may also be dry or have a low water content.

[0733] Generally speaking, suspension vehicles for use in the present invention can vary in composition based on the desired performance characteristics. In one embodiment, the suspension vehicle can comprise about 40% to about 80% by weight of polymer(s) and about 20% to about 60% by weight of solvent(s). Preferred embodiments of the suspension vehicle include vehicles formed from polymer(s) and solvent(s) mixed in the following ratios: about 25% by weight of solvent and about 75% by weight of polymer; about 50% by weight of solvent and about 50% by weight of polymer; or about 75% by weight of solvent and about 25% by weight of polymer. Thus, in some embodiments, the suspension vehicle can comprise selected components, and in other embodiments, consists essentially of selected components.

[0734] Suspension vehicle may exhibit Newtonian behavior.Suspension vehicle is typically formulated to provide a viscosity that maintains the particle formulation uniform dispersion for a predetermined period of time.This helps to facilitate the preparation of suspension formulations that are tailored to provide controlled delivery of the drug contained in drug particle formulation.The viscosity of suspension vehicle can vary depending on the desired application, the size and type of particle formulation, and the loading amount of particle formulation in suspension vehicle.The viscosity of suspension vehicle can be changed by changing the type or relative amount of solvent or polymer used.

[0735] The suspension vehicle may have a viscosity ranging from about 100 poise to about 1,000,000 poise, preferably from about 1,000 poise to about 100,000 poise. In a preferred embodiment, the suspension vehicle typically has a viscosity of about 5,000 to about 30,000 poise, preferably about 8,000 to about 25,000 poise, and more preferably about 10,000 to about 20,000 poise at 33°C. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise plus or minus about 3,000 poise at 33°C. Viscosity can be measured using a parallel plate rheometer at 33°C at a shear rate of 10-4 / sec.

[0736] The suspension vehicle may exhibit phase separation upon contact with an aqueous environment; however, the suspension vehicle typically exhibits substantially no phase separation as a function of temperature. For example, at temperatures ranging from approximately 0° C. to approximately 70° C., and upon temperature cycling, such as cycling from 4° C. to 37° C. to 4° C., the suspension vehicle typically exhibits no phase separation.

[0737] The suspension vehicle can be prepared by combining the polymer and solvent under dry conditions, such as in a dry box. The polymer and solvent can be combined at an elevated temperature, such as from about 40°C to about 70°C, and liquefied to form a single phase. The components can be blended under vacuum to remove air bubbles generated from the dry components. The components can be combined using a conventional mixer, such as a double helix or similar mixer, set at a speed of about 40 rpm. However, higher speeds can also be used to mix the components. Once a liquid solution of the components is obtained, the suspension vehicle can be cooled to room temperature. Differential scanning calorimetry (DSC) can be used to confirm that the suspension vehicle is single-phase. Additionally, the vehicle components (e.g., solvent and / or polymer) can be treated to substantially reduce or substantially eliminate peroxides (e.g., by treatment with methionine; see, e.g., U.S. Patent Application Publication No. 2007-0027105).

[0738] The drug particle formulation is added to a suspension vehicle to form a suspension formulation. In some embodiments, the suspension formulation may comprise a drug particle formulation and a suspension vehicle, and in other embodiments, consists essentially of a drug particle formulation and a suspension vehicle.

[0739] The suspension formulation can be prepared by dispersing the particle formulation in a suspension vehicle. The suspension vehicle can be heated and the particle formulation added to the suspension vehicle under dry conditions. The components can be mixed under reduced pressure at an elevated temperature, such as about 40°C to about 70°C. The components can be mixed at a sufficient speed, such as about 40 rpm to about 120 rpm, for a sufficient time, such as about 15 minutes, to achieve uniform dispersion of the particle formulation in the suspension vehicle. The mixer can be a double-spiral blade or other suitable mixer. The resulting mixture can be removed from the mixer, sealed in a dry container to prevent water from contaminating the suspension formulation, and cooled to room temperature before further use, for example, before filling into an implantable drug delivery device, unit-dose container, or multi-dose container.

[0740] Suspension formulations typically have a total water content of less than about 10% by weight, preferably less than about 5% by weight, and more preferably less than about 4% by weight.

[0741] In preferred embodiments, the suspension formulations of the present invention are substantially homogeneous and flowable and provide for delivery of a drug particle formulation from an osmotic delivery device to a subject.

[0742] In summary, the components of the suspension vehicle provide biocompatibility. The components of the suspension vehicle provide suitable chemical and physical properties for forming a stable suspension of the drug particle formulation. These properties include, but are not limited to, the viscosity of the suspension; the purity of the vehicle; the residual moisture content of the vehicle; the density of the vehicle; compatibility with dry powders; compatibility with implantable devices, the molecular weight of the polymer; the stability of the vehicle; and the hydrophobicity and hydrophilicity of the vehicle. These properties can be manipulated and controlled, for example, by changing the vehicle composition and manipulating the ratio of the components used in the suspension vehicle.

[0743] The suspension formulations described herein can be used in implantable osmotic delivery devices to provide zero-order, continuous, controlled, and sustained delivery of compounds over extended periods, for example, for weeks, months, or up to about a year or more.Such implantable osmotic delivery devices can typically deliver drug-containing suspension formulations at a desired flow rate for a desired period.The suspension formulations can be loaded into implantable osmotic delivery devices by conventional techniques. [Example]

[0744] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, and percent changes), but some experimental error and deviation should be accounted for. Unless otherwise specified, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0745] Example 1: Production of long-acting GLP-1 receptor agonist polypeptides The long-acting GLP-1 receptor agonist polypeptides of the invention provided in Table 4 were synthesized using the Prelude peptide synthesizer (Protein Technologies The amino acids were synthesized by solid-phase synthesis using the Fmoc strategy with N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) or 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) activation (5-fold molar excess relative to the amino acid) in N,N-dimethylformamide (DMF). N',N-diisopropylethylamine (DIEA) was used as the base. A 20% piperidine / DMF solution was used for Fmoc deprotection. The resin used was Rink Amide MBHA LL (Novabiochem) with a loading of 0.30–0.40 mmol / g on a 20–400 μmol scale.

[0746] Upon completion of solid-phase synthesis of the linear polypeptide, the resin was washed with dichloromethane (DCM) and dried under vacuum for 30 min. For analogs containing the allyloxycarbonyl (Alloc) protecting group, its removal was achieved via a solution of Pd(PPh3)3 in (chloroform / acetic acid / n-methylmorpholine, 37:2:1). For analogs containing tert-butyloxycarbonyl (BOC)-Lys-fluorenylmethyloxycarbonyl (Fmoc)-OH, the Fmoc protecting group was removed using 20% ​​piperidine / DMF. The resulting Fmoc-deprotected resin was washed with DMF (6 × 30 s). Next, elongation of the spacer region was carried out stepwise by manually adding each building block under preactivation conditions. The addition of the lipophilic substituent (also called the "acyl chain") was carried out under solid-phase peptide synthesis (SPPS) conditions without a preactivation step. Final deprotection of the peptide and cleavage from the solid support was achieved by treating the resin with (95% TFA, 2% water, 2% thioanisole, and 1% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether. The diethyl ether layer was decanted, and the solid was again triturated with cold diethyl ether and pelleted by centrifugation.

[0747] For analogs containing lactam bridges, the appropriate allyl-protected amino acid building blocks were installed under the standard solid-phase conditions described above. Alternatively, Fmoc-Lys-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)-OH was installed as a handle for later incorporation of acyl spacers and side chains. Once the linear peptide was completed, the allyl protecting groups were removed as described above. Lactam bridge formation was achieved via a solid-phase protocol using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 0.5 M) activation and DIEA as the base. Deprotection of the Fmoc and ivDde groups was achieved via a 4% solution of hydrazine in DMF. The resulting deprotected resin was washed with DMF (6 × 30 s). Elongation of the spacer region and addition of lipophilic substituents were performed as described in the previous paragraph. Final deprotection of the peptide and cleavage from the solid support was achieved by treating the resin with (95% TFA, 2% water, 2% thioanisole, and 1% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether. The diethyl ether was decanted, and the solid was again triturated with cold diethyl ether and pelleted by centrifugation.

[0748] The crude products were then dissolved in a solution of acetonitrile (ACN) / HO, 0.1% TFA. A 10% solution of acetic acid was added to each solution of crude peptide product and stirred until analysis by LC / MS indicated removal of the CO adduct. The solutions were frozen and lyophilized. Purification was performed as described in Example 2.

[0749] Example 2: Purification and characterization of long-acting GLP-1 receptor agonist polypeptides, i.e., linear polypeptides, free of lipophilic substituents and optional spacers The product of Example 1 was lyophilized and analyzed by electrospray ionization-liquid chromatography / mass spectrometry (ESI-LC / MS) and analytical high-pressure liquid chromatography (HPLC) and was found to be pure (>98%). Mass results were consistent with calculated values.

[0750] Characterization of peptide analogs was performed using either Method A, Method B, Method C, or Method D by C18 HPLC and LC / MS analysis (Acquity SQD Waters Corp, Milford, MA) and UV detection obtained by dual absorbance signals at 215 nm and 280 nm.

[0751] Method A, LC / MS conditions: Phenomenex HPLC Aeris™ Peptide XB Cl8 35 column, 1.7 pm, 2.1 x 100 mm, or ACQUiTY BEH300 or BEH130 CT8 column, 1.77 pm, 2.1 x 100 mm, over 30 minutes at a flow rate of 0.5 mL / min, λ -215 nm, 280 nm, using 5-65% acetonitrile / water with 0.05% TFA.

[0752] Method B, C18 HPLC conditions: HPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column at 25 °C for 30 min, with a flow rate of 0.5 mL / min, λ -215 nm, 280 nm, in 5-65% acetonitrile / water containing 0.05% TFA.

[0753] Method C, HPLC conditions: HPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column at 25 °C for 20 min, with a flow rate of 0.5 mL / min, λ -215 nm, 280 nm, in 5-65% acetonitrile / water containing 0.05% TFA.

[0754] Method D, HPLC conditions: HPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column, with a flow rate of 0.5 mL / min, λ -215 nm, 280 nm, over 10 min at 25°C, in 5-65% acetonitrile / water containing 0.05% TFA. A 5.0 μL sample was injected using PLNO (partial loop with needle overfill) injection mode.

[0755] Table 10 provides exemplary long-acting GLP-1 receptor agonist polypeptides of the present disclosure.

[0756] Polypeptide analogs that do not have a lipophilic substituent and an optional spacer may be referred to herein as "linear polypeptides." Polypeptide analogs that have at least one covalently attached lipophilic substituent and an optional spacer may be referred to herein as "conjugated polypeptides."

[0757] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5]

[0758] Example 3: Stability of long-acting GLP-1 receptor agonist polypeptides Several long-acting GLP-1 receptor agonist polypeptides described herein were tested for stability as trifluoroacetate salts in 1 mg / ml solutions in DMSO (i.e., an organosulfur solvent) or water (i.e., DI water). These analog polypeptides were incubated at 37°C, and samples were withdrawn at various time intervals and analyzed by LC / MS and HPLC to determine the purity and mass of the parent peptide and the extent of degradation products. The purity results from these analyses are presented in Table 11 and are considered indicators of stability.

[0759] Example 11: Stability of long-acting GLP-1 receptor agonist polypeptides [Table 13-1] [Table 13-2]

[0760] Example 4: Solubility of long-acting GLP-1 receptor agonist polypeptides Certain long-acting GLP-1 receptor agonist polypeptides described herein were tested for solubility in saline or DI water at room temperature. Samples were visually inspected for clarity and for the appearance of turbidity or cloudiness. The results of these analyses are shown in Table 12. [Table 14]

[0761] Example 5: Functional Assays of Long-Acting GLP-1 Receptor Agonist Polypeptides: Human and Rat Receptors (GLP-1R) Activation of the human and rat GLP-1 receptor (GLP-1R) leads to an increase in cellular cyclic adenosine monophosphate (cAMP). Accumulated cAMP can be measured in vitro using standard detection methods in the presence of the nonspecific cAMP / cGMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Therefore, fitted dose-response curves of cAMP accumulation can be used to estimate the in vitro potency (pEC50) of peptides that activate each of these receptors.

[0762] Cell treatment and cAMP accumulation assay CHO-K1 cells stably expressing human GLP-1R (Genebank accession number NM_002062) were grown in 90% F12-K medium supplemented with 10% FBS and 250 μg / ml G418. For rat GLP-1R expression, a cDNA fragment encoding the full-length open reading frame of rat GLP-1R (Genebank accession number NM012728) was subcloned into the expression vector pcDNA3.1+ to allow transient receptor expression in CHO-K1 cells.

[0763] On day 1, CHO-K1 cells were plated at 1.5 million cells per T75 flask in 90% F12-K medium supplemented with 10% FBS. On day 3, cells were transfected with 40 mcg of rat GLP1R expression plasmid using Lipofectamine 2000 transfection reagent. On day 5, human or rat GLP-1R-expressing cells were dispensed into white 384-well OptiPlates at 1000 cells per well in 5 mcL of assay buffer consisting of 1x HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% casein.

[0764] Peptides were serially diluted 4-fold in assay buffer to a final concentration of 1 × 10 -9 M~9.5~10 -16Two assay controls consisting of 50 mcM forskolin (cAMP system maximum) or assay buffer only (cAMP system minimum) were also prepared. Five microliters of each peptide concentration or assay control was added to triplicate wells and incubated for 30 minutes at room temperature. During this incubation step, 4x europium-labeled cAMP tracer solution and 4x Ulight®-anti-cAMP solution (consisting of anti-cAMP monoclonal antibody labeled with Ulight™ dye) were prepared according to the manufacturer's protocol (PerkinElmer LANCE Ultra cAMP Kit). Following this incubation, 5 mcL of europium-labeled cAMP and 5 mcL of Ulight anti-cAMP antibody were added to each well. The plate was covered to prevent evaporation and incubated in the dark at room temperature for 60 minutes. The plate was read on an Envision fluorescent plate reader (PerkinElmer).

[0765] Data analysis Test values ​​were first normalized to the system maximum and system minimum mean values ​​in Excel using the formula: (Test Value - System Minimum Mean) / (System Maximum Mean - System Minimum Mean) * 100. Normalized test values ​​represent the baseline-corrected percentage of the system maximum cAMP response induced by forskolin. Data from up to three replicates were analyzed for each peptide. Potency values ​​were estimated by fitting the data using a four-parameter logistic curve model: Y = Base + (Top - Base) / (1 + 10^((LogEC50-X))) in GraphPad Prism software (v7.04). The Hill slope was constrained to 1.0. EC50 values ​​were converted to pEC50 values ​​using the formula: pEC50 = -Log(EC50). Mean pEC50 values ​​and 95% confidence intervals are reported in Table 13. [Table 15]

[0766] Example 6: Intravenous infusion of a long-acting GLP-1 receptor agonist polypeptide: A study to assess the intravenous pharmacokinetics of the polypeptide The peptides were dissolved in 0.05% Tween-20 in phosphate-buffered saline and administered to nonfasted male Sprague-Dawley rats (n=3 per group) as a 1-hour intravenous infusion via the femoral vein cannula at a final dose of 0.033 mg / kg. The formulation was administered at a rate of 1.67 mL / kg / hour. Blood samples (approximately 250 μL) were collected via the jugular vein cannula into microtiter tubes containing K2EDTA as an anticoagulant and 25 μL of protease inhibitor cocktail for pharmacokinetic analysis at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 8, 24, 48, 72, and 96 hours after the start of the infusion. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 14.

[0767] Example 7: Subcutaneous bolus injection of a long-acting GLP-1 receptor agonist polypeptide: A study to assess the bioavailability of the polypeptide The peptides were dissolved in 0.05% Tween-20 in phosphate-buffered saline and administered to nonfasted male Sprague-Dawley rats (n=3 per group) via a single bolus injection into the subcutaneous space between the shoulder blades at a dose of 0.100 mg / kg. Blood samples (approximately 250 μL) were collected via the jugular vein cannula into microtiter tubes containing K2EDTA as an anticoagulant and 25 μL of protease inhibitor cocktail for pharmacokinetic analysis at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, and 96 hours post-dose. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are also shown in Table 14. [Table 16]

[0768] Example 8: Methods for plasma sample preparation for pharmacokinetic studies Protein precipitation A 60 μL aliquot of each plasma sample was placed into an Impact Protein Precipitation 96-well filtration plate (Phenomenex, Torrance, CA). 6 μL of 0.5% Tween-20 was added to each well. The plate was then vortex-mixed at 1200 rpm for 10 minutes, after which 180 μL of 0.1% TFA in 2:1 ethanol:acetonitrile containing the appropriate internal standard was added to each well. The plate was vortex-mixed at 1400 rpm for 5 minutes and then centrifuged at 500 × g for 10 minutes. The filtrate was evaporated under a stream of nitrogen at 45°C. The residue was reconstituted in 80 μL of 20% acetonitrile (aqueous) containing 0.1% formic acid.

[0769] Example 9: LC / MS quantification of long-acting GLP-1 receptor agonist polypeptides in plasma All calibration standards were prepared in control rat plasma containing K2EDTA and a protease inhibitor cocktail.

[0770] Samples and standards were analyzed by TurboIonSpray™ UPLC-MS / MS using a system consisting of a CTC HTS PAL autoinjector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with column oven (Palo Alto, CA), a Valco switching valve (Houston, TX), and either an AB Sciex API 5600 TripleTOF™ or a Sciex API 4000 QTrap mass spectrometer (Framingham, MA). Samples were injected onto a 2.1 x 50 mm reversed-phase C18 analytical column, typically a Waters CORTECS UPLC C18+, 1.6 μm (Waters Corporation, Milford, MA) or similar. Chromatographic separation was performed using a gradient method with water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) as the mobile phase. Initial conditions consisted of 90% A and 10% B. The organic fraction was increased to 95% B over 2–3 min, depending on the peptide. Typical flow rates were 600 μL / min. Column temperature was kept constant at 50°C. Peptides were quantified by monitoring one or more products generated from multiply charged parent ions.

[0771] Example 10: In vivo efficacy of long-acting GLP-1 receptor agonist polypeptides for food intake inhibition in rats To determine the amount of food intake inhibition exhibited by these long-acting GLP-1 receptor agonist polypeptides, short-term food intake was measured continuously for 96 hours using a BioDAQ food monitoring system (Research Diets, New Brunswick, NJ). Long-Evans rats were obtained at approximately 8 weeks of age. Rats were housed individually and acclimated to a 45% high-fat diet for at least 2 weeks prior to dosing. After 1 week of acclimation, all rats were individually housed in BioDAQ cages (Research Diets, New Brunswick, NJ) and maintained at a constant temperature (approximately 22°C) and relative humidity of 30-70% on a 12-hour light-dark cycle (lights on from 5:00 AM to 5:00 PM). Rats had free access to water and pelleted food (Research Diets D12451i, 45 kcal% fat, Research Diets, New Brunswick, NJ). All procedures were performed in accordance with USDA regulations under the Animal Welfare Act, and the procedures were approved by the Mispro Institutional Animal Care and Use Committee. Animals were randomized into treatment groups according to body weight (n = 8 rats / group). Animals were administered (SC bolus injection) either a long-acting GLP-1 receptor agonist polypeptide (30 mcg / kg) or vehicle control (saline) between 4:30 and 5:00 AM before lights out. The hopper gate was closed while the animals were being administered. Immediately after administration was completed, the hopper gate was opened and continuous data collection began. Data were initially analyzed using BioDAQ Viewer software (version 2.3.07), with bout filters set as needed to reduce noise in the data related to non-feeding behavior. All data are expressed as % inhibition from vehicle control and summarized as mean values. Data were analyzed for statistical significance using two-sample t-tests using Microsoft Excel (Redmond, WA). A P value <0.05 was considered to indicate a significant difference between treatment groups. The results of short-term food intake inhibition % from vehicle control for long-acting GLP-1 receptor agonist polypeptides are shown in Table 15. [Table 17]

[0772] Example 11: In vivo efficacy of long-acting GLP-1 receptor agonist polypeptides on body weight change in LE rats following bolus SC administration. To examine the efficacy and durability of long-acting GLP-1 receptor agonist polypeptides on weight loss following bolus SC administration (30 mcg / kg), body weights were measured in the same LE rats used to measure food intake in Example 11. Data were analyzed in Excel and / or Prism (GraphPad Software, Inc., La Jolla, CA) using one-way ANOVA to compare each group to the appropriate control group. A P value of <0.05 was considered to indicate a significant difference between treatment groups. The mean weight loss (%) from baseline and vehicle control (ΔΔ%) from the food intake study is shown in Table 16. [Table 18]

[0773] Example 12: Weight loss efficacy of long-acting GLP-1 receptor agonist polypeptides in LE DIO rats A weight loss efficacy study was conducted in a rodent model of obese Long-Evans (LE) diet-induced obese (DIO) rats to investigate the efficacy and duration of a long-acting GLP-1 receptor agonist polypeptide after subcutaneous bolus (SC) administration. Male LE DIO rats (Envigo Laboratories, Inc., Indianapolis, IN) were used. From the onset of weaning, rats were fed a high-fat diet (Teklad TD 95217, fat-based, 40% kcal; Harlan Laboratories, Madison, WI). Rats were 15–17 weeks old at the start of the study. Rats were housed individually per cage and provided with free access to a high-fat diet (Harlan TD.95217, 4.3 kcal / g) and water. They were maintained at 21°C and 50% relative humidity on a 12-h light / dark cycle from 5:00 AM to 5:00 PM and allowed to acclimate for at least 10 days before use. Eighteen-week-old male LE DIO rats (14 weeks on a high-fat diet) were administered a bolus SC injection of a long-acting GLP-1 receptor agonist polypeptide (30 mcg / kg; n=6 animals / treatment group). All other procedures were the same as those described in the previous examples. The mean weight loss (%) (ΔΔ%) results from baseline and vehicle control are shown in Table 17. [Table 19]

[0774] Example 13: Efficacy of long-acting GLP-1 receptor agonist polypeptides on food intake in LE DIO rats To examine the efficacy and durability of long-acting GLP-1 receptor agonist polypeptides in inhibiting food intake after bolus SC administration (30 mcg / kg), food intake was measured in the same LE DIO rats used for body weight measurements in Example 13. Food hoppers were weighed daily. All other procedures were the same as those described in the previous example. The results of the mean percent reduction in food intake from vehicle controls (ΔΔ%) are shown in Table 18. [Table 20]

[0775] Example 14: Long-term weight loss efficacy of long-acting GLP-1 receptor agonist polypeptides in LE DIO rats A long-term study was conducted to investigate the effect and persistence of continuous administration of a long-acting GLP-1 receptor agonist polypeptide on body weight after 27 days of treatment in LE DIO rats. Male LE DIO rats aged 18 weeks (14 weeks on a high-fat diet) were treated as described in Example 13. Before the start of drug treatment (day -9) and on day 27, rats were weighed and fat and lean body composition measurements were performed using quantitative magnetic resonance (QMR) (Echo Medical Systems, Houston, TX) according to the manufacturer's protocol. Body weight was measured throughout the study. Rats were randomly assigned to treatment groups (n=8 / group) with similar average body weights and fat masses.

[0776] LE DIO rats were administered either a long-acting GLP-1 receptor agonist polypeptide or vehicle (saline) by SC injection every other day (eod). All other procedures were the same as those described in the previous examples. The mean weight loss (%) (ΔΔ%) results from baseline and vehicle control are shown in Figure 1 and Table 19.

[0777] Example 15: Long-term antidiabetic efficacy of long-acting GLP-1 receptor agonist polypeptides in ZDF rats A longitudinal study was conducted to determine the antidiabetic effects of continuous administration of a long-acting GLP-1 receptor agonist polypeptide on HbA1c (the primary antidiabetic parameter) after 27 days of treatment in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were obtained at 6 weeks of age (Charles River, Raleigh, NC) and used in the study at 8 weeks of age. Upon receipt, rats were housed one animal per cage, provided with Purina 5008 chow (Lab Diet, St. Louis, MO) and water ad libitum, maintained at 21°C and 50% relative humidity, and on a 12-h light / dark cycle from 5:00 AM to 5:00 PM. They were allowed to acclimate for 9 days before the start of the study. Blood samples were collected from the tail vein before blood collection (day -3) and blood glucose and HbA1c were measured. ZDF rats were randomized into treatment groups (n = 9–10 / group) with similar mean HbA1c and glucose levels. The long-acting GLP-1 receptor agonist polypeptide was administered every other day (eod) via SC injection. Blood samples were collected again on days 14 and 27 (end of study) to measure blood glucose and HbA1c. A final whole blood sample was collected by cardiac puncture under isoflurane anesthesia (day 27). HbA1c analysis was performed using a Carolina Chemistries CLC720i clinical chemistry analyzer (Mindray Inc., Mahwah, NY) using the protocol and method parameters described by the manufacturer. HbA1c results, expressed as mean percent change (ΔΔ%) from baseline and vehicle control, are shown in Table 19. [Table 21]

[0778] Example 16: Comparison of weight loss and antidiabetic efficacy of GLP-1 receptor agonist polypeptides in ZDF rats In one embodiment, the GLP-1 receptor agonist polypeptide can be administered to a patient via an implantable drug delivery device. The Medici Drug Delivery System™ (Intarcia Therapeutics, Boston, MA) includes an osmotic drug delivery minipump (shown in FIG. 3) for subcutaneous placement in a patient, delivering the drug subcutaneously at a continuous rate for an extended period of time, such as 3, 6, 12, 18, or 24 months. The osmotic drug delivery minipump in FIG. 3 is 44 mm long and 4 mm in diameter. It contains a volume-limited drug reservoir. Therefore, high in vivo efficacy is required to enable delivery of the drug via this minipump.

[0779] The in vivo efficacy of a representative long-acting GLP-1 receptor agonist (A120) of the present invention against weight loss and type 2 diabetes in DIO rats was tested and compared with that of semaglutide (Novo Nordisk A / S Bagsvaerd, Denmark). Comparison of in vivo efficacy of both compounds against weight loss in DIO rats is shown in Figure 2A (% of baseline vs. vehicle over 28 days, n=6 / dose). Comparison of in vivo efficacy (i.e., HbA1c reduction) against type 2 diabetes in DIO rats is shown in Figure 2B (Δ-HbA1c vs. vehicle over 28 days, n=9 / dose). Antidiabetic efficacy was compared with the ZDF rat model of type 2 diabetes.

[0780] Compound A120 was found to be approximately 2.3-fold more potent than semaglutide for weight loss and approximately 2.6-fold more potent than semaglutide for HbA1c reduction. Adjusting for the pharmacokinetic parameters of its cognate counterparts, the human efficacy of compound A120 is predicted to be approximately 3.1-fold that of semaglutide.

[0781] Example 17: In vitro metabolic stability pharmacokinetic study (T1 / 2) of GLP-1 receptor agonists Rat and human kidney brush border membranes In vitro incubation with renal brush border membrane (kBBM) preparations was used to characterize the ability of peptides to resist degradation by proteases and peptidases in the systemic circulation. kBBM was chosen because it contains high concentrations of a diverse set of proteases and peptidases, many of which are present throughout the body. In general, peptides with low in vivo CL are stable in this assay, while peptides with high in vivo CL are unstable.

[0782] Brush border membranes from rat and human kidney tissue were prepared by centrifugation and stored at -70°C. Thawed stocks of rat or human kBBM were diluted to the appropriate concentration with 25 mM HEPES buffer (pH 7.4) containing 1% casein and dispensed into a 96-well plate. The kBBM solution was preheated to 37°C for 10 minutes. The reaction was initiated by the addition of test peptide (final concentration 1 mcM), also dissolved in 25 mM HEPES buffer (pH 7.4) containing 1% casein. The final concentration of kBBM in each incubation was 50 mcg protein / mL. The reaction was maintained at 37°C in a shaking water bath. At 0, 0.25, 0.5, 1.0, 2.0, and 4.0 hours after initiation, 30 mcL of the reaction mixture was removed and placed into a 96-well plate containing 120 mcL of ice-cold methanol containing 2.5% formic acid. The quenched samples were centrifuged at 2178×g for 10 minutes, after which a portion of the supernatant was transferred to a clean 96-well plate and diluted 1:1 with water. The samples were analyzed by UPLC-MS / MS. The results of this analysis for compound A120 (SEQ ID NO: 120) are shown in Table 20.

[0783] Human subcutaneous tissue homogenate In vitro incubation with subcutaneous (SC) tissue homogenates was used to characterize the ability of peptides to resist pre-systemic degradation by proteases and peptidases after SC administration. In vivo nonclinical studies have shown that peptidase activity in the SC space can limit the bioavailability of peptides after SC administration. Peptides with high SC bioavailability are stable in this assay, whereas peptides with low SC bioavailability are unstable in this assay.

[0784] Human SC tissue was homogenized in cold 25 mM HEPES buffer (pH 7.4, 10 times the sample weight) and filtered through a double layer of cheesecloth. The filtrate was aliquoted, flash-frozen in a methanol / dry ice bath, and stored at -80°C. The protein concentration of each pooled batch was determined using a BCA protein assay. Thawed human SC tissue homogenate stock was diluted to 1.0 mg protein / mL with 25 mM HEPES buffer (pH 7.4) and dispensed into a 96-well plate. The diluted SC homogenate was preheated to 37°C for 10 minutes. The reaction was initiated by the addition of test peptide (final concentration 10 mcM), also dissolved in 25 mM HEPES buffer (pH 7.4). The reaction was maintained at 37°C in a shaking water bath. At 0, 0.25, 0.5, 1.0, 2.0, and 4.0 hours after initiation, 50 mcL of the reaction mixture was removed and placed in a 96-well plate containing 150 mcL of ice-cold methanol containing 2.5% formic acid. The quenched samples were centrifuged at 2178 x g for 10 minutes, after which a portion of the supernatant was transferred to a clean 96-well plate and diluted 1:10 with water. The samples were analyzed by UPLC-MS / MS. The results of this analysis for compound A120 (SEQ ID NO: 120) are shown in Table 20.

[0785] The unbound fraction in plasma (f u ) Because peptides tend to adsorb to the surfaces of plastic tubing, dialysis membranes, and molecular weight cutoff filters, traditional methods for measuring plasma protein binding, such as equilibrium dialysis, ultrafiltration, and ultracentrifugation, are unreliable. Therefore, the extent to which acylated peptides bind to serum albumin was assessed using surface plasmon resonance (SPR). The usefulness of this technique for reasonably estimating the percentage of drug bound to plasma proteins has been demonstrated in the literature. The results of this analysis for compound A120 (SEQ ID NO: 120) are shown in Table 20. Based on this data, the estimated half-life of compound A120 in humans is approximately 6 days. [Table 22]

[0786] Example 18: In vivo pharmacokinetic parameters of compound A120 in rat and monkey models The pharmacokinetic profiles of Compound A120 in rat and monkey models were determined after intravenous infusion, subcutaneous bolus injection, or subcutaneous infusion of the compound. The results of these assays are presented in Table 21. [Table 23]

[0787] Example 19: Albumin binding of long-acting GLP-1 receptor agonists The potency shifts of the conjugated GLP-1 receptor agonists are shown in Table 22. Data were obtained using a cAMP accumulation assay at the GLP-1 receptor for each of GLP-1(7-36), semaglutide, exenatide, and compound A120 in the presence of various concentrations of human serum albumin (HSA). The results of this analysis are also shown in Figure 4. [Table 24]

[0788] Example 20: Efficacy of long-acting GLP-1 receptor agonists across species The potencies of GLP-1(7-36), semaglutide, exenatide, and Compound A120 were determined against the human (h), cynomolgus monkey (c), and rat (r) orthologues of the GLP-1 receptor (GLP-1R), glucagon receptor (GCGR), and gastric inhibitory polypeptide receptor (GIPR). Values ​​were obtained by cAMP accumulation assays. The results of these assays are presented in Table 23. [Table 25]

[0789] The data demonstrate that compound A120 is consistently potent at GLP-1R across three related species, and that this compound is highly selective for GLP-1R over GCGR and GIPR.

[0790] Example 21: Comparative data of compound A120 to semaglutide [Table 26] [Table 27]

[0791] Other embodiments While the present invention has been described with reference to its detailed description, the foregoing description is illustrative, not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Amino acid sequence of SEQ ID NO: 200: HX ２ X ３ GTX ６ X ７ X ８ X ９ X １０ S.X. １２ X １３ X １４ EX １６ X １７ X １８ X １９ X ２０ X ２１ FIX ２４ W.L.K.X. ２８ GGPX ３２ SGAPPPS-(OH / NH ２ ) (SEQ ID NO:200), or a pharma- ceutically acceptable salt thereof, X ２ is A, 2-aminoisobutyric acid (Aib), or G; X ３ is E or N-methyl Glu; X ６ is F or Y; X ７ is S or T; X ８ is diaminopimelic acid (Dap), E, ​​K, N, N-methyl Ser, Q, S, s, or Y; X ９ is D or E; X １０ is I, L, N-methylLeu, or V; X １２ is E, K, Q, or S; X １３ is Aib, E, K, Q, S, W, or Y; X １４ is E, R, I, K, L, M, Q, or Y; X １６ is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn); X １７ is E, K, or Q; X １８ is A, K, S, or Y; X １９ is A, K, or V; X ２０ is E, K, or R; X ２１ is Aib, E, H, K, L, Q, or Y; X ２４ is A, Aib, E, K, Q, S, or Y; X ２８ is D, E, K, N, Q, S, or Y; and X ３２ is Dap, H, K, R, or S; Here, X １６ is Dab, Dap, K, or Orn, it is covalently linked, optionally via a spacer, to a lipophilic substituent; Here, X １６ If E, then X １４ , X １７ , X １８ , X １９ , X ２０ , or X ２１ at least one of is selected to be lysine, said lysine residue being covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the peptide comprises X17 and X ２１ At position; X 21 and X １７ At position; X 21 and X ２８ At position; X 28 and X ２１ At position; X 20 and X ２４ At position; X 24 and X ２０ or X and X １６ position, optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain.

2. Amino acid sequence of SEQ ID NO: 204: HX ２ EGTFTX ８ D.X. １０ S.X. １２ QX １４ EX １６ X １７ X １８ X １９ X ２０ X ２１ FIX ２４ W.L.K.X. ２８ GGPX ３２ SGAPPPS-(OH / NH ２ 20. The isolated polypeptide of claim 1, comprising: X ２ is 2-aminoisobutyric acid (Aib), or G; X ８ is N or S; X １０ is I, L, or V; X １２ is E, K, or Q; X １４ is M or Y; X １６ is (diaminopimelic acid) Dap, optionally covalently linked to a lipophilic substituent via a spacer, or is K, optionally covalently linked to a lipophilic substituent via a spacer; X １７ is E or K; X １８ is A or Y; X １９ is A or V; X ２０ is E, K, or R; X ２１ is E, K, L, or Q; X ２４ is E, K, or S; X ２８ is N or Q; and X ３２ is H or S; wherein the peptide comprises X17 and X ２１ At position; X 21 and X １７ At position; X 20 and X ２４ At position; X 24 and X ２０ position, optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain.

3. Amino acid sequence of SEQ ID NO: 208: HAibEGTFTSDX １０ SKQYEX １６ EAX １９ X ２０ X ２１ FIX ２４ WLKNGGPSSGAPPPS-(OH / NH ２ 209. The isolated polypeptide of claim 1, comprising: X １０ is L or V; X １６ is K covalently linked to the lipophilic substituent, optionally via a spacer; X １９ is A or V; X ２０ is E, K, or R; X ２１ is K or Q; X ２４ is E, K, or S; and wherein the peptide comprises X21 and X １７ At position; X 20 and X ２４ or X24 and X ２０ position, optionally further comprising a lactam bridge formed via an amide bond between a lysine side chain and a glutamic acid side chain.

4. X １４ When M is X １６ 3. The isolated polypeptide of claim 1 or 2, wherein is K covalently attached, optionally via a spacer, to a lipophilic substituent, or a pharma- ceutically acceptable salt thereof.

5. X １６ If E, then X ２１ is K or E, or a pharma- ceutically acceptable salt thereof.

6. X １４ The isolated polypeptide of claim 1 or 2, wherein is Y, or a pharma- ceutically acceptable salt thereof.

7. X １６ 3. The isolated polypeptide of claim 1 or 2, or a pharma- ceutically acceptable salt thereof, wherein is a lysine covalently linked, optionally via a spacer, to a lipophilic substituent.

8. X ２４ If S, then X １０ The isolated polypeptide of any one of claims 1 to 3, or a pharma- ceutically acceptable salt thereof, wherein:

9. 4. The isolated polypeptide of claim 1, or a pharma- ceutically acceptable salt thereof, comprising: ２０ and X ２４ or a pharma- ceutically acceptable salt thereof, further comprising a lactam bridge formed through an amide bond between a lysine side chain and a glutamic acid side chain at positions A1 and B1, respectively.

10. X ２ The isolated polypeptide of any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, wherein is Aib.

11. The lipophilic substituent is covalently attached to the isolated polypeptide via a spacer, The lipophilic substituent and the spacer are represented by formula II: -(Y) ｎ -CO-(CH ２ ) ｍ -Z Formula II Or, Formula IV: -(Y1) n1 -(dpeg) r -(Y2) n2 -CO-(CH 2 ) m -Z Formula IV The During the ceremony, dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; Y is selected from the group consisting of γGlu, Asp, Lys, and Gly; Y1 is selected from the group consisting of γGlu, Asp, and Gly; Y2 is selected from the group consisting of γGlu, Asp, and Gly; Z is -CH ３ Or -CO ２ H; m is 4 to 24; n is 1 to 10; n1 is 0 to 10; n2 is 0 to 10; and 11. The isolated polypeptide of any one of claims 1 to 10, or a pharma- ceutically acceptable salt thereof, wherein r is 1 to 8.

12. An isolated polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 1-162, or a pharma- ceutically acceptable salt thereof.

13. The isolated polypeptide of claim 12, comprising the amino acid sequence of SEQ ID NO:55, or a pharma- ceutically acceptable salt thereof.

14. 13. The isolated polypeptide of claim 12, comprising the amino acid sequence of SEQ ID NO: 115, or a pharma- ceutically acceptable salt thereof.

15. 13. The isolated polypeptide of claim 12, comprising the amino acid sequence of SEQ ID NO: 120, or a pharma- ceutically acceptable salt thereof.

16. The isolated polypeptide of claim 12, comprising the amino acid sequence of SEQ ID NO: 132, or a pharma- ceutically acceptable salt thereof.

17. A pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable adjuvant, carrier, or vehicle.

18. An osmotic delivery device comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to claim 18.

19. A pharmaceutical composition for use in treating obesity in a human subject in need thereof, reducing weight in a human subject in need thereof, or suppressing appetite in a human subject in need thereof, said pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

20. A pharmaceutical composition for use in the treatment of diabetes in a human subject in need thereof, said pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

21. A pharmaceutical composition for use in the treatment of non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH) in a human subject in need thereof, said pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

22. A pharmaceutical composition for use in the treatment of a neurological disease or disorder in a human subject in need thereof, said pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.