Human amylin analog peptides and methods of use

Abstract

The present invention relates to isolated polypeptides that are analogs of human amylin. The disclosed amylin analog polypeptides have a longer elimination half-life (t 1 / 2 The amylin analog polypeptides have advantageous physicochemical properties compared to endogenous amylin, such as improved solubility and thermal stability, and improved solubility and thermal stability. The present invention also relates to methods of using the disclosed amylin analog polypeptides in various therapeutic indications, as well as methods of producing the amylin analog polypeptides. The disclosed amylin analog polypeptides are particularly useful in methods of treating metabolic diseases or disorders, such as type 1 and type 2 diabetes, and in methods of providing weight loss.

Description

[Technical field]

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 744,236, filed October 11, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 10, 2019, is named 616782_102487-055PC_SL-10-10-2019.txt and is 123,647 bytes in size.

[0003] The present invention relates to isolated polypeptides that are analogs of human amylin. The disclosed amylin analog polypeptides have a longer elimination half-life (t 1 / 2 ) and have beneficial physicochemical properties compared to endogenous amylin, such as improved solubility and thermal stability. The present invention also relates to methods of using the presently disclosed amylin analog polypeptides in a variety of therapeutic indications, as well as methods of making the amylin analog polypeptides. As described in more detail below, the disclosed amylin analog polypeptides are particularly useful in methods of treating metabolic diseases or disorders, such as type 1 and type 2 diabetes, and providing weight loss. [Background technology]

[0004] 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, 7404 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 type 1 diabetes and may occur later in type 2 diabetes, leads to insufficient secretion of insulin and amylin.

[0005] Amylin functions as part of the pancreatic endocrine tissue, which are cells in the pancreas that synthesize and secrete hormones. Amylin contributes to glycemic control and is secreted from pancreatic islets into the blood circulation and cleared by peptidases in the kidney. The metabolic function of amylin is well characterized as an inhibitor of the appearance of nutrients such as glucose in the plasma. Thus, amylin functions as a synergistic partner to insulin, a peptide that regulates blood glucose 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 glucose levels from becoming too high, especially after meals.

[0006] 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 excess amounts of glucagon, which raises blood sugar immediately after eating.

[0007] For many reasons, human amylin, with a half-life in serum of about 13 minutes, is not suitable for use as a therapeutic agent. Instead, pramlintide (Symlin®, developed by Amylin Pharmaceuticals, Inc., San Diego, CA, USA, and marketed by AstraZeneca plc, Cambridge, UK) was developed as a synthetic analogue of human amylin for the treatment of type 1 or type 2 diabetes patients who use prandial insulin but cannot achieve the 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.

[0008] In the treatment of patients with type 1 diabetes, pramlintide is administered by subcutaneous injection before meals up to four times per day as an adjunct to insulin therapy administered after meals. Pramlintide cannot be mixed with insulin; a separate syringe is used. Reported side effects of pramlintide include nausea and vomiting. Adverse reactions can include severe hypoglycemia, especially in patients with type 1 diabetes. As a result, patients who begin taking pramlintide will receive a reduced prandial insulin dose.

[0009] For the treatment of patients with type 2 diabetes, pramlintide is administered by subcutaneous injection before each meal at a recommended starting dose that is gradually increased to a target maintenance dose. Another investigational analog of human amylin, davalintide (AC2307; also developed by Amylin Pharmaceuticals, Inc.), has a half-life of approximately 26 minutes.

[0010] Thus, there is a need for improved amylin analog polypeptides that mimic amylin activity but have greater therapeutic potential than both endogenous human amylin and existing amylin analogs such as pramlintide and davalintide. Summary of the Invention

[0011] It has now been discovered that the polypeptides of the present invention, and pharma- ceutically acceptable compositions thereof, are useful as amylin analogs. Such polypeptides are represented by SEQ ID NO:199: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TX 37 -(OH / NH2) (SEQ ID NO: 199), X1 is S, K, k, H or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; 10 is Q or S;X 12 is L or K;X 13 is A, S, E or K; X 14 is N, n, d, Y or Q; X 15 is E, F, f, Y, I, k, K or α-aminoisobutyric acid (Aib); X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib;X 20 is S or Aib;X 22 is N or E;X 29 is P, R, or K; X 31 is k, K, N, or H; X 35 is e, E, N, K, G, A, Y, or P; X 37 is Y or P; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; X 31 If N, then X 35 is E, or X 35 If N, then X 31 and K, or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0012] [Figure 1] 3 is a table showing comparative sequence alignments of certain reference polypeptides: human amylin, SEQ ID NO: 300; rat amylin, SEQ ID NO: 301; pramlintide, SEQ ID NO: 302; davalintide, SEQ ID NO: 303; hCT (human calcitonin), SEQ ID NO: 304; sCT (salmon calcitonin), SEQ ID NO: 305; and beta calcitonin gene-related peptide (β-CGRP), SEQ ID NO: 306. [Diagram 2] A shows a dose-response curve of acylated amylin analog A73 at hAMY3R. B shows a dose-response curve of acylated amylin analog A73 at hCTR. C shows a dose-response curve of pramlintide at hAMY3R. D shows a dose-response curve of human calcitonin (hCalcitonin) at hCTR. The term "acylation" 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. [Figure 3A] FIG. 1 shows data from a pharmacokinetic study to assess renal polypeptide clearance (CL) following intravenous infusion of a linear, ie, non-acylated, polypeptide. [Figure 3B]FIG. 1 shows data from a pharmacokinetic study to assess polypeptide clearance (CL) from the kidney following intravenous infusion of the conjugate, an acylated polypeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1. General Description of Certain Embodiments of the Invention The present invention relates to isolated polypeptides that are amylin analogs, as well as pharmaceutical compositions comprising these polypeptides. The present invention also relates to methods of making and using such amylin analog polypeptides. These amylin analog polypeptides are particularly useful in methods of treating metabolic diseases or disorders, including type 1 and type 2 diabetes, obesity, and in methods of providing weight loss.

[0014] 2.Definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "solvent" includes a combination of two or more such solvents, reference to "peptide" includes one or more peptides or mixtures of peptides, reference to "drug" includes one or more drugs, reference to "osmotic delivery device" includes one or more osmotic delivery devices, etc. Unless specifically stated or clear from the context, the term "or" as used herein is understood to be inclusive and encompasses both "or" and "and".

[0015] 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 2 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".

[0016] Unless specifically stated or clear from the context, the term "substantially" as used herein is understood to be within a narrow range of variation or otherwise normal tolerance in the art. Substantially may be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value.

[0017] 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.

[0018] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0019] The terms "drug," "therapeutic agent," and "beneficial agent" are used interchangeably to refer to any therapeutically active substance 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 invention are well suited for the delivery of proteins, small molecules, and combinations thereof.

[0020] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and typically refer to molecules that include 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).

[0021] In some embodiments, naturally occurring L-amino acids are represented by either the conventional three letter or uppercase single letter amino acid symbols in Table 1. In other embodiments, both naturally occurring L-amino acids and D-amino acids are represented by either the conventional three letter or uppercase single letter amino acid symbols in Table 1. In still other embodiments, D-amino acids are represented by lowercase single letter amino acid symbols that correspond to the single letter symbols in Table 1, i.e., g, a, l, m, f, w, k, q, e, s, p, v, i, c, y, h, r, n, d, and t.

[0022] [Table 1]

[0023] 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 via 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, selenization, and C-terminal amidation. The term peptide also includes peptides that contain modifications of the amino and / or carboxy termini. Modifications of the terminal amino group include, but are not limited to, the desamino modification, the N-lower alkyl modification, the N-di-lower alkyl modification, and the N-acyl modification. Modifications of the terminal carboxy group include, but are not limited to, the amide modification, the lower alkyl amide modification, the dialkyl amide modification, and the lower alkyl ester modification (e.g., the lower alkyl is a C1-C4 alkyl). The term peptide also includes modifications of the amino acids located 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.

[0024] 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 in order starting from the amino terminus and increasing toward the carboxy terminus of the peptide.

[0025] 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.

[0026] The term "insulinotropic" as used herein 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.

[0027] The term "insulinotropic peptide" as used herein includes, but is not limited to, glucagon-like peptide 1 (GLP-1), and its derivatives and analogs, GLP-1 receptor agonists, such as exenatide, having the amino acid sequence of SEQ ID NO: 307, and its derivatives and analogs.

[0028] The term "acylation" 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, and "lipophilic substituent" and "spacer" are defined herein. Certain lipophilic substituents can bind to albumin, each optionally via a spacer, to confer affinity to the resulting acylated polypeptide. The extent to which lipophilic substituents bind to albumin, each optionally via a spacer, to confer affinity 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.

[0029] The terms "linear" or "linear polypeptide" as used herein refer to "non-acylated" polypeptides, in other words, the disclosed amylin analog polypeptides that do not have a lipophilic substituent, optionally connected via a spacer, respectively, where "lipophilic substituent" and "spacer" are defined herein.

[0030] As used herein, the term "conjugate" or conjugated polypeptide refers to an "acylated" polypeptide, in other words, the disclosed amylin analog polypeptides having one or more lipophilic substituents, each optionally connected via a spacer.

[0031] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, trifluoroacetic acid (TFA), oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Acid salts include, for example, acid salts, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0032] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharma-ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0033] As used herein, the phrase "incretin mimetic" includes, but is not limited to, GLP-1 peptides, GLP-1 receptor agonists, peptide derivatives of GLP-1, peptide analogs of GLP-1; exenatide, exenatide having the amino acid sequence of SEQ ID NO: 307, exenatide peptides, peptide derivatives of exenatide, and peptide analogs of exenatide.

[0034] Examples of preferred incretin mimetics include exenatide, exenatide having the amino acid sequence of exendin-4 (the naturally occurring form of exenatide), exenatide-LAR, lixisenatide, GLP-1(7-36), 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.

[0035] The term "exenatide" as used herein includes exenatide, (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2), exenatide having the amino acid sequence of SEQ ID NO: 307, native exendin-4, exenatide peptides, exenatide peptide analogs, and exenatide peptide derivatives.

[0036] The term "GLP-1" refers to a polypeptide produced primarily by L-cells located in the ileum and colon, and to a lesser extent by L-cells located in the duodenum and jejunum. 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]. GLP-1R agonistic effects are diverse.GLP-1 maintains glucose homeostasis by enhancing endogenous glucose-dependent insulin secretion, sensitizing beta cells to glucose and GLP-1, inhibiting 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.

[0037] As described in more detail below, in some embodiments, the amylin analog polypeptides disclosed herein are provided in methods for the treatment of type 1 diabetes as an adjunct to insulin treatment. As used herein, the term "insulin" refers to human insulin or any insulin analog. Exemplary, non-limiting insulin analogs include those listed in Table 2:

[0038] [Table 2] TIFF0007636321000003.tif236170TIFF0007636321000004.tif190170

[0039] The term "prandial insulin" as used herein refers to a fast-acting insulin preparation that reaches peak blood concentrations approximately 45-90 minutes after administration and peak activity approximately 1-3 hours, and is administered at or around the time of a meal.

[0040] The term "vehicle" as used herein refers to a medium used to transport 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 including a drug particle formulation.

[0041] 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 invention, the suspension vehicle is formulated to exhibit phase separation upon contact with an aqueous environment having less than approximately 10% water.

[0042] As used herein, the phrase "single-phase" refers to a homogeneous system of a solid, semi-solid, or liquid that is physically and chemically uniform throughout.

[0043] The term "dispersion" as used herein refers to dissolving, dispersing, suspending, or otherwise distributing a compound, eg, a drug particle formulation, in a suspension vehicle.

[0044] 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.

[0045] The phrase "physically stable" as used herein refers to a formulation in which aggregates (e.g., dimers and other high molecular weight products) are formed in an acceptable percentage. Furthermore, a physically stable formulation does not change its physical state, for example, from liquid to solid or from amorphous to crystalline form.

[0046] 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, DM & Considine, GD, 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).

[0047] 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, which is performed under selected conditions (e.g., at a temperature of about 37°C). Other methods for determining viscosity include measuring the dynamic viscosity using a viscometer, such as a Cannon-Fenske viscometer, a Cannon-Fenske Ubbelohde viscometer for opaque solutions, or an Ostwald viscometer. In general, 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.

[0048] The term "non-aqueous" as used herein typically refers to a total water content of, for example, a suspension formulation, of 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.

[0049] 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 animals 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; birds, including domestic birds, wild birds and game birds, such as chickens, turkeys and other poultry, ducks and geese. The term does not indicate a particular age or sex. Thus, it is intended to include both adults and newborns.

[0050] 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 in the absence of symptoms. For example, treatment may be administered to susceptible individuals prior to the onset of symptoms (e.g., in light of a history of the condition and / or in light of genetic or other susceptibility factors). Treatment may be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0051] The term "osmotic delivery device" as used herein typically refers to a device used to deliver a drug (e.g., a disclosed amylin analog polypeptide) to a subject, the device including, 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 amylin analog 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 the subject, for example, subdermally or subcutaneously (e.g., in the inner, outer or back of the 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.

[0052] The term "continuous delivery" as used herein typically refers to the substantially continuous release of a drug from an osmotic delivery device into tissues near the implantation site, such as subdermal and subcutaneous tissues. For example, an osmotic delivery device releases a drug at an essentially predetermined rate based on the principle of osmosis. Extracellular fluid passes directly through a semipermeable membrane into the osmotic engine of the osmotic delivery device, which expands to move a piston at a slow and constant moving rate. The movement of the piston forces the drug formulation to be released 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.

[0053] The term "substantially steady-state delivery" as used herein 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., the disclosed amylin analog polypeptides) means that the rate of drug delivered is constant and independent of the drug available in the delivery system, e.g., 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 straight line is fitted to the data, the line has a slope of approximately zero.

[0054] The phrase "drug half-life" as used herein 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) assay, enzyme-linked immunosorbent assay (ELISA), or immunoenzyme sandwich assay (IEMA).

[0055] 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."

[0056] 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."

[0057] Baseline is defined as the last assessment on or before the day of initial placement of the osmotic delivery device (containing drug or placebo).

[0058] 3. Endogenous amylin, related peptides, and amylin receptors Human amylin, a 37-residue polypeptide hormone, is co-secreted with insulin from pancreatic beta cells. Loss of beta cell function, which occurs early in type 1 diabetes and may occur later in type 2 diabetes, leads to insufficient secretion of insulin and amylin. Amylin is thought to play a role in blood glucose regulation by slowing gastric emptying and promoting satiety, thereby preventing a rapid rise in blood glucose levels after eating. The overall effect is to slow the rate of appearance of glucose in the blood after eating.

[0059] The amino acid sequence of amylin is most closely related to that of calcitonin gene-related peptide (CGRP), which shares similarly positioned disulfide bonds and an amidated C-terminus. This is also true for calcitonin, adrenomedullin, and adrenomedullin 2. Together, these peptides form a small family united by these properties. As a result, there is some overlap in the binding and pharmacological activity of each peptide's cognate receptor. The table in Figure 1 shows a comparative sequence alignment of amylin and certain related reference polypeptides.

[0060] Peptides commonly designated as calcitonin (CT) peptide family members include calcitonin gene-related peptide (CGRP), calcitonin (CT), amylin (AMY), adrenomedullin 1, and adrenomedullin 2 / intermidin (ADM1, ADM2, respectively). Two G protein-coupled receptor proteins (calcitonin receptor; CTR, and calcitonin receptor-like receptor; CALCRL) and three receptor activity-modifying proteins (RAMP1, RAMP2, RAMP3) constitute pharmacologically distinct receptors within this entire peptide family (CTR, AMY1, AMY2, AMY3, CGRPR, AM1, AM2). There appear to be at least five distinct receptors (AMY1, AMY2, AMY3, CTR, CGRPR) to which amylin binds with significant affinity. CTR dimerizes with RAMP1, 2, or 3 to reconstitute the AMY1, AMY2, or AMY3 receptor with pharmacological selectivity for amylin over calcitonin. In the absence of RAMPs, CTR pharmacology is selective for calcitonin over amylin. CALCRL dimerizes with RAMP1 to generate CGRPR with high affinity for CGRP and reduced affinity for all other peptide family members, including amylin. CALCRL and RAMP2 or RAMP3 reconstitute the pharmacology of AM1 and AM2, respectively, with very low to no affinity for amylin.

[0061] Amylin analog polypeptides have been developed that have binding affinity to the amylin receptor complex. For example, pramlintide was developed by Amylin Pharmaceuticals and approved by the US Food and Drug Administration (FDA) as a synthetic analog of human amylin for the treatment of type 1 and type 2 diabetes patients who use prandial insulin but fail to achieve the desired glycemic control despite optimal insulin therapy. Pramlintide is an amylinomimetic agent that is at least as potent as human amylin. It is also a 37 amino acid polypeptide, differing in amino acid sequence from human amylin by the substitution of amino acids at positions 25 (alanine), 28 (serine), and 29 (serine) with proline. As a result of these substitutions, pramlintide is soluble, non-adhesive, and non-aggregating, thereby overcoming many of the physicochemical disadvantages of native human amylin. The half-life of pramlintide is approximately 48 minutes in humans, which is longer than the half-life of native human amylin (approximately 13 minutes). Pramlintide requires frequent and inconvenient administration.

[0062] In the treatment of patients with type 1 diabetes, pramlintide is administered by subcutaneous injection before meals up to four times per day as an adjunct to insulin therapy administered after meals. Pramlintide cannot be mixed with insulin, and a separate syringe is used. Pramlintide is administered with or before each meal or snack consisting of at least 250 calories or 30 g of carbohydrate. A typical starting dose for patients with type 1 diabetes is 15 μg subcutaneous pramlintide before each meal, then titrated up to a target dose of 60 μg before each meal. Reported side effects of pramlintide include nausea and vomiting. Adverse reactions can include severe hypoglycemia, especially in patients with type 1 diabetes. As a result, prandial insulin doses are reduced in diabetic patients who begin taking pramlintide.

[0063] For the treatment of patients with type 2 diabetes, pramlintide is administered by subcutaneous injection at a recommended starting dose of 60 μg, with a target maintenance dose of 120 μg administered before each meal.

[0064] Davalintide (AC2307) is another analog of human amylin. Davalintide is an investigational drug with a half-life of approximately 26 minutes. Like pramlintide, davalintide also requires frequent administration by injection.

[0065] Certain disclosed amylin analog polypeptides, including those in Table 3 below, exhibit one or more of superior solubility, stability, biological activity and specificity, and longer half-life than endogenous human amylin and known synthetic amylin analog polypeptides. Certain disclosed amylin analog polypeptides have been developed to accommodate less frequent administration than required for pramlintide. Certain disclosed amylin analog polypeptides have been developed for administration by weekly or monthly injections. Certain disclosed amylin analog polypeptides have been developed for administration via implantation of a delivery device comprising the amylin analog polypeptide, the delivery device comprising a dose of the amylin analog polypeptide for up to 3 months, 6 months, 9 months, 1 year, 18 months, or 2 years.

[0066] 4. Description of Exemplary Embodiments In certain embodiments, the present invention relates to isolated polypeptides that are amylin analogs.

[0067] In some embodiments, the isolated polypeptide of the disclosure has the sequence of SEQ ID NO:199: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TX 37 (OH / NH2) (SEQ ID NO: 199), X1 is S, K, k, H or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; X 10 is Q or S; X 12 is L or K; X 13 is A, S, E or K; X 14 is N, n, d, Y or Q; X 15 is E, F, f, Y, I, k, K or α-aminoisobutyric acid (Aib); X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib; X 20 is S or Aib; X 22 is N or E; X 29 is P, R, or K; X 31 is k, K, N, or H; X 35 is e, E, N, K, G, A, Y, or P; X 37 is Y or P; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; X 31 If N, then X 35 is E, or X35 If N, then X 31 (subject to the condition that K) or a pharmaceutically acceptable salt thereof.

[0068] In some embodiments, X 31 is K. In some embodiments, X 31 is N.

[0069] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0070] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:199 include:

[0071] In some embodiments, the carboxy-terminal X 37 is Y-(NH). In some embodiments, the carboxy-terminal X 37 is Y-(OH). In some embodiments, the carboxy-terminal X 37 is P-(NH). In some embodiments, the carboxy-terminal X 37 is P-(OH).

[0072] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k. In some embodiments, X1 is H. In some embodiments, X1 is I.

[0073] As used herein, k refers to D-lysine.

[0074] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0075] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0076] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0077] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0078] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0079] In some embodiments, X 12 is L. In some embodiments, X 12 is K.

[0080] In some embodiments, X 13 is A. In some embodiments, X 13 is S. In some embodiments, X 13 is E. In some embodiments, X 13 is K.

[0081] In some embodiments, X 14 is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X is d. 14 is Y. In some embodiments, X 14 is Q.

[0082] As used herein, n refers to D-asparagine.

[0083] As used herein, d refers to D-aspartic acid.

[0084] In some embodiments, X 15 is E. In some embodiments, X 15is F. In some embodiments, X 15 is f. In some embodiments, X 15 is Y. In some embodiments, X 15 is I. In some embodiments, X 15 is K. In some embodiments, X 15 In some embodiments, X is 15 is Aib.

[0085] As used herein, f refers to D-phenylalanine.

[0086] As used herein, Aib alternatively refers to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine.

[0087] In some embodiments, X 16 is L. In some embodiments, X 16 In some embodiments, X is 1. 16 is K. In some embodiments, X 16 In some embodiments, X is 16 In some embodiments, X is Aib. 16 is N-MeL.

[0088] As used herein, l refers to D-leucine.

[0089] As used herein, N-MeL refers to N-methylleucine.

[0090] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q. In some embodiments, X 17 is R. In some embodiments, X 17 is K. In some embodiments, X 17 In some embodiments, X is17 is Aib.

[0091] In some embodiments, X 18 is K. In some embodiments, X 18 is H. In some embodiments, X 18 is R.

[0092] In some embodiments, X 19 is S. In some embodiments, X 19 is Aib.

[0093] In some embodiments, X 20 is S. In some embodiments, X 20 is Aib.

[0094] In some embodiments, X 22 is N. In some embodiments, X 22 is E.

[0095] In some embodiments, X 29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 is K.

[0096] In some embodiments, X 31 In some embodiments, X is 31 is K. In some embodiments, X 31 is N. In some embodiments, X 31 is H.

[0097] In some embodiments, X 35 is e. In some embodiments, X 35 is E. In some embodiments, X 35 is N. In some embodiments, X 35 is K. In some embodiments, X 35 is G. In some embodiments, X 35is A. In some embodiments, X 35 is Y. In some embodiments, X 35 is P.

[0098] As used herein, e refers to D-glutamic acid.

[0099] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:199 include:

[0100] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 is Q. In some embodiments, X is S and X 15 is E. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E. In some embodiments, X is S and X 37 is Y.

[0101] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X 10 is Q. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 18 In some embodiments, X is K and X 31In some embodiments, X is K. 35 is E. In some embodiments, X is K and X 37 is Y.

[0102] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X 10 is Q. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 17 is H. In some embodiments, X is k and X 18 In some embodiments, X is k and X 31 In some embodiments, X is k and X 35 is E. In some embodiments, X is k and X 37 is Y.

[0103] In some embodiments, X5 is S and X 15 is E. In some embodiments, X5 is S and X 10 is Q. In some embodiments, X5 is S and X 16 is L. In some embodiments, X5 is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X5 is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E. In some embodiments, X5 is S and X 37 is Y.

[0104] In some embodiments, X 10 is Q and X 15is E. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X 18 is K. In some embodiments, X 10 is Q and X 31 is K. In some embodiments, X 10 is Q and X 35 is E. In some embodiments, X 10 is Q and X 37 is Y.

[0105] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 18 is K. In some embodiments, X 15 is E and X 31 is K. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 37 is Y.

[0106] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 18 is K. In some embodiments, X 16 is L and X 31 is K. In some embodiments, X 16 is L and X35 is E. In some embodiments, X 16 is L and X 37 is Y.

[0107] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 18 is K. In some embodiments, X 16 is k and X 31 is K. In some embodiments, X 16 is k and X 35 is E. In some embodiments, X 16 is k and X 37 is Y.

[0108] In some embodiments, X 17 is H and X 18 is K. In some embodiments, X 17 is H and X 31 is K. In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 37 is Y.

[0109] In some embodiments, X 18 is K and X 31 is K. In some embodiments, X 18 is K and X 35 is E. In some embodiments, X 18 is K and X 37 is Y.

[0110] In some embodiments, X 31 is K and X 35 is E. In some embodiments, X 31 is K and X 37 is Y.

[0111] In some embodiments, X 35 is E and X 37 is Y.

[0112] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:199 include:

[0113] In some embodiments, X1 is S, X5 is S, and X 15 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 17 is H. In some embodiments, X1 is S, X5 is S, and X 18 In some embodiments, X is S, X is S, and X is K. 31 In some embodiments, X is S and X 35 In some embodiments, X is S, X is S, and X 37 is Y.

[0114] In some embodiments, X1 is K, X5 is S, and X 15 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 17 is H. In some embodiments, X1 is K, X5 is S, and X 18 In some embodiments, X is K, X is S, and X 31 In some embodiments, X is K, X is S, and X 35 In some embodiments, X is K, X is S, and X 37 is Y.

[0115] In some embodiments, X1 is k, X5 is S, and X 15 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 17 is H. In some embodiments, X1 is k, X5 is S, and X 18 In some embodiments, X is k, X is S, and X 31 In some embodiments, X is k, X is S, and X 35 In some embodiments, X is k, X is S, and X 37 is Y.

[0116] In some embodiments, X5 is S and X 15 is E and X 16 is L. In some embodiments, X5 is S and X 15 is E and X 16 In some embodiments, X is S and X 15 is E and X 17 is H. In some embodiments, X5 is S and X 15 is E and X 18 In some embodiments, X is S and X 15 is E and X 31 In some embodiments, X is S and X 15 is E and X 35 is E. In some embodiments, X5 is S and X 15 is E and X 37 is Y.

[0117] In some embodiments, X 10 is Q and X 15 is E and X 16 is L. In some embodiments, X10 is Q and X 16 is k and X 17 is H. In some embodiments, X 10 is Q and X 18 is K and X 31 is K. In some embodiments, X 10 is Q and X 31 is K and X 35 is E. In some embodiments, X 10 is Q and X 31 is K and X 37 is Y.

[0118] In some embodiments, X 15 is E and X 16 is L and X 17 is H. In some embodiments, X 15 is E and X 16 is L and X 18 is K. In some embodiments, X 15 is E and X 16 is L and X 31 is K. In some embodiments, X 15 is E and X 16 is L and X 35 is E. In some embodiments, X 15 is E and X 16 is L and X 37 is Y.

[0119] In some embodiments, X 15 is E and X 16 is k and X 17 is H. In some embodiments, X 15 is E and X 16 is k and X 18 is K. In some embodiments, X 15 is E and X 16 is k and X 31 is K. In some embodiments, X 15 is E and X 16 is k and X 35is E. In some embodiments, X 15 is E and X 16 is k and X 37 is Y.

[0120] In some embodiments, X 16 is L and X 17 is H and X 18 is K. In some embodiments, X 16 is L and X 17 is H and X 31 is K. In some embodiments, X 16 is L and X 17 is H and X 35 is E. In some embodiments, X 16 is L and X 17は H and X 37 is Y.

[0121] In some embodiments, X 16 is k and X 17 is H and X 18 is K. In some embodiments, X 16 is k and X 17 is H and X 31 is K. In some embodiments, X 16 is k and X 17 is H and X 35 is E. In some embodiments, X 16 is k and X 17は H and X 37 is Y.

[0122] In some embodiments, X 17 is H and X 18 is K and X 31 is K. In some embodiments, X 17 is H and X 18 is K and X 35 is E. In some embodiments, X 17 is H and X 18 is K and X 37 is Y.

[0123] In some embodiments, X 18 is K and X 31 is K and X 35 is E. In some embodiments, X 18 is K and X 31 is K and X 37 is Y.

[0124] In some embodiments, X 31 is K and X 35 is E and X 37 is Y.

[0125] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH). In some embodiments, the carboxy terminal amino acid 37 is P-(NH2). In some embodiments, the carboxy terminal amino acid 37 is P-(OH).

[0126] In some embodiments, the isolated polypeptide of the disclosure has the sequence of SEQ ID NO:200: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200), X1 is S, K, k, H or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; X 10 is Q or S; X 12is L or K; X 13 is A, S, E or K; X 14 is N, n, d, Y or Q; X 15 is E, F, f, Y, I, k, K or α-aminoisobutyric acid (Aib); X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib; X 20 is S or Aib; X 22 is N or E; X 29 is P, R, or K; X 31 is k, K, or N; X 35 is e, E, or N; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; X 31 If N, then X 35 is E, or X 35 If N, then X 31 (subject to the condition that K) or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is selected from the group consisting of the amino acid sequence represented by the consensus sequence of:

[0127] In some embodiments, X 31is K. In some embodiments, X 31 is N.

[0128] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0129] In some embodiments, the isolated polypeptide of the disclosure has the sequence of SEQ ID NO: 201: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TKVGSETY-(OH / NH2) (SEQ ID NO:201), X1 is S, K, k, H or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; X 10 is Q or S; X 12 is L or K; X 13 is A, S, E or K; X 14 is N, n, d, Y or Q; X 15 is E, F, f, Y, I, k, K, or Aib; X 16 is k, K, L, Aib, N-MeL or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib; X 20 is S or Aib; X 22 is N or E; X 29 is P, R, or K; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0130] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:200 or SEQ ID NO:201 include:

[0131] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0132] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k. In some embodiments, X1 is H. In some embodiments, X1 is I.

[0133] As used herein, k refers to D-lysine.

[0134] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0135] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0136] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0137] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0138] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0139] In some embodiments, X 12 is L. In some embodiments, X 12 is K.

[0140] In some embodiments, X 13 is A. In some embodiments, X 13 is S. In some embodiments, X 13 is E. In some embodiments, X 13 is K.

[0141] In some embodiments, X 14 is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X is d. 14 is Y. In some embodiments, X 14 is Q.

[0142] As used herein, n refers to D-asparagine.

[0143] As used herein, d refers to D-aspartic acid.

[0144] In some embodiments, X 15 is E. In some embodiments, X 15 is F. In some embodiments, X 15 is f. In some embodiments, X 15 is Y. In some embodiments, X 15 is I. In some embodiments, X 15is K. In some embodiments, X 15 In some embodiments, X is 15 is Aib.

[0145] As used herein, f refers to D-phenylalanine.

[0146] As used herein, Aib alternatively refers to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine.

[0147] In some embodiments, X 16 is L. In some embodiments, X 16 In some embodiments, X is 1. 16 is K. In some embodiments, X 16 In some embodiments, X is 16 In some embodiments, X is Aib. 16 is N-MeL.

[0148] As used herein, l refers to D-leucine.

[0149] As used herein, N-MeL refers to N-methylleucine.

[0150] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q. In some embodiments, X 17 is R. In some embodiments, X 17 is K. In some embodiments, X 17 In some embodiments, X is 17 is Aib.

[0151] In some embodiments, X 18 is K. In some embodiments, X 18is H. In some embodiments, X 18 is R.

[0152] In some embodiments, X 19 is S. In some embodiments, X 19 is Aib.

[0153] In some embodiments, X 20 is S. In some embodiments, X 20 is Aib.

[0154] In some embodiments, X 22 is N. In some embodiments, X 22 is E.

[0155] In some embodiments, X 29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 is K.

[0156] In some embodiments, X 31 In some embodiments, X is 31 is K. In some embodiments, X 31 is N.

[0157] In some embodiments, X 35 is e. In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0158] As used herein, e refers to D-glutamic acid.

[0159] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:200 or SEQ ID NO:201 include:

[0160] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 is Q. In some embodiments, X is S and X 15 is E. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E.

[0161] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X 10 is Q. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 18 In some embodiments, X is K and X 31 In some embodiments, X is K and X 35 is E.

[0162] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X 10 is Q. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 17is H. In some embodiments, X is k and X 18 In some embodiments, X is k and X 31 In some embodiments, X is k and X 35 is E.

[0163] In some embodiments, X5 is S and X 15 is E. In some embodiments, X5 is S and X 10 is Q. In some embodiments, X5 is S and X 16 is L. In some embodiments, X5 is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X5 is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E.

[0164] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X 18 is K. In some embodiments, X 10 is Q and X 31 is K. In some embodiments, X 10 is Q and X 35 is E.

[0165] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 18 is K. In some embodiments, X 15 is E and X 31 is K. In some embodiments, X 15 is E and X 35 is E.

[0166] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 18 is K. In some embodiments, X 16 is L and X 31 is K. In some embodiments, X 16 is L and X 35 is E.

[0167] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 18 is K. In some embodiments, X 16 is k and X 31 is K. In some embodiments, X 16 is k and X 35 is E.

[0168] In some embodiments, X 17 is H and X 18 is K. In some embodiments, X 17 is H and X 31 is K. In some embodiments, X 17 is H and X 35 is E.

[0169] In some embodiments, X 18is K and X 31 is K. In some embodiments, X 18 is K and X 35 is E.

[0170] In some embodiments, X 31 is K and X 35 is E.

[0171] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:200 or SEQ ID NO:201 include:

[0172] In some embodiments, X1 is S, X5 is S, and X 15 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 17 is H. In some embodiments, X1 is S, X5 is S, and X 18 In some embodiments, X is S, X is S, and X is K. 31 In some embodiments, X is S and X 35 is E.

[0173] In some embodiments, X1 is K, X5 is S, and X 15 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 17 is H. In some embodiments, X1 is K, X5 is S, and X 18 In some embodiments, X is K, X is S, and X 31 In some embodiments, X is K, X is S, and X35 is E.

[0174] In some embodiments, X1 is k, X5 is S, and X 15 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 17 is H. In some embodiments, X1 is k, X5 is S, and X 18 In some embodiments, X is k, X is S, and X 31 In some embodiments, X is k, X is S, and X 35 is E.

[0175] In some embodiments, X5 is S and X 15 is E and X 16 is L. In some embodiments, X5 is S and X 15 is E and X 16 In some embodiments, X is S and X 15 is E and X 17 is H. In some embodiments, X5 is S and X 15 is E and X 18 In some embodiments, X is S and X 15 is E and X 31 In some embodiments, X is S and X 15 is E and X 35 is E.

[0176] In some embodiments, X 10 is Q and X 15 is E and X 16 is L. In some embodiments, X 10 is Q and X 16 is k and X 17 is H. In some embodiments, X 10is Q and X 18 is K and X 31 is K. In some embodiments, X 10 is Q and X 31 is K and X 35 is E.

[0177] In some embodiments, X 15 is E and X 16 is L and X 17 is H. In some embodiments, X 15 is E and X 16 is L and X 18 is K. In some embodiments, X 15 is E and X 16 is L and X 31 is K. In some embodiments, X 15 is E and X 16 is L and X 35 is E.

[0178] In some embodiments, X 15 is E and X 16 is k and X 17 is H. In some embodiments, X 15 is E and X 16 is k and X 18 is K. In some embodiments, X 15 is E and X 16 is k and X 31 is K. In some embodiments, X 15 is E and X 16 is k and X 35 is E.

[0179] In some embodiments, X 16 is L and X 17 is H and X 18 is K. In some embodiments, X 16 is L and X 17 is H and X 31 is K. In some embodiments, X 16 is L and X 17is H and X 35 is E.

[0180] In some embodiments, X 16 is k and X 17 is H and X 18 is K. In some embodiments, X 16 is k and X 17 is H and X 31 is K. In some embodiments, X 16 is k and X 17 is H and X 35 is E.

[0181] In some embodiments, X 17 is H and X 18 is K and X 31 is K. In some embodiments, X 17 is H and X 18 is K and X 35 is E.

[0182] In some embodiments, X 18 is K and X 31 is K and X 35 is E.

[0183] In some embodiments, the isolated polypeptide of the disclosure has the sequence of SEQ ID NO: 202: X1CNTX5TCATX 10 RLANX 15 X 16 X 17 X 18 SSNNFGPILPPTX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 202), X1 is S, k, or K; X5 is S or A; X 10 is Q or S; X 15 is E or F; X 16 is k, K, or L; X 17are H, V, and Q; X 18 is K, H, or R; X 31 is K or N; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTX5TC (SEQ ID NO: 308) are optionally further linked by a disulfide bridge; X 31 If N, then X 35 is E, or X 35 If N, then X 31 and m is K), or a pharma- ceutically acceptable salt thereof.

[0184] In some embodiments, X 31 is K. In some embodiments, X 31 is N.

[0185] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0186] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 203: X1CNTX5TCATX 10 RLANX 15 X 16 X 17 X 18 SSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 203), X1 is S, k, or K; X5 is S or A; X 10 is Q or S; X 15 is E or F; X 16 is k, K, or L; X 17 are H, V, and Q; X 18 is K, H, or R; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTX5TC (SEQ ID NO: 308) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0187] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:202 or SEQ ID NO:203 include:

[0188] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0189] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0190] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0191] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0192] In some embodiments, X 15 is E. In some embodiments, X15 is F. In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0193] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q.

[0194] In some embodiments, X 18 is K. In some embodiments, X 18 is H. In some embodiments, X 18 is R.

[0195] In some embodiments, X 31 is K. In some embodiments, X 31 is N.

[0196] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0197] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:202 or SEQ ID NO:203 include: In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 is E. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 18 In some embodiments, X is S and X 31In some embodiments, X is S and X 35 is E.

[0198] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X 15 is E. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 18 In some embodiments, X is K and X 31 In some embodiments, X is K and X 35 is E.

[0199] In some embodiments, X5 is S and X 15 is E. In some embodiments, X5 is S and X 16 is L. In some embodiments, X5 is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X5 is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E.

[0200] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X18 is K. In some embodiments, X 10 is Q and X 31 is K. In some embodiments, X 10 is Q and X 35 is E.

[0201] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 18 is K. In some embodiments, X 15 is E and X 31 is K. In some embodiments, X 15 is E and X 35 is E.

[0202] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 18 is K. In some embodiments, X 16 is L and X 31 is K. In some embodiments, X 16 is L and X 35 is E.

[0203] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 18 is K. In some embodiments, X 16 is k and X 31 is K. In some embodiments, X 16 is k and X 35 is E.

[0204] In some embodiments, X 17 is H and X 18 is K. In some embodiments, X 17 is H and X 31 is K. In some embodiments, X 17 is H and X 35 is E.

[0205] In some embodiments, X 18 is K and X 31 is K. In some embodiments, X 18 is K and X 35 is E.

[0206] In some embodiments, X 31 is K and X 35 is E.

[0207] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:202 or SEQ ID NO:203 include:

[0208] In some embodiments, X1 is S, X5 is S, and X 10 In some embodiments, X is S, X is S, and X 15 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 17 is H. In some embodiments, X1 is S, X5 is S, and X 18 In some embodiments, X is S, X is S, and X is K. 31 In some embodiments, X is S and X 35 is E.

[0209] In some embodiments, X1 is K, X5 is S, and X 10In some embodiments, X is K, X is S, and X 15 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 17 is H. In some embodiments, X1 is K, X5 is S, and X 18 In some embodiments, X is K, X is S, and X 31 In some embodiments, X is K, X is S, and X 35 is E.

[0210] In some embodiments, X5 is S and X 10 is Q and X 15 is E. In some embodiments, X5 is S and X 10 is Q and X 16 is L. In some embodiments, X5 is S and X 10 is Q and X 16 In some embodiments, X is S and X 10 is Q and X 17 is H. In some embodiments, X5 is S and X 10 is Q and X 18 In some embodiments, X is S and X 10 is Q and X 31 In some embodiments, X is S and X 10 is Q and X 35 is E.

[0211] In some embodiments, X 10 is Q and X 15 is E and X 16 is L. In some embodiments, X 10 is Q and X 15 is E and X 16 In some embodiments, X is10 is Q and X 15 is E and X 17 is H. In some embodiments, X 10 is Q and X 15 is E and X 18 is K. In some embodiments, X 10 is Q and X 15 is E and X 31 is K. In some embodiments, X 10 is Q and X 15 is E and X 35 is E.

[0212] In some embodiments, X 15 is E and X 16 is L and X 17 is H. In some embodiments, X 15 is E and X 16 is L and X 18 is K. In some embodiments, X 15 is E and X 16 is L and X 31 is K. In some embodiments, X 15 is E and X 16 is L and X 35 is E.

[0213] In some embodiments, X 15 is E and X 16 is k and X 17 is H. In some embodiments, X 15 is E and X 16 is k and X 18 is K. In some embodiments, X 15 is E and X 16 is k and X 31 is K. In some embodiments, X 15 is E and X 16 is k and X 35 is E.

[0214] In some embodiments, X 16 is L and X17 is H and X 18 is K. In some embodiments, X 16 is L and X 17 is H and X 31 is K. In some embodiments, X 16 is L and X 17 is H and X 35 is E.

[0215] In some embodiments, X 16 is k and X 17 is H and X 18 is K. In some embodiments, X 16 is k and X 17 is H and X 31 is K. In some embodiments, X 16 is k and X 17 is H and X 35 is E.

[0216] In some embodiments, X 17 is H and X 18 is K and X 31 is K. In some embodiments, X 17 is H and X 18 is K and X 35 is E.

[0217] In some embodiments, X 18 is K and X 31 is K and X 35 is E.

[0218] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 204: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 204), X1 is S, K, or k; X 10 is Q or S; X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0219] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0220] In some embodiments, the isolated polypeptide of the disclosure has the sequence of SEQ ID NO: 205: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 205), X1 is S, K, or k; X 10 is Q or S; X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0221] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:204 or SEQ ID NO:205 include:

[0222] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0223] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0224] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0225] In some embodiments, X 15 is E. In some embodiments, X 15 is F.

[0226] In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0227] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q.

[0228] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0229] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:204 or SEQ ID NO:205 include:

[0230] In some embodiments, X is S and X 10 is Q. In some embodiments, X is S and X 10 is S. In some embodiments, X is S and X 15 is E. In some embodiments, X is S and X 15 is F. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 17 is V. In some embodiments, X is S and X 17 is Q. In some embodiments, X is S and X 35 is E. In some embodiments, X is S and X 35 is N.

[0231] In some embodiments, X is K and X 10 is Q. In some embodiments, X is K and X 10 is S. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 15 is F. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 17 is V. In some embodiments, X is K and X 17 is Q. In some embodiments, X is K and X 35 is E. In some embodiments, X is K and X 35 is N.

[0232] In some embodiments, X is k and X 10 is Q. In some embodiments, X is k and X 10 is S. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 15 is F. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 16 In some embodiments, X is k and X 17 is H. In some embodiments, X is k and X 17 is V. In some embodiments, X is k and X 17 is Q. In some embodiments, X is k and X 35 is E. In some embodiments, X is k and X 35 is N.

[0233] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 15 is F. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X16 is K. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X 17 is V. In some embodiments, X 10 is Q and X 17 is Q. In some embodiments, X 10 is Q and X 35 is E. In some embodiments, X 10 is Q and X 35 is N.

[0234] In some embodiments, X 10 is S and X 15 is E. In some embodiments, X 10 is S and X 15 is F. In some embodiments, X 10 is S and X 16 is L. In some embodiments, X 10 is S and X 16 is K. In some embodiments, X 10 is S and X 16 In some embodiments, X is 10 is S and X 17 is H. In some embodiments, X 10 is S and X 17 is V. In some embodiments, X 10 is S and X 17 is Q. In some embodiments, X 10 is S and X 35 is E. In some embodiments, X 10 is S and X 35 is N.

[0235] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X15 is E and X 16 is K. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 17 is V. In some embodiments, X 15 is E and X 17 is Q. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 35 is N.

[0236] In some embodiments, X 15 is F and X 16 is L. In some embodiments, X 15 is F and X 16 is K. In some embodiments, X 15 is F and X 16 In some embodiments, X is 15 is F and X 17 is H. In some embodiments, X 15 is F and X 17 is V. In some embodiments, X 15 is F and X 17 is Q. In some embodiments, X 15 is F and X 35 is E. In some embodiments, X 15 is F and X 35 is N.

[0237] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 17 is V. In some embodiments, X 16 is L and X 17is Q. In some embodiments, X 16 is L and X 35 is E. In some embodiments, X 16 is L and X 35 is N.

[0238] In some embodiments, X 16 is K and X 17 is H. In some embodiments, X 16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is K and X 35 is N.

[0239] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 17は V. In some embodiments, X 16 is k and X 17 is Q. In some embodiments, X 16 is k and X 35 is E. In some embodiments, X 16 is k and X 35 is N.

[0240] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 is N.

[0241] In some embodiments, X 17 is V and X 35 is E. In some embodiments, X 17 is V and X 35 is N.

[0242] In some embodiments, X 17 is Q and X 35 is E. In some embodiments, X 17 is Q and X 35 is N.

[0243] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 206: SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 206), X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of SCNTSTC (SEQ ID NO: 310) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0244] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0245] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 207: SCNTSTCATQRLANX 15 X 16 X 17KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 207), X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of SC*NTSTC* are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0246] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:206 or SEQ ID NO:207 include:

[0247] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0248] In some embodiments, X 15 is E. In some embodiments, X 15 is F.

[0249] In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0250] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17is Q.

[0251] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 is K. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 17 is V. In some embodiments, X 15 is E and X 17 is Q. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 35 is N.

[0252] In some embodiments, X 15 is F and X 16 is L. In some embodiments, X 15 is F and X 16 is K. In some embodiments, X 15 is F and X 16 In some embodiments, X is 15 is F and X 17 is H. In some embodiments, X 15 is F and X 17 is V. In some embodiments, X 15 is F and X 17 is Q. In some embodiments, X 15 is F and X 35 is E. In some embodiments, X 15 is F and X 35 is N.

[0253] In some embodiments, X 16 is L and X 17is H. In some embodiments, X 16 is L and X 17 is V. In some embodiments, X 16 is L and X 17 is Q. In some embodiments, X 16 is l and X 35 is E. In some embodiments, X 16 is L and X 35 is N.

[0254] In some embodiments, X 16 is K and X 17 is H. In some embodiments, X 16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is K and X 35 is N.

[0255] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 17 is V. In some embodiments, X 16 is k and X 17 is Q. In some embodiments, X 16 is k and X 35 is E. In some embodiments, X 16 is k and X 35 is N.

[0256] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 is N.

[0257] In some embodiments, X 17 is V and X 35 is E. In some embodiments, X 17 is V and X 35 is N.

[0258] In some embodiments, X 17 is Q and X 35 is E. In some embodiments, X 17 is Q and X 35 is N.

[0259] In some embodiments, the isolated polypeptide of the present disclosure comprises: SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 127), which is also referred to herein as compound A127; SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:57), which is also referred to herein as compound A57; SC*NTSTC*ATQRLANEKHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 128), which is also referred to herein as compound A128; SC*NTSTC*ATQRLANEkHKSSNNFGPILPPTKVGSETY-(NH2) SEQ ID NO: 129), which is also referred to herein as compound A129; and SC*NTSTC*ATQRLANFLVKSSNEFGPILPPTKVGSETY-(NH2) (SEQ ID NO:43), which is also referred to herein as compound A43.

[0260] In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence: SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:57), which is also referred to herein as compound A57.

[0261] In some embodiments, the isolated polypeptide of the disclosure comprises the amino acid sequence: SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 127), also referred to herein as compound A127.

[0262] In some embodiments, the isolated polypeptide of the disclosure has the amino acid sequence: SC*NTSTC*ATQRLANEk*((γGlu)2-CO(CH2) 14 CH3)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 27), which is also referred to herein as compound A27.

[0263] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 208: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 208), X1 is K or k; X 10 is Q or S; X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 318) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0264] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0265] In some embodiments, the isolated polypeptide of the disclosure is SEQ ID NO: 209: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 209), X1 is K or k; X 10 is Q or S; X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; each K independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; wherein the two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 318) are optionally further linked by a disulfide bridge), or a pharma- ceutically acceptable salt thereof.

[0266] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:208 or SEQ ID NO:209 include:

[0267] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0268] In some embodiments, X1 is K. In some embodiments, X1 is k.

[0269] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0270] In some embodiments, X 15 is E. In some embodiments, X 15 is F.

[0271] In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0272] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q.

[0273] In some embodiments, X is K and X 10 is Q. In some embodiments, X is K and X 10 is S. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 15 is F. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 17 is V. In some embodiments, X is K and X 17 is Q. In some embodiments, X is K and X 35is E. In some embodiments, X is K and X 35 is N.

[0274] In some embodiments, X is k and X 10 is Q. In some embodiments, X is k and X 10 is S. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 15 is F. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 16 In some embodiments, X is k and X 17 is H. In some embodiments, X is k and X 17 is V. In some embodiments, X is k and X 17 is Q. In some embodiments, X is k and X 35 is E. In some embodiments, X is k and X 35 is N.

[0275] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 15 is F. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 is K. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X 17 is V. In some embodiments, X 10 is Q and X17 is Q. In some embodiments, X 10 is Q and X 35 is E. In some embodiments, X 10 is Q and X 35 is N.

[0276] In some embodiments, X 10 is S and X 15 is E. In some embodiments, X 10 is S and X 15 is F. In some embodiments, X 10 is S and X 16 is L. In some embodiments, X 10 is S and X 16 is K. In some embodiments, X 10 is S and X 16 In some embodiments, X is 10 is S and X 17 is H. In some embodiments, X 10 is S and X 17 is V. In some embodiments, X 10 is S and X 17 is Q. In some embodiments, X 10 is S and X 35 is E. In some embodiments, X 10 is S and X 35 is N.

[0277] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 is K. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 17 is V. In some embodiments, X15 is E and X 17 is Q. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 35 is N.

[0278] In some embodiments, X 15 is F and X 16 is L. In some embodiments, X 15 is F and X 16 is K. In some embodiments, X 15 is F and X 16 In some embodiments, X is 15 is F and X 17 is H. In some embodiments, X 15 is F and X 17 is V. In some embodiments, X 15 is F and X 17 is Q. In some embodiments, X 15 is F and X 35 is E. In some embodiments, X 15 is F and X 35 is N.

[0279] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 17 is V. In some embodiments, X 16 is L and X 17 is Q. In some embodiments, X 16 is L and X 35 is E. In some embodiments, X 16 is L and X 35 is N.

[0280] In some embodiments, X 16 is K and X 17 is H. In some embodiments, X16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is k and X 35 is N.

[0281] In some embodiments, X 16 is K and X 17 is H. In some embodiments, X 16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is k and X 35 is N

[0282] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 is N.

[0283] In some embodiments, X 17 is V and X 35 is E. In some embodiments, X 17 is V and X 35 is N.

[0284] In some embodiments, X 17 is Q and X 35 is E. In some embodiments, X 17 is Q and X 35 is N.

[0285] In some embodiments, the isolated polypeptide of the present disclosure comprises: KC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 130), which is also referred to herein as compound A130; and KC*NTSTC*ATQRLANFLQKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 131), also referred to herein as compound A131.

[0286] In some embodiments, X 31 is K. In some embodiments, X 31 is N.

[0287] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0288] 5. Conjugation of a lipophilic substituent to any of the peptides, optionally via a spacer In some embodiments, any of the disclosed polypeptides are 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 of SEQ ID NO:1 to SEQ ID NO:143 may be modified or further modified by comprising one or more lipophilic substituents, each optionally via a spacer, or by covalently linking to one or more lipophilic substituents, each optionally via a spacer. In some embodiments, the lipophilic substituent may be attached to an amino group of the polypeptide (e.g., the ε-amino group of a lysine residue) by a carboxyl group of the lipophilic substituent, or may be attached to an amino group of the spacer, optionally, where the carboxyl group of the spacer forms an amide bond with the ε-amino group of the lysine residue.

[0289] lipophilic substituent Conjugation of one or more "lipophilic substituents" to any of the disclosed polypeptides of the present invention, each optionally via a "spacer", is intended to promote binding to serum albumin and thus prolong the action of the polypeptide by retarding renal clearance of the conjugated polypeptide. As used herein, a "lipophilic substituent" includes a substituent containing 4-40 carbon atoms, 8-25 carbon atoms, 12-22 carbon atoms, or 6-20 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) by a 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, which is defined in more detail below.

[0290] In some embodiments, the lipophilic substituent comprises a straight or branched chain alkyl group. In some embodiments, the lipophilic substituent is an acyl group of a straight or branched chain fatty acid. In some embodiments, the lipophilic substituent is an acyl group of a straight or branched chain fatty acid, which is further substituted with one or more carboxylic acid and / or hydroxamic acid groups.

[0291] 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 (In the formula, Z is -CH3, or -CO2H; m is a monovalent group of 4 to 24; The lipophilic substituent forms an amide bond between an amino group of a disclosed polypeptide (eg, the ε-amino group of lysine) and a CO-group of the lipophilic substituent.

[0292] 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.

[0293] In some embodiments, Z is -CO2H and the lipophilic substituent is of the formula -CO-(CH2) m In some embodiments, the compound has -COH. 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 -COH.

[0294] In some embodiments, the lipophilic substituent is -CO-(CH) 18 -CO2H.

[0295] In some embodiments, Z is -CH3 and the lipophilic substituent is of the formula -CO-(CH2) m In some embodiments, the compound has a -CO-(CH2) 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.

[0296] Spacer 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 of SEQ ID NO:1-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.

[0297] In some embodiments, the spacer comprises one or more amino acids, e.g., a single amino acid, e.g., Glu, Asp, Gly or Lys, a dipeptide, e.g., 2(Glu), Glu-Gly, or a polypeptide, e.g., 3(Glu), 4(Glu) (SEQ ID NO: 317), 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 the disclosed polypeptide, and an amino group of the spacer can form an amide bond with a carboxyl group of the lipophilic substituent.

[0298] In some embodiments, when the spacer comprises Glu or Asp further comprising 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 the amino group of the 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. In some embodiments, the spacer is γGlu. In some embodiments, the spacer is 2(γGlu). In some embodiments, the spacer is 3(γGlu).

[0299] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by Formula II: -(Y) n -CO-(CH2) m -Z Formula II (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).

[0300] 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.

[0301] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by formula III: -(γGlu) n -CO-(CH2) m -Z("(γGlu) n " is disclosed as SEQ ID NO: 311) Formula III (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0302] In some embodiments, Z is -CH. In some embodiments, Z is -COH.

[0303] 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.

[0304] 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.

[0305] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by formula IV: -(γGlu) n -(Gly)-CO-(CH2) m -Z("(γGlu )n-(Gly)" is disclosed as SEQ ID NO: 312) Formula IV (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0306] In some embodiments, (γGlu) n is selected from the group consisting of γGlu; ​​2(γGlu); 3(γGlu); 4(γGlu) (SEQ ID NO: 313); and 5(γGlu) (SEQ ID NO: 314). In some embodiments, -(γGlu) n -(Gly)-(γGlu) n -(Gly)" (disclosed as SEQ ID NO:312) is selected from the group consisting of: 2(γGlu),Gly; and 3(γGlu),Gly (SEQ ID NO:315).

[0307] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with an optional spacer. In some embodiments, the lipophilic substituent is represented by formula V: -(Gly)-(γGlu) n -(CO-(CH2) m -Z("(Gly)-(γGlu) n " is disclosed as SEQ ID NO: 316) Formula V (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10).

[0308] In some embodiments, certain variables represented by Formula II, Formula III, Formula IV, or Formula V include:

[0309] In some embodiments, Z is -CH. In some embodiments, Z is -COH.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by formula VI: -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2) m -Z Equation VI (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(O(CH2)2) t OCH2NH]-, where t is 1 to 8; r is 1 to 8; n1 is 0 to 10; n2 is a monovalent group ranging from 0 to 10.

[0324] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by Formula VII: -(Y1) n1 -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z Formula VII (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; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; r is 1 to 8; n1 is 0 to 10; n2 is a monovalent group ranging from 0 to 10.

[0325] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - is selected from the group consisting of γGlu,dpeg,dpeg,γGlu; ​​γGlu,dpeg,dpeg,2(γGlu); γGlu,dpeg,dpeg,3(γGlu); γGlu,dpeg,dpeg,4(γGlu); 2(γGlu),dpeg,dpeg,γGlu; ​​and 2(γGlu),dpeg,γGlu.

[0326] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with an optional spacer. In some embodiments, the lipophilic substituent is represented by Formula VIII: -(V)r -(Y2) n2 -CO-(CH2) m -Z Formula VIII (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; Y2 is selected from the group consisting of γGlu, Asp, and Gly; V is -[COCH2(O(CH2)2) t OCH2NH]-, where t is 1 to 8; r is 1 to 8; n2 is a monovalent group ranging from 0 to 10.

[0327] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by formula IX: -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z Formula IX (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 a monovalent group ranging from 0 to 10.

[0328] In some embodiments, -(dpeg) r -(Y2) n2 - is selected from the group consisting of γGlu; ​​and dpeg, dpeg, γGlu.

[0329] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent is represented by the formula X: -(Y1) n1 -(dpeg) r -CO-(CH2) m -Z formula (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 a monovalent group ranging from 0 to 10.

[0330] In some embodiments, -(Y1) n1 -(dpeg) r - is 2(γGlu),dpeg.

[0331] In some embodiments, the spacer is of formula XI: -N(R1)(CHR2) p CO-[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r Formula XI (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; and r is 0 or 1.

[0332] This spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[0333] In some embodiments, the spacer has formula XII: [-N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r Formula XII (In the formula, Each R3 is hydrogen or C1-C4 alkyl; q is 1, 2 or 3; and r is 0 or 1.

[0334] This spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[0335] In some embodiments, certain variables represented by formula XI or formula XII include: In some embodiments, each R1 is hydrogen. In some embodiments, each R3 is hydrogen. In some embodiments, each R1 and each R3 is hydrogen.

[0336] In some embodiments, at least one R2 is CO2H. In some embodiments, one R2 is CO2H.

[0337] 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.

[0338] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[0339] In some embodiments, r is 0. In some embodiments, r is 1.

[0340] In some embodiments, the spacer is γ-glutamyl, i.e., -NH(CHCO2H)(CH2)2CO--. In some embodiments, the spacer is γ-aminobutanoyl, i.e., -NH(CH2)3CO--. In some embodiments, the spacer is β-asparagyl, i.e., -NH(CHCO2H)(CH2)CO--. In some embodiments, the spacer is -NH(CH2)2CO--. In some embodiments, the spacer is glycyl. In some embodiments, the spacer is β-alanyl.

[0341] 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-] r In 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 It is.

[0342] In some embodiments, the spacer is represented by Formula XIII: -(Y) n - Formula XIII (In the formula, Y is selected from the group consisting of γGlu, Asp, Lys, and Gly; n is 1 to 10).

[0343] 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.

[0344] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0345] In some embodiments, the spacer is represented by formula XIV: -(γGlu) n -("(γGlu) n " is disclosed as SEQ ID NO: 311) Formula XIV (In the formula, n is 1 to 10).

[0346] 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.

[0347] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0348] In some embodiments, the spacer is represented by formula XV: -(γGlu) n -(Gly)-(γGlu) n-(Gly)" is disclosed as SEQ ID NO: 312) Formula XV (In the formula, n is 1 to 10).

[0349] In some embodiments, (γGlu) n is selected from the group consisting of γGlu; ​​2(γGlu); 3(γGlu); 4(γGlu) (SEQ ID NO: 313); and 5(γGlu) (SEQ ID NO: 314). In some embodiments, -(γGlu) n -(Gly)-((γGlu) n -(Gly)" (disclosed as SEQ ID NO:312) is selected from the group consisting of: 2(γGlu),Gly; and 3(γGlu),Gly (SEQ ID NO:315).

[0350] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0351] In some embodiments, the spacer is represented by Formula XVI: -(Gly)-(γGlu) n -("(Gly)-(γGlu) n " is disclosed as SEQ ID NO: 316) Formula XVI (In the formula, n is 1 to 10).

[0352] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0353] In some embodiments of Formula XIII, Formula XIV, Formula XV, or Formula XVI, 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.

[0354] In some embodiments, the spacer is represented by Formula XVII: -(Y1) n1 -(V) r -(Y2) n2 - Formula XVII (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; V is -[COCH2(O(CH2)2) t OCH2NH]-, where t is 1 to 8; r is 1 to 8; n1 is 0 to 10; n2 is 0 to 10).

[0355] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0356] In some embodiments, the spacer is represented by Formula XVIII: -(Y1) n1 -(dpeg) r -(Y2) n2 - Formula XVIII (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).

[0357] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - is selected from the group consisting of γGlu,dpeg,dpeg,γGlu; ​​γGlu,dpeg,dpeg,2(γGlu); γGlu,dpeg,dpeg,3(γGlu); γGlu,dpeg,dpeg,4(γGlu); 2(γGlu),dpeg,dpeg,γGlu; ​​and 2(γGlu),dpeg,γGlu.

[0358] In some embodiments, the spacer forms a bridge between an amino group of the disclosed polypeptide and a CO-group of the lipophilic substituent, in some embodiments, one end of the spacer forms a covalent bond with an amino group of the disclosed polypeptide and the other end of the spacer forms a covalent bond with a hydrogen atom or a protecting group.

[0359] Thus, in some embodiments, the isolated polypeptide provided herein comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by a consensus sequence selected from the group consisting of SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209, or a pharma- ceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and optionally a spacer.

[0360] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent of formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer selected from the group consisting of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VII.

[0361] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:200, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent of formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:200, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer selected from the group consisting of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:200, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:200, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:200, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VII.

[0362] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:204, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent of formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:204, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer selected from the group consisting of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:204, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:204, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:204, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VII.

[0363] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:206, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent of formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:206, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer selected from the group consisting of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:206, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:206, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:206, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VII.

[0364] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:208, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent of formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:208, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer selected from the group consisting of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:208, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:208, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:208, or a pharma- ceutically acceptable salt thereof, and further comprises a lipophilic substituent and spacer of formula VII.

[0365] In some embodiments, the isolated polypeptide provided herein has the sequence of SEQ ID NO:200: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200), X1 is S, K, k, H, or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; 10 is Q or S;X 12 is L or K;X 13 is A, S, E, or K; X 14 is N, n, d, Y, or Q; X 15 is E, F, f, Y, I, k, K, or Aib; X 16is k, K, L, Aib, N-MeL, or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib;X 20 is S or Aib;X 22 is N or E;X 29 is P, R, or K; X 31 is k, K, or N; and X 35 is e, E, or N; each K independently represents an L-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; each k independently represents a D-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; 31 If N, then X 35 is E, or X 35 If N, then X 31 or a pharma- ceutically acceptable salt thereof; the isolated peptide comprises an amino acid sequence selected from the group consisting of the amino acid sequence represented by the consensus sequence of: -CO-(CH2) m -Z Formula I (In the formula, Z is -CH3, or -CO2H; m is 4 to 24).

[0366] In some embodiments, the isolated polypeptide provided herein has the sequence of SEQ ID NO:200: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200), X1 is S, K, k, H, or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; 10 is Q or S;X 12 is L or K;X 13 is A, S, E, or K; X 14 is N, n, d, Y, or Q; X 15 is E, F, f, Y, I, k, K, or Aib; X 16 is k, K, L, Aib, N-MeL, or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib;X 20 is S or Aib;X 22 is N or E;X 29 is P, R, or K; X 31 is k, K, or N; and X 35 is e, E, or N; each K independently represents an L-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; each k independently represents a D-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; 31 If N, then X 35 is E, or X 35 If N, then X 31 or a pharma- ceutically acceptable salt thereof; and the isolated peptide comprises an amino acid sequence selected from the group consisting of an amino acid sequence represented by the consensus sequence of: -(γGlu) n -CO-(CH2) m -Z("(γGlu) n " is disclosed as SEQ ID NO: 311) Formula III (In the formula, Z is -CH3 or -CO2H; m is 4 to 24; n is 1 to 10) and a spacer.

[0367] In some embodiments, the isolated polypeptide provided herein has the sequence of SEQ ID NO:200: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200), X1 is S, K, k, H, or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; 10 is Q or S;X 12 is L or K;X 13 is A, S, E, or K; X 14 is N, n, d, Y, or Q; X 15 is E, F, f, Y, I, k, K, or Aib; X 16 is k, K, L, Aib, N-MeL, or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib;X 20 is S or Aib;X 22 is N or E;X 29 is P, R, or K; X 31 is k, K, or N; and X 35is e, E, or N; each K independently represents an L-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; each k independently represents a D-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; 31 If N, then X 35 is E, or X 35 If N, then X 31 or a pharma- ceutically acceptable salt thereof; and the isolated peptide comprises an amino acid sequence selected from the group consisting of an amino acid sequence represented by the consensus sequence of: -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2) m -Z Equation VI (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(O(CH2)2) t OCH2NH]-, where t is 1 to 8; r is 1 to 8; n1 is 0 to 10; and n2 is 0 to 10), and further includes a lipophilic substituent and a spacer.

[0368] In some embodiments, the isolated polypeptide provided herein has the sequence of SEQ ID NO:200: X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200), X1 is S, K, k, H, or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; 10 is Q or S;X 12 is L or K;X 13 is A, S, E, or K; X 14 is N, n, d, Y, or Q; X 15 is E, F, f, Y, I, k, K, or Aib; X 16 is k, K, L, Aib, N-MeL, or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib;X 20 is S or Aib;X 22 is N or E;X 29 is P, R, or K; X 31 is k, K, or N; and X 35 is e, E, or N; each K independently represents an L-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; each k independently represents a D-lysine optionally covalently linked to a lipophilic substituent, optionally via a spacer; the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; 31 If N, then X 35 is E, or X 35 If N, then X 31 or a pharma- ceutically acceptable salt thereof; and the isolated peptide comprises an amino acid sequence selected from the group consisting of the amino acid sequence represented by the consensus sequence of: -(Y1) n1 -(dpeg) r -(Y2)n2 -CO-(CH2) m -Z Formula VII (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; dpeg is -[CO(CH2)O(CH2)2O(CH2)NH]-; r is 1 to 8; n1 is 0 to 10; and n2 is 0 to 10), and further includes a lipophilic substituent and a spacer.

[0369] In some embodiments, the isolated polypeptide has positions X1, X 15 , or X 16 contains a lipophilic substituent.

[0370] In some embodiments, positions X1, X 15 , or X 16 The isolated polypeptide comprising a lipophilic substituent at is conjugated via a lysine at that position.

[0371] In some embodiments, the isolated polypeptide comprises a lipophilic substituent at position X1.

[0372] In some embodiments, the isolated polypeptide has a structure at position X 15 contains a lipophilic substituent.

[0373] In some embodiments, the isolated polypeptide has a structure at position X 16 contains a lipophilic substituent.

[0374] In some embodiments, the isolated polypeptide comprising a lipophilic substituent at position X1 is conjugated via a lysine at that position.

[0375] In some embodiments, position X15 The isolated polypeptide comprising a lipophilic substituent at is conjugated via a lysine at that position.

[0376] In some embodiments, position X 16 The isolated polypeptide comprising a lipophilic substituent at is conjugated via a lysine at that position.

[0377] In some embodiments, the isolated polypeptide of the disclosure has the amino acid sequence: SC*NTSTC*ATQRLANEk*((γGlu)2-CO(CH2) 14 CH3)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 27), which is also referred to herein as compound A27.

[0378] In some embodiments, the isolated polypeptide of the disclosure has the amino acid sequence: K*((γGlu)2(CO(CH2) 18 CO2H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:64), which is also referred to herein as compound A64.

[0379] In some embodiments, the isolated polypeptide of the disclosure has the amino acid sequence: K*((γGlu)2(CO(CH2) 16 CO2H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:65), which is also referred to herein as compound A65.

[0380] In some embodiments, the isolated polypeptide of the present disclosure has the amino acid sequence: K*(γGlu-CO(CH2) 16 CO2H)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 109), which is also referred to herein as compound A109.

[0381] 6. Exemplary Compounds: Amylin Analog Polypeptides In some embodiments, the isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Table 3:

[0382] [Table 3] TIFF0007636321000006.tif239170TIFF0007636321000007.tif239170TIFF000763632100 0008.tif238170TIFF0007636321000009.tif239170TIFF0007636321000010.tif238170TI FF0007636321000011.tif238170TIFF0007636321000012.tif238170TIFF00076363210000 13.tif239170TIFF0007636321000014.tif239170TIFF0007636321000015.tif204170 Points to note: each K* independently represents an L-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; each k* independently represents a D-lysine optionally covalently linked, optionally via a spacer, to a lipophilic substituent; the two cysteine ​​residues (C*) in positions 2 and 7 are optionally further linked by a disulfide bridge; As used herein, (γGlu)2 and 2(γGlu) both mean -(γGlu)-(γGlu)-; (γGlu)3 and 3(γGlu) both mean -(γGlu)-(γGlu)-(γGlu)-; etc.; and When a variable occurs multiple times in a given formula, each occurrence of that variable is determined independently. For example, in the case of a -(Y)3- group, where Y can be γGlu, Asp, Lys, or Gly, each Y is independently selected to be one of the four amino acids. Thus, by way of non-limiting example, -(Y)3- can be -(γGlu)-(γGlu)-(γGlu)-, -(γGlu)-(Asp)-(γGlu)-, -(Gly)-(Asp)-(γGlu)-, or -(Gly)-(γGlu)-(γGlu)-.

[0383] In some embodiments, the invention provides a compound set forth in Table 3 above, or a pharma- ceutically acceptable salt thereof. In some embodiments, the pharma- ceutically acceptable salt is an acetate salt. In some embodiments, the pharma- ceutically acceptable salt is a trifluoroacetic acid (TFA) salt. In some embodiments, the pharma- ceutically acceptable salt is a hydrochloric acid (HCl) salt. In one embodiment, the compound is A27. In some embodiments, the compound is an acetate salt of compound A27. In some embodiments, the compound is a TFA salt of compound A27. In some embodiments, the compound is an HCl salt of compound A27. In one embodiment, the compound is A57. In some embodiments, the compound is an acetate salt of compound A57. In some embodiments, the compound is a TFA salt of compound A57. In some embodiments, the compound is an HCl salt of compound A57. In one embodiment, the compound is A64. In some embodiments, the compound is an acetate salt of compound A64. In some embodiments, the compound is a TFA salt of compound A64. In some embodiments, the compound is an HCl salt of compound A64.

[0384] 7. Polypeptide intermediates In certain embodiments, the present invention also relates to synthetic intermediates of isolated polypeptides that are amylin analogs. In some embodiments, the polypeptide intermediates of the present disclosure are represented by SEQ ID NO:210: X1CX3TX5X6CX8TX 10 RX 12 X 13 X14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO:210), X1 is S, K, k, H or I; X3 is N or S; X5 is S or A; X6 is T or S; X8 is A or K; X 10 is Q or S; X 12 is L or K; X 13 is A, S, E or K; X 14 is N, n, d, Y or Q; X 15 is E, F, f, Y, I, k, K or α-aminoisobutyric acid (Aib); X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l; X 17 is H, V, Q, R, k, K, or Aib; X 18 is K, H, or R; X 19 is S or Aib; X 20 is S or Aib; X 22 is N or E; X 29 is P, R, or K; X 31 is k, K, or N; X 35 is e, E, or N; each K independently represents an L-lysine optionally covalently linked to a protecting group, or a spacer optionally linked to a protecting group; each k independently represents a D-lysine optionally covalently attached to a protecting group, or a spacer optionally attached to a protecting group; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further linked by a disulfide bridge; X 31 If N, then X 35 is E, or X 35 If N, then X 31 and wherein x is K.

[0385] In some embodiments, X 31 is K. In some embodiments, X 31 is N.

[0386] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0387] In some embodiments, X 31 is K and X 35 is E.

[0388] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:210 include:

[0389] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0390] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k. In some embodiments, X1 is H. In some embodiments, X1 is I.

[0391] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0392] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0393] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0394] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0395] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0396] In some embodiments, X 12 is L. In some embodiments, X 12 is K.

[0397] In some embodiments, X 13 is A. In some embodiments, X 13 is S. In some embodiments, X 13 is E. In some embodiments, X 13 is K.

[0398] In some embodiments, X 14 is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X is d. 14 is Y. In some embodiments, X 14 is Q.

[0399] In some embodiments, X 15 is E. In some embodiments, X 15 is F. In some embodiments, X 15 is f. In some embodiments, X15 is Y. In some embodiments, X 15 is I. In some embodiments, X 15 is K. In some embodiments, X 15 In some embodiments, X is 15 is Aib.

[0400] In some embodiments, X 16 is L. In some embodiments, X 16 In some embodiments, X is 1. 16 is K. In some embodiments, X 16 In some embodiments, X is 16 In some embodiments, X is Aib. 16 is N-MeL.

[0401] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q. In some embodiments, X 17 is R. In some embodiments, X 17 is K. In some embodiments, X 17 In some embodiments, X is 17 is Aib.

[0402] In some embodiments, X 18 is K. In some embodiments, X 18 is H. In some embodiments, X 18 is R.

[0403] In some embodiments, X 19 is S. In some embodiments, X 19 is Aib.

[0404] In some embodiments, X 20 is S. In some embodiments, X 20 is Aib.

[0405] In some embodiments, X 22 is N. In some embodiments, X 22 is E.

[0406] In some embodiments, X 29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 is K.

[0407] In some embodiments, X 31 In some embodiments, X is 31 is K. In some embodiments, X 31 is N.

[0408] In some embodiments, X 35 is e. In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0409] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:210 include:

[0410] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 is Q. In some embodiments, X is S and X 15 is E. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E.

[0411] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X 10 is Q. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 18 In some embodiments, X is K and X 31 In some embodiments, X is K and X 35 is E.

[0412] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X 10 is Q. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 17 is H. In some embodiments, X is k and X 18 In some embodiments, X is k and X 31 In some embodiments, X is k and X 35 is E.

[0413] In some embodiments, X5 is S and X 15 is E. In some embodiments, X5 is S and X 10 is Q. In some embodiments, X5 is S and X 16 is L. In some embodiments, X5 is S and X 16 In some embodiments, X is S and X 17is H. In some embodiments, X5 is S and X 18 In some embodiments, X is S and X 31 In some embodiments, X is S and X 35 is E.

[0414] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X 18 is K. In some embodiments, X 10 is Q and X 31 is K. In some embodiments, X 10 is Q and X 35 is E.

[0415] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 18 is K. In some embodiments, X 15 is E and X 31 is K. In some embodiments, X 15 is E and X 35 is E.

[0416] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16is L and X 18 is K. In some embodiments, X 16 is L and X 31 is K. In some embodiments, X 16 is L and X 35 is E.

[0417] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 18 is K. In some embodiments, X 16 is k and X 31 is K. In some embodiments, X 16 is k and X 35 is E.

[0418] In some embodiments, X 17 is H and X 18 is K. In some embodiments, X 17 is H and X 31 is K. In some embodiments, X 17 is H and X 35 is E.

[0419] In some embodiments, X 18 is K and X 31 is K. In some embodiments, X 18 is K and X 35 is E.

[0420] In some embodiments, X 31 is K and X 35 is E.

[0421] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:210 include:

[0422] In some embodiments, X1 is S, X5 is S, and X 15In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 16 In some embodiments, X is S, X is S, and X 17 is H. In some embodiments, X1 is S, X5 is S, and X 18 In some embodiments, X is S, X is S, and X is K. 31 In some embodiments, X is S and X 35 is E.

[0423] In some embodiments, X1 is K, X5 is S, and X 15 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 16 In some embodiments, X is K, X is S, and X 17 is H. In some embodiments, X1 is K, X5 is S, and X 18 In some embodiments, X is K, X is S, and X 31 In some embodiments, X is K, X is S, and X 35 is E.

[0424] In some embodiments, X1 is k, X5 is S, and X 15 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 16 In some embodiments, X is k, X is S, and X 17 is H. In some embodiments, X1 is k, X5 is S, and X 18 In some embodiments, X is k, X is S, and X 31In some embodiments, X is k, X is S, and X 35 is E.

[0425] In some embodiments, X5 is S and X 15 is E and X 16 is L. In some embodiments, X5 is S and X 15 is E and X 16 In some embodiments, X is S and X 15 is E and X 17 is H. In some embodiments, X5 is S and X 15 is E and X 18 In some embodiments, X is S and X 15 is E and X 31 In some embodiments, X is S and X 15 is E and X 35 is E.

[0426] In some embodiments, X 10 is Q and X 15 is E and X 16 is L. In some embodiments, X 10 is Q and X 16 is k and X 17 is H. In some embodiments, X 10 is Q and X 18 is K and X 31 is K. In some embodiments, X 10 is Q and X 31 is K and X 35 is E.

[0427] In some embodiments, X 15 is E and X 16 is L and X 17 is H. In some embodiments, X 15 is E and X 16 is L and X 18 is K. In some embodiments, X 15 is E and X16 is L and X 31 is K. In some embodiments, X 15 is E and X 16 is L and X 35 is E.

[0428] In some embodiments, X 15 is E and X 16 is k and X 17 is H. In some embodiments, X 15 is E and X 16 is k and X 18 is K. In some embodiments, X 15 is E and X 16 is k and X 31 is K. In some embodiments, X 15 is E and X 16 is k and X 35 is E.

[0429] In some embodiments, X 16 is L and X 17 is H and X 18 is K. In some embodiments, X 16 is L and X 17 is H and X 31 is K. In some embodiments, X 16 is L and X 17 is H and X 35 is E.

[0430] In some embodiments, X 16 is k and X 17 is H and X 18 is K. In some embodiments, X 16 is k and X 17 is H and X 31 is K. In some embodiments, X 16 is k and X 17 is H and X 35 is E.

[0431] In some embodiments, X17 is H and X 18 is K and X 31 is K. In some embodiments, X 17 is H and X 18 is K and X 35 is E.

[0432] In some embodiments, X 18 is K and X 31 is K and X 35 is E.

[0433] In some embodiments, a polypeptide intermediate of the disclosure has the structure of SEQ ID NO:211: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO:211), X1 is S, K, or k; X 10 is Q or S; X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked to a protecting group, or a spacer optionally linked to a protecting group; each k independently represents a D-lysine optionally covalently attached to a protecting group, or a spacer optionally attached to a protecting group; wherein the two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked by a disulfide bridge).

[0434] In some embodiments, X 35is E. In some embodiments, X 35 is N.

[0435] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:211 include:

[0436] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0437] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0438] In some embodiments, X 10 is Q. In some embodiments, X 10 is S.

[0439] In some embodiments, X 15 is E. In some embodiments, X 15 is F.

[0440] In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0441] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q.

[0442] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0443] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:211 include:

[0444] In some embodiments, X is S and X 10 is Q. In some embodiments, X is S and X 10 is S. In some embodiments, X is S and X 15 is E. In some embodiments, X is S and X 15 is F. In some embodiments, X is S and X 16 is L. In some embodiments, X is S and X 16 In some embodiments, X is S and X 16 In some embodiments, X is S and X 17 is H. In some embodiments, X is S and X 17 is V. In some embodiments, X is S and X 17 is Q. In some embodiments, X is S and X 35 is E. In some embodiments, X is S and X 35 is N.

[0445] In some embodiments, X is K and X 10 is Q. In some embodiments, X is K and X 10 is S. In some embodiments, X is K and X 15 is E. In some embodiments, X is K and X 15 is F. In some embodiments, X is K and X 16 is L. In some embodiments, X is K and X 16 In some embodiments, X is K and X 16 In some embodiments, X is K and X 17 is H. In some embodiments, X is K and X 17 is V. In some embodiments, X is K and X17 is Q. In some embodiments, X is K and X 35 is E. In some embodiments, X is K and X 35 is N.

[0446] In some embodiments, X is k and X 10 is Q. In some embodiments, X is k and X 10 is S. In some embodiments, X is k and X 15 is E. In some embodiments, X is k and X 15 is F. In some embodiments, X is k and X 16 is L. In some embodiments, X is k and X 16 In some embodiments, X is k and X 16 In some embodiments, X is k and X 17 is H. In some embodiments, X is k and X 17 is V. In some embodiments, X is k and X 17 is Q. In some embodiments, X is k and X 35 is E. In some embodiments, X is k and X 35 is N.

[0447] In some embodiments, X 10 is Q and X 15 is E. In some embodiments, X 10 is Q and X 15 is F. In some embodiments, X 10 is Q and X 16 is L. In some embodiments, X 10 is Q and X 16 is K. In some embodiments, X 10 is Q and X 16 In some embodiments, X is 10 is Q and X 17 is H. In some embodiments, X 10 is Q and X17 is V. In some embodiments, X 10 is Q and X 17 is Q. In some embodiments, X 10 is Q and X 35 is E. In some embodiments, X 10 is Q and X 35 is N.

[0448] In some embodiments, X 10 is S and X 15 is E. In some embodiments, X 10 is S and X 15 is F. In some embodiments, X 10 is S and X 16 is L. In some embodiments, X 10 is S and X 16 is K. In some embodiments, X 10 is S and X 16 In some embodiments, X is 10 is S and X 17 is H. In some embodiments, X 10 is S and X 17 is V. In some embodiments, X 10 is S and X 17 is Q. In some embodiments, X 10 is S and X 35 is E. In some embodiments, X 10 is S and X 35 is N.

[0449] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 is K. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X15 is E and X 17 is V. In some embodiments, X 15 is E and X 17 is Q. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 35 is N.

[0450] In some embodiments, X 15 is F and X 16 is L. In some embodiments, X 15 is F and X 16 is K. In some embodiments, X 15 is F and X 16 In some embodiments, X is 15 is F and X 17 is H. In some embodiments, X 15 is F and X 17 is V. In some embodiments, X 15 is F and X 17 is Q. In some embodiments, X 15 is F and X 35 is E. In some embodiments, X 15 is F and X 35 is N.

[0451] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 17 is V. In some embodiments, X 16 is L and X 17 is Q. In some embodiments, X 16 is L and X 35 is E. In some embodiments, X 16 is L and X 35 is N.

[0452] In some embodiments, X16 is K and X 17 is H. In some embodiments, X 16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is K and X 35 is N.

[0453] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 17 is V. In some embodiments, X 16 is k and X 17 is Q. In some embodiments, X 16 is k and X 35 is E. In some embodiments, X 16 is k and X 35 is N.

[0454] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 is N.

[0455] In some embodiments, X 17 is V and X 35 is E. In some embodiments, X 17 is V and X 35 is N.

[0456] In some embodiments, X 17 is Q and X 35 is E. In some embodiments, X 17 is Q and X 35 is N.

[0457] In some embodiments, a polypeptide intermediate of the disclosure has the sequence of SEQ ID NO:212: SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO:212), X 15 is E or F; X 16 is L, K, or k; X 17 is H, V, or Q; X 35 is E or N; each K independently represents an L-lysine optionally covalently linked to a protecting group, or a spacer optionally linked to a protecting group; each k independently represents a D-lysine optionally covalently linked to a protecting group, or a spacer optionally linked to a protecting group; wherein the two cysteine ​​residues of SCNTSTC (SEQ ID NO: 310) are optionally further linked by a disulfide bridge) is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SCNTSTC (SEQ ID NO: 310).

[0458] In some embodiments, X 35 is E. In some embodiments, X 35 is N.

[0459] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO:212 include:

[0460] In some embodiments, the carboxy terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy terminal amino acid 37 is Y-(OH).

[0461] In some embodiments, X 15 is E. In some embodiments, X 15is F.

[0462] In some embodiments, X 16 is L. In some embodiments, X 16 is K. In some embodiments, X 16 is k.

[0463] In some embodiments, X 17 is H. In some embodiments, X 17 is V. In some embodiments, X 17 is Q.

[0464] In some embodiments, X 15 is E and X 16 is L. In some embodiments, X 15 is E and X 16 is K. In some embodiments, X 15 is E and X 16 In some embodiments, X is 15 is E and X 17 is H. In some embodiments, X 15 is E and X 17 is V. In some embodiments, X 15 is E and X 17 is Q. In some embodiments, X 15 is E and X 35 is E. In some embodiments, X 15 is E and X 35 is N.

[0465] In some embodiments, X 15 is F and X 16 is L. In some embodiments, X 15 is F and X 16 is K. In some embodiments, X 15 is F and X 16 In some embodiments, X is 15 is F and X 17 is H. In some embodiments, X15 is F and X 17 is V. In some embodiments, X 15 is F and X 17 is Q. In some embodiments, X 15 is F and X 35 is E. In some embodiments, X 15 is F and X 35 is N.

[0466] In some embodiments, X 16 is L and X 17 is H. In some embodiments, X 16 is L and X 17 is V. In some embodiments, X 16 is L and X 17 is Q. In some embodiments, X 16 is L and X 35 is E. In some embodiments, X 16 is L and X 35 is N.

[0467] In some embodiments, X 16 is K and X 17 is H. In some embodiments, X 16 is K and X 17 is V. In some embodiments, X 16 is K and X 17 is Q. In some embodiments, X 16 is K and X 35 is E. In some embodiments, X 16 is K and X 35 is N.

[0468] In some embodiments, X 16 is k and X 17 is H. In some embodiments, X 16 is k and X 17 is V. In some embodiments, X 16 is k and X 17 is Q. In some embodiments, X16 is k and X 35 is E. In some embodiments, X 16 is k and X 35 is N.

[0469] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 is N.

[0470] In some embodiments, X 17 is V and X 35 is E. In some embodiments, X 17 is V and X 35 is N.

[0471] In some embodiments, X 17 is Q and X 35 is E. In some embodiments, X 17 is Q and X 35 is N.

[0472] In some embodiments, the isolated peptide comprising the amino acid sequence of SEQ ID NO:210, SEQ ID NO:211, or SEQ ID NO:212 further comprises a protecting group or a spacer optionally linked to a protecting group. In some embodiments, the isolated peptide further comprises a protecting group. In some embodiments, the isolated peptide further comprises a spacer. In some embodiments, the isolated peptide further comprises a spacer linked to a protecting group.

[0473] In some embodiments, the isolated polypeptide has positions X1, X 15 , or X 16 and optionally comprising a protecting group or a spacer attached to the protecting group.

[0474] In some embodiments, positions X1, X 15 , or X 16and an isolated polypeptide comprising a protecting group or a spacer optionally linked to a protecting group is conjugated via a lysine at that position.

[0475] In some embodiments, the isolated polypeptide comprises a protecting group or a spacer optionally linked to a protecting group at position X1.

[0476] In some embodiments, the isolated polypeptide has a structure at position X 15 and optionally comprising a protecting group or a spacer attached to the protecting group.

[0477] In some embodiments, the isolated polypeptide has a structure at position X 16 and optionally comprising a protecting group or a spacer attached to the protecting group.

[0478] In some embodiments, the isolated polypeptide comprising a protecting group or a spacer optionally linked to a protecting group at position X1 is conjugated via a lysine at that position.

[0479] In some embodiments, position X 15 and an isolated polypeptide comprising a protecting group or a spacer optionally linked to a protecting group is conjugated via a lysine at that position.

[0480] In some embodiments, position X 16 and an isolated polypeptide comprising a protecting group or a spacer optionally linked to a protecting group is conjugated via a lysine at that position.

[0481] In some embodiments, the protecting group is selected from the group consisting of acetyl, allyloxycarbonyl, benzyl, Boc, Cbz, Dmb, (dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Fmoc, tert-butyl, or trityl. In some embodiments, the protecting group is acetyl, allyloxycarbonyl, dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Fmoc, tert-butyl, or trityl. In some embodiments, the protecting group is acetyl, allyloxycarbonyl, dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, or Fmoc.

[0482] In some embodiments, the spacer is selected from the group consisting of Formula XI, Formula XII, Formula XIII, Formula XIV, Formula XV, Formula XVI, Formula XVII, and Formula XVIII. In some embodiments, the isolated polypeptide further comprises a spacer of Formula XIII. In some embodiments, the isolated peptide further comprises a spacer of Formula XIV. In some embodiments, the isolated peptide further comprises a spacer of Formula XVII. In some embodiments, the isolated peptide further comprises a spacer of Formula XVIII. In some embodiments, the spacer is bound to a protecting group. In some embodiments, the protecting group is selected from the group consisting of acetyl, allyloxycarbonyl, benzyl, Boc, Cbz, Dmb, (dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Fmoc, tert-butyl, or trityl. In some embodiments, the protecting group is acetyl, allyloxycarbonyl, dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Fmoc, tert-butyl, or trityl. In some embodiments, the protecting group is acetyl, trityl, or tert-butyl. In some embodiments, the spacer is not bound to a protecting group.

[0483] Exemplary Polypeptide Intermediates In some embodiments, the isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Table 4:

[0484] [Table 4] TIFF0007636321000017.tif234170

[0485] In some embodiments, the invention provides a peptide intermediate as set forth in Table 4 above. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO:129, SEQ ID NO:144, SEQ ID NO:145, or SEQ ID NO:156. In some embodiments, the peptide intermediate has an amino acid sequence of SEQ ID NO:129. In some embodiments, the peptide intermediate has an amino acid sequence of SEQ ID NO:145. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO:129, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, or SEQ ID NO:150. In some embodiments, the peptide intermediate has an amino acid sequence of SEQ ID NO:147. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, or SEQ ID NO:154. In some embodiments, the peptide intermediate has an amino acid sequence of SEQ ID NO:151.

[0486] In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 155 or SEQ ID NO: 156. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 155. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 157. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, or SEQ ID NO: 164. In some embodiments, the peptide intermediate is a peptide having an amino acid sequence of SEQ ID NO: 164.

[0487] 8. Uses, Formulation and Administration How to use According to another embodiment, the present invention relates to a method of treating a metabolic disease or disorder in a subject in need thereof, comprising providing to the subject an effective amount of an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof. Metabolic diseases or disorders include type 1 diabetes, type 2 diabetes, and obesity. Additionally, the present invention relates to a method of effecting weight loss in a subject, including a diabetic subject, comprising providing to the subject an effective amount of an amylin analog polypeptide of the present disclosure.

[0488] The disclosed amylin analog polypeptides are particularly useful for treating diabetes, and the methods include providing an effective amount of the amylin analog polypeptide to a diabetic subject. In some embodiments, the disclosed amylin analog polypeptides are used to treat a subject with type 1 or type 2 diabetes to control or reduce the concentration of blood glucose in the subject, where blood glucose levels can be monitored or estimated based on measured blood levels of glycated hemoglobin (hemoglobin A1c, HbA1c).

[0489] (i) in some embodiments, the amylin analog polypeptides of the disclosure are used to treat a subject with type 1 diabetes; (ii) in some embodiments, the amylin analog polypeptides of the disclosure are used to treat a subject with type 2 diabetes; (iii) in some embodiments, the amylin analog polypeptides of the disclosure are used to treat obesity; (iv) in some embodiments, the amylin analog polypeptides of the disclosure are used to effect weight loss in a subject, such as a diabetic subject; The amylin analog polypeptide of use (i), (ii), (iii) or (iv) comprises any amino acid sequence of the present disclosure, including those selected from the group consisting of SEQ ID NOs: 199, 200, 204, 206, 127, 57, 128, 129, 43, 209, 130, 64, 65, 131, 109 and 27.

[0490] In some embodiments, an amylin analog polypeptide is used to treat a subject with type 1 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, an amylin analog polypeptide is used to treat a subject with type 1 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, an amylin analog polypeptide is used to treat a subject with type 1 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, an amylin analog polypeptide is used to treat a subject with type 1 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0491] In some embodiments, an amylin analog polypeptide is used to treat a subject having type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, an amylin analog polypeptide is used to treat a subject having type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, an amylin analog polypeptide is used to treat a subject having type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, an amylin analog polypeptide is used to treat a subject having type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0492] In some embodiments, an amylin analog polypeptide is used to treat a subject with obesity, and the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, an amylin analog polypeptide is used to treat a subject with obesity, and the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, an amylin analog polypeptide is used to treat a subject with obesity, and the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, an amylin analog polypeptide is used to treat a subject with obesity, and the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0493] In some embodiments, an amylin analog polypeptide is used to effect weight loss in a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, an amylin analog polypeptide is used to effect weight loss in a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, an amylin analog polypeptide is used to effect weight loss in a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, an amylin analog polypeptide is used to effect weight loss in a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0494] The amylin analog polypeptide of the present disclosure, such as insulin, is provided (i.e., administered) to a diabetic subject to maintain, control, or reduce blood glucose concentration in the subject. Diabetic patients treated with the amylin analog polypeptide of the present disclosure as an adjunct to insulin therapy are at risk of hypoglycemia (i.e., low blood sugar), especially severe hypoglycemia. Therefore, when treated with the amylin analog polypeptide of the present disclosure, reducing the dose of mealtime insulin in a diabetic subject is intended to reduce the risk of hypoglycemia, especially severe hypoglycemia.

[0495] As used herein, severe hypoglycemia refers to an episode of hypoglycemia that requires the assistance of another individual (including help with the administration of oral carbohydrates) or requires the administration of glucagon, intravenous glucose, or other medical intervention.

[0496] Thus, administration of the amylin analog polypeptides of the present disclosure as an adjunct to insulin therapy, particularly prandial insulin therapy, generally requires a reduction in the dose of prandial insulin required to adequately maintain healthy blood glucose concentrations in the subject. In other words, in type 1 or type 2 diabetes patients who already self-administer prandial insulin at a particular dose before initiating treatment with the amylin analog polypeptides of the present disclosure, initiating treatment with the amylin analog polypeptides of the present disclosure will result in a reduction (e.g., 25%, 50%, 75%, or 100%) in the dose of prandial insulin that they continue to self-administer.

[0497] In some embodiments, the method comprises providing to a subject in need of treatment an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof via injection, hi some embodiments, the method comprises providing to a subject in need of treatment an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof formulated for oral administration.

[0498] In some embodiments, the method includes providing an amylin analog 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 a continuous delivery of an amylin analog polypeptide from an osmotic delivery device to a subject in need of treatment. A delivery device, such as an osmotic delivery device, includes sufficient amylin analog polypeptide of the present disclosure for continuous administration for up to 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months. Thus, continuous administration of an amylin analog polypeptide of the present disclosure via an osmotic delivery device eliminates daily dosing or multiple daily dosing of an existing amylin analog polypeptide, such as pramlintide. Diabetic patients treated with pramlintide need to adjust dosing of pramlintide before a meal with prandial insulin administered after a meal. In contrast, diabetic patients treated with an amylin analog polypeptide of the present disclosure via an osmotic delivery device receive continuous delivery of the amylin analog polypeptide and only need to administer reduced amounts of prandial insulin.

[0499] The substantially steady state delivery of the amylin analog polypeptide from the osmotic delivery device is continuous over the administration period, hi some embodiments, the subject or patient is a human subject or patient.

[0500] 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.

[0501] In a further embodiment, the therapeutic method of the present invention provides a significant reduction in the fasting plasma glucose concentration of the subject after implantation of the osmotic delivery device in the subject (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 days or less after implantation of the osmotic delivery device in the subject. The significant reduction in fasting plasma glucose is typically statistically significant or considered significant to the subject by the physician, as demonstrated by application of an appropriate statistical test. The significant reduction in fasting plasma glucose compared to the pre-implant baseline is typically maintained over the administration period.

[0502] 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 a continuous delivery of a drug from an osmotic delivery device, whereby a substantially steady-state delivery of the drug at a therapeutic concentration 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 3 months. The drug has a known or determined half-life in a typical subject. Humans are the preferred subjects for the practice of the present invention. The present invention includes a drug effective for treating a disease or condition, as well as an osmotic delivery device containing the drug for use in the present 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.

[0503] 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.

[0504] The 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. The methods include providing delivery of an isolated amylin analog polypeptide. In some embodiments, the isolated amylin analog polypeptide is delivered continuously from an implantable osmotic delivery device. In some embodiments, a substantially steady state delivery of the amylin analog polypeptide from the osmotic delivery device is achieved and is substantially continuous over the administration period. In some embodiments, the subject is a human.

[0505] The present invention includes an isolated amylin analog polypeptide, as well as an osmotic delivery device comprising the isolated amylin analog 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.

[0506] In further embodiments, the therapeutic methods of the invention result in a significant reduction 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 prior to implantation of the osmotic delivery device), which significant reduction 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, within about 1 day or less after implantation of the osmotic delivery device in the subject. In preferred embodiments of the invention, the 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 prior to implantation is achieved within about 2 days or less after implantation of the osmotic delivery device in the subject, preferably within about 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 or considered significant to a subject by a physician when demonstrated by application of an appropriate statistical test. A significant reduction in fasting plasma glucose compared to pre-transplant baseline is typically maintained over the course of administration.

[0507] In embodiments of all aspects of the invention relating to a method of treating a disease or condition in a subject, an exemplary osmotic delivery device includes an impermeable reservoir having an inner and outer surface and a first and second open end; a semipermeable membrane in sealing relationship with the first open end of the reservoir; an osmotic engine in the reservoir and adjacent the semipermeable membrane; a piston adjacent 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 drug formulation or suspension formulation comprising a drug, the second chamber comprising 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 the suspension formulation. In a preferred embodiment, the reservoir comprises titanium or a titanium alloy.

[0508] In embodiments of all aspects of the invention relating to methods of 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 including a drug and a vehicle formulation. A vehicle formulation for use in forming a suspension formulation of the invention can include, for example, a solvent and a polymer.

[0509] The reservoir of the osmotic delivery device can comprise, for example, titanium or a titanium alloy.

[0510] In embodiments of all aspects of the invention, an implanted osmotic delivery device can be used to provide subcutaneous delivery.

[0511] In embodiments of all aspects of the invention, the continuous delivery can be, for example, a zero order controlled continuous delivery.

[0512] combination In some embodiments, the amylin analog polypeptide of the present disclosure is combined with a second drug to form a combination. In some embodiments, the amylin analog polypeptide of the present disclosure is combined with a second drug to form a combination, and the second drug is an insulinotropic peptide. In some embodiments, the amylin analog polypeptide of the present disclosure is combined with a second drug to form a combination, and the second drug is a GLP-1 receptor agonist. In some embodiments, the amylin analog polypeptide of the present disclosure is combined with a second drug to form a combination, and the second drug is a (GLP-1) agonist, such as exenatide, a derivative of exenatide, an analog of exenatide, or semaglutide. In some embodiments, the GLP-1 receptor agonist is exenatide. In some embodiments, the GLP-1 receptor agonist is semaglutide.

[0513] In some embodiments, the amylin analog polypeptide of the present disclosure is administered to a subject in combination with a second agent without being combined with the second agent, and the second agent is a (GLP-1) agonist such as exenatide, a derivative of exenatide, an analog of exenatide, or semaglutide.

[0514] In some embodiments, the amylin analog polypeptides of the disclosure are combined with insulin or an insulin derivative, hi some embodiments, the amylin analog polypeptides of the disclosure are combined with a long-acting basal insulin or a long-acting basal insulin derivative.

[0515] In some embodiments, the amylin analog polypeptide of the present disclosure is administered to the subject in combination with insulin or an insulin derivative, i.e., as an adjunct to insulin therapy, without being combined with insulin or an insulin derivative. In some embodiments, the amylin analog peptide of the present disclosure is administered to the subject in combination with prandial insulin, without being combined with insulin or an insulin derivative. In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 2 diabetes.

[0516] In some embodiments, the amylin analog polypeptides of the present disclosure are co-administered to a human patient with insulin or an insulin derivative to provide a so-called dual hormone "artificial pancreas" therapy. In some embodiments, the amylin analog polypeptides of the present disclosure are co-administered to a subject in combination with insulin or an insulin derivative, without being co-administered with insulin or an insulin derivative, to provide a dual hormone "artificial pancreas" therapy. In some embodiments, the amylin analog polypeptides of the present disclosure are co-administered to a subject in combination with insulin or an insulin derivative, thus providing a dual hormone "artificial pancreas" therapy. In some embodiments, the artificial pancreas therapy includes a fast-acting insulin or a fast-acting insulin derivative. In some embodiments, the artificial pancreas therapy includes a long-acting or basal insulin, or a long-acting or basal insulin derivative.

[0517] In some embodiments, any of the disclosed amylin analogs is formulated in combination with exenatide.In some embodiments, any of the disclosed amylin analogs is formulated in combination with a GLP-1 receptor agonist.

[0518] Some embodiments of the present invention include the use of an amylin analog polypeptide in combination with a second therapeutic agent, including but not limited to insulinotropic peptides, peptide hormones, such as glucagon and incretin mimetics (e.g., GLP-1 receptor agonists such as exenatide), and their peptide analogs and peptide derivatives; PYY (also known as peptide YY, peptide tyrosine tyrosine), and its peptide analogs and peptide derivatives, such as PYY(3-36); oxyntomodulin, and its peptide analogs and peptide derivatives; and gastric inhibitory peptide (GIP), and its peptide analogs and peptide derivatives. In some embodiments, a pharmaceutical composition comprising an amylin analog polypeptide in combination with a GLP-1 receptor agonist is used to treat type 2 diabetes.

[0519] GLP-1, including the 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 induce glucose uptake by cells and stimulate insulin secretion (i.e., are insulinotropic), resulting in a lowering of serum glucose concentrations (see, e.g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).

[0520] A number of 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; 6,593,2 No. 95; No. 6,703,359; No. 6,706,689; No. 6,720,407; No. 6,821,949; No. 6,849,708 ; Same No. 6,849,714; Same No. 6,887,470; Same No. 6,887,849; Same No. 6,903,186; Same No. 7,022,674; Same No. 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). One 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). Victoza® (liraglutide) for once-daily injection is commercially available in the United States, Europe, and Japan. Another example of a GLP-1 receptor agonist is Ozempic® (Novo Nordisk A / S, Bagsvaerd DK) (semaglutide). For ease of reference herein, the family of GLP-1 receptor agonists, GLP-1 peptides, GLP-1 peptide derivatives and GLP-1 peptide analogues with insulinotropic activity will be collectively referred to as "GLP-1".

[0521] The molecule exenatide has the amino acid sequence of exendin-4 (Kolterman OG,et al., J. Clin. Endocrinol. Metab. 88(7):3082-9 (2003)) and is produced by chemical synthesis or recombinant expression. For ease of reference herein, the family of exenatide peptides (including, for example, exendin-3, exendin-4, and exendin-4-amide), exenatide peptide derivatives, and exenatide peptide analogs are collectively referred to as "exenatide."

[0522] Peptide YY (PYY) is a peptide amide of 36 amino acid residues. 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 (1997)). (1989)). The sequence of PYY, as well as its peptide analogs and derivatives, are known in the art (e.g., U.S. Patent Nos. 5,574,010 and 5,552,520).

[0523] 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, as well as its peptide analogs and peptide 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).

[0524] Gastric inhibitory peptide (GIP) is an insulinotropic peptide hormone (Efendic, S.,et al., Horm Metab Res. 36:742-6 (2004)) that is 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 42 amino acid biologically active 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 JJ, Diabetes Metab Res Rev. 21(2):91-117 (2005) and Efendic S., Horm Metab Res. 36(11-12):742-6 (2004)).

[0525] Glucagon is a peptide hormone produced by alpha cells in the pancreas that increases the concentration of glucose in the bloodstream. Its effect is opposite to that of insulin, which lowers glucose levels. When the concentration of glucose in the bloodstream becomes 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, which allows glucose to be taken up and used by insulin-dependent tissues. Thus, glucagon and insulin are part of a feedback system that keeps blood glucose levels at a stable level.

[0526] Pharmaceutically acceptable compositions According to another embodiment, the present invention provides a composition comprising a compound of the present invention, i.e., an isolated polypeptide or a pharma- ceutically acceptable derivative thereof, and a pharma- ceutically acceptable carrier, adjuvant, or vehicle. The amount of the compound in the composition of the present invention is such that it is effective to measurably activate one or more amylin and / or calcitonin receptors in a biological sample or in a patient. In certain embodiments, the amount of the compound in the composition of the present invention is such that it is effective to measurably activate human amylin 3 receptor (hAMY3) and / or human calcitonin receptor (hCTR) in a biological sample or in a patient in the absence or presence of human serum albumin. In certain embodiments, the composition of the present invention is formulated for administration to a patient in need of such a composition. In some embodiments, the composition of the present invention is formulated for injectable administration to a patient. In some embodiments, the composition of the present invention is formulated for administration to a patient via an implantable delivery device, such as an osmotic delivery device.

[0527] The term "subject", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0528] "Pharmaceutically acceptable derivative" means 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 providing, directly or indirectly, a compound of the invention, or an inhibitory active metabolite or residue thereof.

[0529] 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 polypeptides or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" is intended to include any and all solvents, 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 pharma- ceutical 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 compounds can also be incorporated into the compositions.

[0530] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of 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, the pharmaceutical composition or isolated polypeptide of the present disclosure is formulated for administration by topical administration. In some embodiments, the pharmaceutical composition or isolated polypeptide of the present disclosure is formulated for administration by inhalation administration. In some embodiments, the pharmaceutical composition is suitable for subdermal or subcutaneous implantation and is formulated for administration by a device or other suitable delivery mechanism that delivers the pharmaceutical composition subcutaneously. In some embodiments, the pharmaceutical composition is suitable for subdermal or subcutaneous implantation and is formulated for administration by an implanted device that delivers the pharmaceutical composition subcutaneously. In some embodiments, the pharmaceutical composition is suitable for subdermal or subcutaneous placement or other implantation and is 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, subcutaneous application, or combinations thereof may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents 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.

[0531] 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. The composition 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. A suitable fluidity can be, for example, lecithin, or the like. This can be achieved by using a coating such as , by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0532] Sterile injection can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components required from above-listed.For the preparation of sterile powder for sterile injection, the preparation method is vacuum drying and freeze-drying, which makes the powder containing active ingredient and any additional desired ingredients from its previously sterile filtered solution.

[0533] Oral compositions generally include 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, where 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.

[0534] 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, e.g., a gas such as carbon dioxide, or a nebulizer.

[0535] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. 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.

[0536] In one embodiment, the active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharma-ceutically 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.

[0537] 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 dosage for treating a subject, each unit containing 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, as well as the inherent limitations of the art of compounding such active compound for treating an individual.

[0538] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0539] Drug Particle Formulations In some embodiments, a pharmaceutical composition is provided that includes any of the disclosed polypeptides formulated as a trifluoroacetate, acetate, or hydrochloride salt. In some embodiments, a pharmaceutical composition is provided that includes any of the disclosed polypeptides formulated as a trifluoroacetate salt. In some embodiments, a pharmaceutical composition is provided that includes any of the disclosed polypeptides formulated as an acetate salt. In some embodiments, a pharmaceutical composition is provided that includes any of the disclosed polypeptides formulated as a hydrochloride salt.

[0540] The compound for use in carrying out the present invention, i.e., the isolated polypeptide or a pharma- ceutically acceptable salt thereof, 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 to form a suspension formulation. In some embodiments, the amylin analog polypeptide is formulated into a particle formulation and converted to particles (e.g., spray-dried). In some embodiments, the particles containing the amylin analog polypeptide are suspended in a vehicle formulation, resulting in a suspension formulation of vehicle and suspended particles containing the amylin analog polypeptide.

[0541] 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.

[0542] In some embodiments, the present invention provides a drug particle formulation for pharmaceutical use. The particle formulation typically includes 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.

[0543] In any of the embodiments, the particle formulation may contain about 50% to about 90% by weight of the drug, about 50% to about 85% by weight of the drug, about 55% to about 90% by weight of the drug, about 60% to about 90% by weight of the drug, about 65% to about 85% by weight of the drug, about 65% to about 90% by weight of the drug, about 70% to about 90% by weight of the drug, about 70% to about 85% by weight of the drug, about 70% to about 80% by weight of the drug, or about 70% to about 75% by weight of the drug.

[0544] Typically, the amount of carbohydrate in a particle formulation is determined by aggregation concerns. In general, the amount of carbohydrate should not be too high to avoid excess carbohydrate not bound to the drug promoting crystal growth in the presence of water.

[0545] Typically, the amount of antioxidant in a particle formulation is dictated by oxidation concerns, while the amount of amino acid in the formulation is dictated by oxidation concerns and / or moldability of the particles during spray drying.

[0546] Typically, the amount of buffer in a particle formulation is determined by pre-processing concerns, stability concerns, and moldability 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.

[0547] 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 sorbitol), disaccharides (e.g., lactose, sucrose, trehalose, and cellobiose), polysaccharides (e.g., raffinose, melezitose, maltodextrin, dextran, and starch), and alditols (non-cyclic polyols; e.g., mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosylsorbitol, and myoinsitol).Suitable carbohydrates include disaccharides and / or non-reducing sugars, e.g., sucrose, trehalose, and raffinose.

[0548] 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 hydroxanisol, butylated hydroxyltoluene, and propyl gallate.Furthermore, easily oxidized amino acids, such as cysteine, methionine, and tryptophan, can be used as antioxidants.

[0549] 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.

[0550] 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.

[0551] 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.

[0552] 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.

[0553] Particles are typically sized to be deliverable through an implantable osmotic delivery device. A uniform shape and size of the particles typically helps provide a consistent and 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%, 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.

[0554] Those skilled in the art will understand that a population of particles follows the principles of particle size distribution. Widely used art-recognized methods for describing particle size distribution include, for example, the mean diameter and D-values, such as the D50 value, which are commonly used to represent the mean diameter of the range of particle sizes of a given sample.

[0555] The particles of the particulate formulation have a diameter 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 between about 2 microns and about 50 microns.

[0556] The particles of the particle formulation comprising the isolated amylin analog polypeptide have an average diameter between about 0.3 microns and about 150 microns. The particles of the particle formulation comprising the isolated amylin analog polypeptide have an average diameter between about 2 microns and about 150 microns, for example, an average diameter less than 150 microns, an average diameter less than 100 microns, an average diameter less than 50 microns, an average diameter less than 30 microns, an average diameter less than 10 microns, an average diameter less than 5 microns, and an average diameter of about 2 microns. In some embodiments, the particles have an average diameter between about 0.3 microns and 50 microns, for example, between about 2 microns and about 50 microns. In some embodiments, the particles have an average diameter between 0.3 microns and 50 microns, for example, between about 2 microns and about 50 microns, and each particle is less than about 50 microns in diameter.

[0557] Typically, the particles of the particle formulation, when incorporated into a suspension vehicle, do not settle in less than about 3 months at the delivery temperature, preferably do not settle in less than about 6 months, more preferably do not settle in less than about 12 months, more preferably do not settle in less than about 24 months at the delivery temperature, and most preferably do not settle in less than about 36 months at the delivery temperature. The suspension vehicle typically has a viscosity between about 5000 and about 30,000 poise, preferably between about 8000 and about 25,000 poise, 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 3000 poise. Generally speaking, smaller particles tend to have a slower settling rate in a viscous suspension vehicle than larger particles. Thus, micron- to nano-sized particles are typically desirable. In a viscous suspension formulation, the about 2 micron to about 7 micron particles of the present invention will not settle out at room temperature for at least 20 years based on simulation modeling studies. One embodiment of the particle formulation of the present invention includes particles with a size range of less than about 50 microns, more preferably less than about 10 microns, more preferably from about 2 microns to about 7 microns, for use in implantable osmotic delivery devices.

[0558] In summary, the disclosed polypeptide, or its pharma- ceutically 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 the delivery of stable and biologically effective drugs to subjects over long periods of time.The particles are administered to patients suspended in a suspension vehicle.

[0559] Suspension of particles in a vehicle In one aspect, 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 includes 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 include additional components, including but not limited to surfactants, antioxidants, and / or other compounds that are soluble in the vehicle.

[0560] The viscosity of the suspension vehicle is typically sufficient to prevent the drug particle formulation from settling during storage and sufficient for use in a delivery method, such as an implantable osmotic delivery device. The suspension vehicle is biodegradable in that it disintegrates or degrades 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.

[0561] In embodiments, the suspension vehicle is a "single-phase" suspension vehicle, which is a homogenous system of a solid, semi-solid, or liquid that is physically and chemically uniform throughout.

[0562] 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 when contacted 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 when contacted with an aqueous environment having less than approximately 10% water.

[0563] The solvent may be any acceptable solvent that is immiscible with water. The solvent may 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 invention include, but are not limited to, lauryl alcohol, benzyl benzoate, benzyl alcohol, lauryl lactate, decanol (also called decyl alcohol), ethylhexyl lactate, and long chain (C8-C24) aliphatic alcohols, esters, or mixtures thereof. Solvents used in suspension vehicles may be "dry" in that they have low water content. Preferred solvents for use in the formulation of suspension vehicles include lauryl lactate, lauryl alcohol, benzyl benzoate, and mixtures thereof.

[0564] Examples of polymers for the formulation of the suspension vehicle 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 with a molecular weight of 2000 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 polymers used in the suspension vehicle may include one or more different polymers, or may include different grades of a single polymer. The polymers used in the suspension vehicle may also be dry or have a low water content.

[0565] Generally speaking, suspension vehicles for use in the present invention may vary in composition based on the desired performance characteristics. In one embodiment, the suspension vehicle may comprise about 40% to about 80% by weight of polymer(s) and about 20% to about 60% by weight of solvent(s). A preferred embodiment of the suspension vehicle comprises a vehicle 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; about 75% by weight of solvent and about 25% by weight of polymer. Thus, in some embodiments, the suspension vehicle may comprise selected components, and in other embodiments, it consists essentially of selected components.

[0566] The suspension vehicle may exhibit Newtonian behavior. The suspension vehicle is typically formulated to provide a viscosity that maintains uniform dispersion of the particle formulation for a given period of time. This helps to facilitate the manufacture of a suspension formulation that is tailored to provide controlled delivery of the drug contained in the drug particle formulation. The viscosity of the suspension vehicle may vary depending on the desired application, the size and type of the particle formulation, and the loading amount of the particle formulation in the suspension vehicle. The viscosity of the suspension vehicle may be varied by changing the type or relative amount of the solvent or polymer used.

[0567] The suspension vehicle may have a viscosity ranging from about 100 poise to about 100,000 poise, preferably from about 1,000 poise to about 100,000 poise. In a preferred embodiment, the suspension vehicle typically has a viscosity between about 5000 and about 30,000 poise, preferably between about 8000 and about 25,000 poise, more preferably between about 10,000 and 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 3000 poise at 33° C. The viscosity can be measured using a parallel plate rheometer at 33° C. at a shear rate of 10−4 / sec.

[0568] The suspension vehicle may exhibit phase separation upon contact with an aqueous environment; however, typically, the suspension vehicle does not exhibit substantial phase separation as a function of temperature. For example, at temperatures ranging from about 0° C. to about 70° C., and upon temperature cycling, such as cycling from 4° C. to 37° C. to 4° C., the suspension vehicle typically does not exhibit phase separation.

[0569] 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 may be blended under reduced pressure to remove air bubbles generated from the dry components. The components can be combined using a conventional mixer, such as a double helical blade or similar mixer, set at a speed of about 40 rpm. However, higher speeds can also be used to mix the components. Once a 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 components of the vehicle (e.g., the solvent and / or the polymer) can be treated to substantially reduce or substantially remove peroxides (e.g., by treatment with methionine; see, e.g., U.S. Patent Application Publication No. 2007-0027105).

[0570] 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.

[0571] The suspension formulation can be prepared by dispersing the particle formulation in the suspension vehicle. The suspension vehicle can be heated and the particle formulation can be added to the suspension vehicle under dry conditions. The ingredients can be mixed under reduced pressure at an elevated temperature, such as about 40°C to about 70°C. The ingredients can be mixed at a sufficient speed, such as about 40 rpm to about 120 rpm, and 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 helix 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, a unit dose container, or a multi-dose container.

[0572] 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.

[0573] In a preferred embodiment, the suspension formulations of the present invention are substantially homogeneous and fluid and provide for delivery of a drug particle formulation from an osmotic delivery device to a subject.

[0574] 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 drug particle formulation. These properties include, but are not limited to, the viscosity of the suspension; the purity of the vehicle; the residual moisture 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.

[0575] 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, weeks, months, or up to about a year or more.Such implantable osmotic delivery devices can typically deliver the suspension formulations containing drugs at desired flow rates over desired periods.The suspension formulations can be loaded into the implantable osmotic delivery devices by conventional techniques.

[0576] Implantable Delivery 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 an 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.

[0577] The implantable osmotic delivery device typically comprises 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 device 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; 6,382,293; 6,401,294; 6,411,295; 6,421,296; 6,430,297; 6,440,297; 6,450,297; 6,460,297; 6,470,297; 6,480,297; 6,490,297; 6,500,297; 6,510,297; 6,520,297; 6,530,297; 6,540,297; 6,550,297; 6,62 ... Nos. 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).

[0578] 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 the 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.

[0579] Osmotic devices release drugs at a predetermined rate based on the principle 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 out 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 for an extended period of time (e.g., about 1, about 3, about 6, about 9, about 10, and about 12 months).

[0580] 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.

[0581] 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.

[0582] 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 rib, for example, at least about 5-8 centimeters below the right rib, and at least 2-3 centimeters to the right of the midline, for example, 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, for example, at least about 5-8 centimeters above the belt line, and at least 2-3 centimeters to the right of the midline, for example, at least about 5-8 centimeters to the right of the midline; The right lower quadrant extends at least about 5-8 centimeters; the left upper quadrant extends at least 2-3 centimeters below the left rib cage, e.g., at least about 5-8 centimeters below the left rib cage, and at least 2-3 centimeters to the left of the midline, e.g., at least about 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 about 5-8 centimeters above the belt line, and at least 2-3 centimeters to the left of the midline, e.g., at least about 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 the osmotic delivery device is generally performed by a medical professional using local anesthesia (e.g., lidocaine).

[0583] Termination of treatment is simply by removal of the osmotic delivery device from the subject, which provides the important advantage of immediately stopping delivery of drug to the subject.

[0584] 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 such 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 required to depressurize the reservoir. In such a scenario, pressure will build up within the device until the device pushes the semipermeable membrane outward at the other end, thereby releasing the 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.

[0585] 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 dosing of laboratory animals (e.g., mice and rats). EXAMPLES

[0586] The following examples are provided to provide those skilled 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.

[0587] Example 1: Production of Amylin Analog Polypeptides The amylin analog polypeptides of the invention provided in Table 3 were synthesized using the Prelude peptide synthesizer (Protein Technologies The amines were synthesized by solid-phase methods 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 amino acid) in N,N-dimethylformamide (DMF) at 1000 nm (Novabiochem, Inc., Tucson, AZ) and 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.

[0588] Final deprotection and cleavage of the peptide from the solid support was accomplished by treating the resin with (92.5% TFA, 2.5% phenol, 2.5% water and 2.5% 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. The crude solid was then dissolved in a 1:1 solution of ACN / water containing 0.01% TFA. Disulfide bridge formation was obtained by adding a solution of iodine / acetic acid (35mg / ml) to the crude product solution until the solution was consistently amber in color. The reaction solution was stirred until analysis by LC / MS indicated the reaction was complete. A 2% solution of ascorbic acid in H2O was added until the solution was clear. The final crude product solution was lyophilized in preparation for final purification.

[0589] The lyophilized solid was redissolved in a 1:1 solution of acetonitrile / water containing 0.1% TFA (10-15 mL) and purified by reverse-phase HPLC on a Waters XBridg™ BEH 130, CIS, 10 pm, 130 Å, 30×250 mm ID column using a 30 gradient ranging from 5-75% acetonitrile / water containing 0.1% TFA over 30-45 min at a flow rate of 30 mL / min and λ-215 nm.

[0590] Example 2: Purification and characterization of amylin analog polypeptides, i.e. linear polypeptides, without lipophilic substituents and optional spacers The purified product was lyophilized and analyzed by ESI-LC / MS and analytical HPLC and was demonstrated to be pure (>98%). All mass results were in agreement with the calculated values.

[0591] 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) with UV detection resulting in dual absorbance signals at 215 nm and 280 nm.

[0592] Method A, LC / MS conditions: Phenomenex UPLC 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.

[0593] Method B, C18 HPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column at 25 °C, 30 min, flow rate of 0.5 mL / min, 1215 nm, 1280 nm, 5-65% acetonitrile / water with 0.05% TFA.

[0594] Method C, UPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column at 25 °C, 20 min, flow rate of 0.5 mL / min, 1215 nm, 1280 nm, 5-65% acetonitrile / water with 0.05% TFA.

[0595] Method D, UPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100 × 2.10 mm column at 5-65% acetonitrile / water with 0.05% TFA at a flow rate of 0.5 mL / min, 1215 nm, 1280 nm, over 10 min at 25 °C. 5.0 μL of sample was injected using PLNO (partial loop with needle overfill) injection mode.

[0596] Table 3 provides exemplary amylin analog polypeptides of the disclosure.

[0597] 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."

[0598] Example 3: Synthesis of Amylin Analog Polypeptide Intermediates Synthesis of polypeptides with modifications at D-Lys16 or L-Lys16 position. For example (compounds B2, A129, B4, and B8) Upon completion of the synthesis of the linear polypeptide as described in Example 1, the resin was washed with dichloromethane (DCM) and dried under reduced pressure for 30 min. For analogs containing an Alloc protecting group, its removal was facilitated via a solution of Pd(PPh3)3 in (chloroform / acetic acid / n-methylmorpholine, 37:2:1). The resulting deprotected resin was washed with 2% sodium diethyldithiodicarbamate trihydrate / DMF (6 x 30 s), 2% DIEA / DMF (6 x 30 s), and finally with DMF (6 x 30 s). The extension of the spacer region was carried out stepwise by manually adding each building block under preactivation conditions. To 1 ml of a 200 mmol solution of Fmoc-γGlu-(OH)-OtBu in DMF, 0.5 ml of DIEA (800 mmol) was added followed by 0.5 ml of HCTU (400 mmol), and the resulting reaction solution was stirred for 5 min, which was then added to the deprotected residues on the linear sequence. The reaction mixture was stirred for approximately 30 min under nitrogen. The resin was drained and washed with DMF (6 × 30 s). Subsequent removal of the Fmoc protecting group was facilitated using 20% ​​piperidine / DMF, followed by a final wash with DMF (6 × 30 s). Final deprotection and cleavage of the peptide from the solid support was achieved by treating the resin with (95% TFA, 2% water, 2% thioanisole, and 1% triisopropylsilane) for 2–3 h. As each building block was incorporated using the methods described above, each intermediate was isolated and characterized by HPLC / MS.

[0599] Synthesis of N-terminally modified polypeptides, e.g. (A130, B22, and B23) The synthesis of the linear sequence was carried out as described in Example 1. The addition of the albumin binding moiety was facilitated by removal of the N-terminal Fmoc protecting group via a 20% solution of piperidine / DMF. The resin was washed with DMF and the incorporation of the side chain building blocks was carried out stepwise under preactivation conditions. To 1 ml of a 200 mmol solution of Fmoc-γGlu-(OH)-OtBu in DMF, 0.5 ml of DIEA (800 mmol) was added followed by 0.5 ml of HCTU (400 mmol). The resulting reaction solution was stirred for 5 min and then added to the deprotected linear sequence. The reaction mixture was stirred under nitrogen for 30 min. The resin was drained and washed with DMF (6 × 30 s). Final deprotection and cleavage of the peptide from the solid support was carried out by treating the resin with (95% TFA, 2% water, 2% thioanisole, and 1% triisopropylsilane) for 2-3 h. Each intermediate was isolated and characterized by HPLC / MS. The chemical data for both the internally modified polypeptide and the N-terminally modified polypeptide intermediates are recorded in Table 5 below.

[0600] [Table 5]

[0601] Example 4: Covalent attachment of a lipophilic substituent and optional spacer to an amylin analog polypeptide, i.e., conversion of a linear polypeptide to a conjugated polypeptide Synthesis of amylin analog polypeptides conjugated with one or more albumin-binding lipophilic substituents and an optional spacer was carried out according to the synthetic method described in Example 1 with modifications.

[0602] Upon completion of the synthesis of the linear polypeptide as described in Example 1, the resin was washed with dichloromethane (DCM) and dried under reduced pressure for 30 min. In the case of analogs containing an alloc protecting group, its removal was performed via a solution of Pd(PPh3)3 in (chloroform / acetic acid / n-methylmorpholine, 37:2:1). In the case of analogs containing BOC-Lys(Fmoc)-OH, the Fmoc protecting group was removed using 20% ​​piperidine / DMF. The resulting deprotected resin was washed with DMF (6 x 30 s). The extension of the spacer region was then 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 regular SPPS conditions without a preactivation step. The final deprotection and cleavage of the peptide from the solid support was carried out by treating the resin with (95% TFA, 2% water, 2% thioanisole, and 1% triisopropylsilane) for 2-3 h. The cleaved peptide was precipitated using cold diethyl ether, which was decanted and the solid was triturated again with cold diethyl ether and pelleted by centrifugation.

[0603] The crude products were then dissolved in a solution of ACN / H2O, 0.1% TFA. A solution of iodine / acetic acid (35 mg / ml) was added to each solution of the crude peptide products until the solutions were consistently amber in color. The reaction solutions were stirred until analysis by LC / MS showed that the desired disulfide bridges had formed. A 2% solution of ascorbic acid in H2O was added to the reaction solutions until the solutions were clear. The solutions were frozen and lyophilized. Purification was obtained by the methods described in Example 1.

[0604] Exemplary syntheses of conjugated peptides are described. Synthesis of A109: The synthesis of the linear sequence was carried out as described in Example 1. The addition of the albumin binding moiety was facilitated by removing the N-terminal Fmoc protecting group with a 20% solution of piperidine / DMF. The resin was then washed with DMF and the incorporation of the side chain building blocks was carried out stepwise under preactivation conditions. To 1 ml of a 200 mmol solution of Fmoc-γGlu-(OH)-OtBu in DMF, 0.5 ml of DIEA (800 mmol) was added followed by 0.5 ml of HCTU (400 mmol). The resulting reaction solution was stirred for 5 min and then added to the deprotected linear sequence. The reaction mixture was stirred under nitrogen for 30 min. The resin was then drained and washed with DMF (6×30 s). The Fmoc protecting group of γGlu was removed with 20% piperidine / DMF. This was followed by coupling with octadecanedioic acid (C18) (200 mmol) in DMF using HATU (400 mmol) and DIEA (800 mmol) under standard solid phase conditions.

[0605] Cleavage was achieved using a solution of 95% TFA / 2% water / 2% thioanisole / 1% TIPS. The crude product was then dissolved in a solution of ACN / H2O, 0.1% TFA. A solution of iodine / acetic acid (35 mg / ml) was added to the solution of the crude peptide product until the solution was consistently amber in color. The reaction solution was stirred until analysis by LC / MS showed that the desired disulfide bridge had formed. A 2% solution of ascorbic acid in H2O was added to the reaction solution until the solution was clear.

[0606] Lyophilization of the crude product gave an off-white solid which was purified by the method described in Example 2.

[0607] Example 5: Stability of Amylin Analog Polypeptides Several amylin analog 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., in DI water). The analog polypeptides were incubated at 37° C. and samples were removed at various time intervals and analyzed by LC / MS and HPLC to determine the purity and mass of the parent peptide, as well as the extent of degradation products. The purity results of these analyses are shown in Tables 6A and 6B and are believed to be indicative of stability.

[0608] [Table 6] TIFF0007636321000020.tif58170

[0609] [Table 7]

[0610] Example 6: Solubility of Amylin Analog Polypeptides The analog polypeptides described herein were tested for solubility in saline containing 20% ​​DMSO in water, (i.e., bioassay buffer) or in water (DI water) at room temperature. Samples were visually inspected for any appearance of sample clarity, turbidity or cloudiness. The results of this analysis are shown in Table 7.

[0611] [Table 8]

[0612] Example 7: Functional Assays: Human Calcitonin Receptor and Amylin 3 Receptor Activation of the human calcitonin receptor (hCTR) or human amylin 3 receptor (hAMY3R) results in an increase in cellular cyclic adenosine monophosphate (cAMP). Accumulated cAMP can be measured in vitro using common detection methods in the presence of the non-specific cAMP / cGMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Thus, it is possible to estimate the in vitro potency (pEC50) of peptides that activate each of these receptors using fitted dose-response curves of cAMP accumulation.

[0613] Cells, culture, and cAMP assay HEK293-CNG cells stably expressing human calcitonin receptor (hCTR) or co-expressing human calcitonin receptor and human receptor activity modifying protein 3 (hAMY3R) (Codex Biosolutions #CB-80200-258 and #CB-80-200-271, respectively) were grown in 90% DMEM, 10% FBS, 250 μg / ml G418, and 1 μg / ml puromycin (hCTR cells) or in 90% DMEM, 10% FBS, 250 μg / ml G418, 1 μg / ml puromycin, 150 μg / ml hygromycin B (hAMY3R cells). Cells were transferred to growth medium no more than 10 passages prior to testing.

[0614] On the day of the assay, cells expressing hCTR or hAMY3R were counted and dispensed into white 384-well OptiPlates (PerkinElmer #6007299) at 500 cells per well in 5 mcL of stimulation buffer consisting of 1x HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% bovine serum albumin (BSA) or 1x HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% casein with 0%, 0.1% or 4% human serum albumin (HSA).

[0615] For each given assay condition, peptides were serially diluted in the same buffer as above. Two assay control solutions consisting of 50 mcM forskolin (cAMP system maximum) or assay buffer only (cAMP system minimum) were also prepared in the appropriate stimulation buffer. 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 the wells. The plate was covered with an adhesive cover to prevent evaporation and incubated for 60 minutes at room temperature in the dark. The plate was read on an Envision fluorescent plate reader.

[0616] Data analysis Test values ​​were first normalized to the forskolin-induced cAMP 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. Normalized data from triplicate tests was analyzed and used to estimate the EC50 of each peptide on each receptor. Data was fitted using a four-parameter logistic curve model: Y=base+(top-base) / (1+10^((LogEC50-X))) in GraphPad Prism software (v7.04). Hill slope was constrained to 1.0. EC50 values ​​were converted to pEC50 values ​​using the formula: pEC50=-Log(EC50).

[0617] Data interpretation For a given receptor, in vitro potency estimates in the absence of HSA (pEC50) are comparable for all peptides, as they reflect albumin-independent binding (free peptide) values ​​(0% HSA, Table 8). However, in the presence of albumin (BSA or HSA), potency measurements across acylated peptides are not easily comparable (Table 8). This is due to the variation in albumin-acyl peptide binding efficiency, which depends jointly on the amino acid sequence, acyl binding motif, binding site, and linker length engineered in each peptide. However, for a given conjugated polypeptide, the reduction in potency (reduced pEC50 value) in the presence versus absence of HSA is a qualitative indication of albumin-peptide interaction (Table 8, Figures 2A and 2B). In contrast, linear polypeptides (pramlintide, hcalcitonin) were not affected by the presence of HSA (Table 8, Figures 2C and 2D), reflecting their low albumin binding efficiency.

[0618] [Table 9]

[0619] Example 8: Intravenous infusion of "linear" (i.e., non-acylated) amylin analog polypeptide: A pharmacokinetic study to assess renal (CL) clearance of linear amylin analog polypeptide Peptides were dissolved in sterile saline and administered to non-fasted male Sprague-Dawley rats (n=3 per group) as a 3-hour intravenous infusion via the femoral vein cannula at a final dose of 0.100 mg / kg. The formulation was administered at a rate of 1.67 mL / kg / hour. Blood samples (approximately 250 μL) were collected for pharmacokinetic analysis via the jugular vein cannula at 0.25, 0.5, 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5, and 6 hours after the start of the infusion (long method) or 1, 1.5, 2, 2.5, and 3 hours after the start of the infusion (steady-state screening method). All samples were collected in microtiter tubes containing K2EDTA as anticoagulant and 25 μL of protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 9, with some illustrative examples shown in Figures 3a-3b.

[0620] Example 9: Intravenous infusion of conjugated (i.e., acylated) amylin analog polypeptides: Pharmacokinetic studies to assess renal (CL) clearance of conjugated amylin analog polypeptides Peptides were dissolved in sterile saline and administered to non-fasted 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 for pharmacokinetic analysis via the jugular vein cannula into microtiter tubes containing K2EDTA as anticoagulant and 25 μL of protease inhibitor cocktail at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24, 30, and 48 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 9, with some illustrative examples shown in Figures 3a-3b.

[0621] [Table 10] TIFF0007636321000025.tif238170TIFF0007636321000026.tif127169

[0622] Example 10: Subcutaneous Infusion: Pharmacokinetic Study to Evaluate Renal (CL) Clearance of Amylin Analog Polypeptides Peptides were dissolved in sterile saline and administered to non-fasted male Sprague-Dawley rats (n=3 per group) as a 1-hour subcutaneous infusion via a cannula placed in the subcutaneous space between the shoulder blades at a final dose of 0.033 mg / kg. The formulation was administered at a rate of 0.145 mL / hr / kg / hr. Blood samples (approximately 250 μL) were collected for pharmacokinetic analysis via the jugular vein cannula into microtiter tubes containing K2EDTA as anticoagulant and 25 μL of protease inhibitor cocktail at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 24, 30 and 48 hours after the start of the infusion. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of the analysis are shown in Table 9.

[0623] Example 11: Subcutaneous bolus injection: Pharmacokinetic study to assess renal (CL) clearance of amylin analog polypeptides Peptides were dissolved in sterile saline and administered to non-fasted 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 0.3 mg / kg. Blood samples (approximately 250 μL) were collected for pharmacokinetic analysis via the jugular cannula into microtiter tubes containing K2EDTA as anticoagulant and 25 μL of protease inhibitor cocktail at 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 24, 30, and 48 hours post-dose. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 9.

[0624] Example 12: Methods for plasma sample preparation for pharmacokinetic studies Protein precipitation A 60 μL aliquot of each plasma sample was placed in a 96-well plate. To each well, 6 μL of 0.5% Tween-20 was added. The plate was then vortex mixed at 1200 rpm for 10 min, 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 1300 rpm for 5 min, then centrifuged at 2844×g for 10 min. The supernatant (180 μL) was placed in a clean 96-well plate and 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.

[0625] solid phase extraction A 60 mL aliquot of each plasma sample was diluted with 180 mL of 10 mM ammonium acetate (pH 6.8) containing the appropriate internal standard and loaded onto an Oasis WCX microElution plate (Waters Corporation, Milford, MA) pretreated with 200 mL of methanol and 200 mL of deionized water. Samples were washed with 200 mL of 5% ammonium hydroxide (aqueous), followed by 200 mL of 20% acetonitrile in water. Analytes were eluted with 200 mL of 5% formic acid in 75:25 acetonitrile:water. The eluate was dried under a stream of nitrogen. The residue was reconstituted in 80 μL of 20% acetonitrile (aqueous) containing 0.1% formic acid.

[0626] Example 13: LC / MS quantification of amylin analog polypeptides in plasma All calibration standards were prepared in control rat plasma containing K2EDTA and a protease inhibitor cocktail.

[0627] Samples and standards were analyzed by TurboIonSpray™ UPLC-MS / MS using a system consisting of a CTC HTS PAL autoinjector (LeapCarrboro, 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 4000QTrap mass spectrometer (Framingham, MA). Samples were injected onto a 2.1×50 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 using 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 component was increased to 95% B over 3-4 min depending on the peptide. Typical flow rate was 600 μL / min. Column temperature was kept constant at 40 or 50 °C. Peptides were quantified by monitoring one or more products generated from multiply charged parent ions.

[0628] Example 14: In vivo efficacy of amylin analog polypeptides with food intake inhibition in rats Short-term food intake was measured continuously for 72 hours using a BioDAQ food monitoring system (Research Diets, New Brunswick, NJ) to determine the amount of food intake inhibition exhibited by these amylin analog polypeptides. Long Evans rats were obtained at approximately 8 weeks of age. Rats were housed singly and acclimated to a 45% high fat diet for at least 2 weeks prior to dosing. After 1 week of acclimation, all rats were housed singly in BioDAQ cages (Research Diets, New Brunswick, NJ) and maintained on a 12-hour light / dark cycle (lights on 7:00 AM to 7:00 PM) at a constant temperature (approximately 22 °C) and 30-70% relative humidity. Rats were allowed free access to water and pelleted chow (Research Diets D12451i, 45 kcal% fat, Research Diets, New Brunswick, NJ). All procedures were performed in compliance with USDA regulations, Animal Welfare Act, and approved by Mispro's Institutional Animal Care and Use Committee. Animals were randomized into treatment groups according to body weight (n=8 rats / group). Animals were dosed (SC bolus injection) with either a specific concentration of amylin analog polypeptide or vehicle control (saline) between 6:00-6:30 AM before lights out with the hopper gated while the animals were receiving the dose. Immediately after dosing was completed, the hopper gate was opened and continuous data collection began. Data were analyzed using BioDAQ Viewer software (version 2.3.07) and, where necessary, time period filters were set to reduce noise in the data related to non-feeding behavior. All data are expressed as % inhibition from vehicle control and summarized as mean. Data were analyzed for statistical significance by two-sample t-test using Microsoft Excel (Redmond, WA). P values ​​<0.05 were considered to indicate significant differences between treatment groups. The % short-term food intake inhibition from the vehicle control results for amylin analog polypeptides are shown in Table 10.

[0629] [Table 11]

[0630] Example 15: In vivo efficacy with body weight change in LE DIO rats after 13 days A long-term (13-day) in vivo dose-response efficacy study was conducted in an obese rodent model (Long Evans (LE) diet-induced obese (DIO) rats) to investigate the efficacy and durability of an amylin analog polypeptide on weight loss. Male LE DIO rats were used (Envigo Laboratories, Inc., Indianapolis, IN) and rats were fed a high-fat chow diet (Teklad TD 95217, 40% kcal from fat, Harlan Laboratories, Madison, WI) during early weaning. Rats were 15-17 weeks of age at the start of the study. Rats were housed individually per cage, had free access to high-fat diet (Harlan TD.95217, 4.3 kcal / g) and water, and were maintained at 21°C and 50% relative humidity on a 12-h light-dark cycle, 5:00 AM to 5:00 PM, and were allowed to acclimate for at least 10 days prior to surgery. All procedures were performed in compliance with USDA regulations, Animal Welfare Act, and approved by the Mispro Institutional Animal Care and Use Committee. Body weight measurements were performed twice / week starting 3 days before surgery. Baseline fat and lean mass measurements were performed 3 days before the start of peptide infusion using a QMR instrument (Echo Medical Systems, Houston, TX). Rats were randomized into different treatment groups (n=4–6 rats / group) according to their fat mass and / or body weight percentage. One day before surgery, Alzet mini-osmotic pumps (2 weeks; model 2002, Durect Corporation, Cupertino, CA) were filled with either vehicle or peptide under sterile conditions. On the day of surgery, rats were anesthetized under isoflurane, and the dorsal skin surface was shaved and cleaned. Rats were injected SC with Flunexin (2.5 mg / kg). A 1–2 cm surgical incision was made between the scapulae. Blunt dissection was used to create a 2-3 cm subcutaneous tunnel into which a sterile prefilled mini-osmotic pump was introduced. The skin opening was closed with skin staples. Each rat was implanted with one or two osmotic pumps containing vehicle or peptide depending on the treatment group.Data was 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 (%) (ΔΔ) from baseline and vehicle control from the 13-day study is shown in Table 9.

[0631] Example 16: Weight loss effects of amylin analog polypeptide in combination with exenatide in LE DIO rats A long-term study was conducted to determine the effect and durability of continuous administration of amylin analog polypeptide in combination with exenatide (GLP-1 receptor agonist) on body weight after 27 days of treatment in LE DIO rats. Male LE DIO rats aged 18 weeks (14 weeks on high fat diet) were subcutaneously (SC) implanted with two (2) Alzet osmotic mini-pumps containing a specific dose of amylin analog polypeptide and / or exenatide (10mcg / kg / d=ED50 for body weight loss) or vehicle (20% DMSO in water) (n=8 animals / treatment group). Amylin analog polypeptide, whose PK supports alternate day dosing (eod), was administered by SC injection eod instead of mini-pump administration. All other procedures were the same as those described in the previous examples. The results of the mean body weight loss (%) (ΔΔ) from baseline and vehicle control from the long-term combination study with exenatide are shown in Table 11.

[0632] Example 17: Antidiabetic efficacy of amylin analog polypeptides in combination with exenatide in ZDF rats A long-term study was conducted to determine the antidiabetic effect of continuous administration of amylin analog polypeptide in combination with exenatide on HbA1c (the main antidiabetic parameter) after 27 days of treatment in Zucker Diabetic Fatty (ZDF) rats. Male ZDF rats were obtained (Charles River, Raleigh, NC) at six (6) weeks of age and used in the study at eight (8) weeks of age. Upon receipt, rats were housed one animal per cage, given free access to Purina 5008 chow (Lab Diet, St. Louis, MO), maintained at 21°C and 50% relative humidity with a 12-h light-dark cycle, 5:00 AM to 5:00 PM, and allowed to acclimate for nine (9) days before the start of the study. Blood samples were taken as pre-bleed (day 3) from the tail vein to measure glucose levels and HbA1c. ZDF rats were randomized into treatment groups with similar mean HbA1c and glucose (n=10 / group) and implanted subcutaneously (SC) with Alzet osmotic minipumps (2 pumps / animal) containing a specific dose of amylin analog polypeptide and / or either exenatide (10mcg / kg / d) or vehicle (20% DMSO in water) (n=10 animals / treatment group). Amylin analog polypeptide, whose PK supports alternate day dosing (eod), was administered by SC injection eod instead of minipump administration. All other procedures were the same as described in the previous examples. Blood samples were taken again on days 14 and 27 (end of study) to measure glucose levels 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 manufacturer's described protocol and method parameters. HbA1c results, expressed as mean % change (ΔΔ) from baseline and vehicle control, from the long-term combination study with exenatide are shown in Table 11.

[0633] Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be 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.

[0634] [Table 12]

[0635] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be 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. SEQ ID NO:209:X １ CNTSTCATX １０ RLANX １５ X １６ X １７ KSSNNFGPILPPTKVGSETY-(OH / NH ２ ) (SEQ ID NO: 209), (wherein X １ is K or k, X １０ is Q or S; X １５ is E or F; X １６ is L, K, or k; X １７ is H, V, or Q; each K independently represents an L-lysine that is covalently attached to a lipophilic substituent or that is not covalently attached to a lipophilic substituent; each k independently represents a D-lysine that is covalently attached to a lipophilic substituent or that is not covalently attached to a lipophilic substituent; Here, X １ CNTSTC (SEQ ID NO: 318), or a pharma- ceutically acceptable salt thereof.

2. 2. The isolated polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 55, 64, 65, 109, 112-120, 126, 130, 131 and 143, or a pharma- ceutically acceptable salt thereof.

3. K((γGlu) ２ (CO(CH ２ ) １６ CO ２ H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH ２ ) (SEQ ID NO:65), The isolated polypeptide of claim 2, or a pharma- ceutically acceptable salt thereof, wherein the two C*s are linked by a disulfide bridge.

4. K((γGlu) ２ (CO(CH ２ ) １８ CO ２ H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH ２ ) (SEQ ID NO:64), The isolated polypeptide of claim 2, or a pharma- ceutically acceptable salt thereof, wherein the two C*s are linked by a disulfide bridge.

5. The isolated polypeptide of claim 1, or a pharma- ceutically acceptable salt thereof, comprising a lipophilic substituent covalently attached to L-lysine or D-lysine at X1 or X16.

6. The lipophilic substituent is Formula VI: -(Y1) ｎ１ -(V) ｒ -(Y2) ｎ２ -CO-(CH ２ ) ｍ -Z (Formula VI) or Formula III: - (γGlu) ｎ -CO-(CH ２ ) ｍ -Z (Formula III) ('(γGlu) ｎ " is disclosed as SEQ ID NO: 311) (In the formula, Z is -CH ３ Or -CO ２ H; 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 -[COCH ２ (O(CH ２ ) ２ ) ｔ OCH ２ NH]-, and t is 1 to 8; r is 1 to 8; n is 1 to 10; n1 is 0 to 10; n2 is 0 to 10.

6. The isolated polypeptide of claim 5 having the structure:

7. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof.

8. An osmotic delivery device comprising the isolated polypeptide of any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of claim 7.

9. A composition for use in a method for treating obesity in a human subject, inducing weight loss in the human subject, or suppressing appetite in the human subject, comprising an isolated polypeptide described in any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof, a pharmaceutical composition described in claim 7, or an osmotic device described in claim 8.

10. A composition for use in a method for treating type 1 or type 2 diabetes in a human subject, the composition comprising an isolated polypeptide described in any one of claims 1 to 6 or a pharma- ceutical acceptable salt thereof, a pharmaceutical composition described in claim 7, or an osmotic device described in claim 8.

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