Human amylin analogue polypeptides and methods of use

Abstract

The present invention relates to isolated polypeptides as human amylin analogs. The disclosed amylin analogue polypeptides have advantageous physicochemical properties relative to endogenous amylin, such as longer elimination half-life (t1 / 2) and improved solubility and thermal stability. The invention also relates to methods of using amylin analogue polypeptides disclosed herein in a variety of therapeutic indications, and methods of making the same. The disclosed amylin analogue polypeptides are particularly useful in methods of treating metabolic diseases or disorders such as type 1 and type 2 diabetes mellitus and providing weight loss.

Description

[0001] This application is a divisional application of the invention patent application with the application date of October 10, 2019, application number 201980081630.7, and invention name “Human Amylin Analog Polypeptide and Method of Use”.

[0002] 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.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 11, 2019, is named 616782_102487-055PC_SL-10-10-2019.txt and is 123,647 bytes in size. Technical Field

[0005] The present invention relates to isolated polypeptides that are analogs of human amylin. The disclosed amylin analog polypeptides have advantageous physicochemical properties relative to endogenous amylin, such as a longer elimination half-life (t 1 / 2 ) and improved solubility and thermal stability. The present invention also relates to methods of using the amylin analog polypeptides disclosed herein in a variety of therapeutic indications, as well as methods of making the same. As explained in more detail below, the disclosed amylin analog polypeptides are particularly useful in treating metabolic diseases or conditions such as type 1 and type 2 diabetes and in providing methods of weight loss. Background Art

[0006] Human amylin, or islet amyloid polypeptide (IAPP), is a 37-residue polypeptide hormone. Amylin is co-secreted from pancreatic beta cells along with insulin at a ratio of approximately 100:1 (insulin:amylin). Pro-islet amyloid polypeptide (i.e., pro-IAPP) is produced in pancreatic beta cells as a 67 amino acid, 7404 dalton pro-peptide that undergoes post-translational modifications (including protease cleavage) to produce the 37-residue amylin. Loss of beta cell function, which occurs in patients with type 1 diabetes early in life and may occur in patients with type 2 diabetes late in life, leads to inadequate secretion of both insulin and amylin.

[0007] Amylin is part of the endocrine pancreas (the cells within the pancreas that synthesize and secrete hormones). Amylin contributes to blood sugar control; it is secreted from the pancreatic islets into the blood circulation and cleared by peptidases in the kidneys. Amylin's metabolic function is well characterized as an inhibitor of the appearance of nutrients such as glucose in the plasma. It thus acts as a co-partner to insulin, a peptide that regulates blood sugar levels and coordinates the body's distribution and uptake of glucose. Insulin's role in the body is, among other things, to prevent blood sugar levels from rising too high, especially after a meal.

[0008] Amylin is thought to play a role in blood sugar regulation by slowing gastric emptying and promoting satiety (i.e., a feeling of fullness), thereby preventing a postprandial (i.e., after a meal) spike in blood sugar levels. The overall effect is to slow the rate at which glucose appears in the blood after a meal. 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 falling too low. This is important because, for example, some people with type 1 diabetes tend to secrete excess amounts of glucagon, which raises blood sugar levels, immediately after a meal.

[0009] For a number of reasons, human amylin, which has a half-life of approximately 13 minutes in serum, is not suitable for use as a therapeutic agent. Instead, pramlintide ( Pramlintide (developed by Amylin Pharmaceuticals, Inc., San Diego, CA, USA, and sold by AstraZeneca plc, Cambridge, UK) is a synthetic analog of human amylin for the treatment of patients with type 1 or type 2 diabetes who, despite optimal insulin therapy, are unable to achieve desired glycemic control using mealtime insulin. Pramlintide differs from human amylin in three of its 37 amino acids. These modifications provide pramlintide with a longer half-life of approximately 48 minutes in humans and reduce its tendency to aggregate, a characteristic found in human amylin.

[0010] For the treatment of patients with type 1 diabetes, pramlintide is administered up to four times daily via subcutaneous injection before meals 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 may include severe hypoglycemia, particularly in patients with type 1 diabetes. Therefore, for patients starting pramlintide, the mealtime insulin dose is reduced.

[0011] For the treatment of patients with type 2 diabetes, pramlintide is administered via subcutaneous injection before each meal at a recommended starting dose that is gradually increased to the target maintenance dose. The other investigational analog of human amylin, davalintide (AC2307; also being developed by Amylin Pharmaceuticals, Inc.), has a half-life of approximately 26 minutes.

[0012] Therefore, there is a need for improved amylin analog polypeptides that mimic the activity of amylin but have greater therapeutic potential than endogenous human amylin and existing amylin analogs such as pramlintide and davalintide. Summary of the Invention

[0013] It has now been found that the polypeptides of the present invention and pharmaceutically acceptable compositions thereof are effective as amylin analogs. These polypeptides have the general formula of SEQ ID NO: 199:

[0014] 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) or a pharmaceutically acceptable salt thereof, wherein:

[0015] 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 N or E; X 29 is P, R or K; X31 is k, K, N, or H; X 35 is e, E, N, K, G, A, Y, or P; and X 37 is Y or P;

[0016] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0017] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0018] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[0019] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a table illustrating 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.

[0021] Figure 2A 、 Figure 2B 、 Figure 2C and Figure 2D Acylated amylin analog A73 ( Figure 2A ), acylated amylin analog A73 under hCTR ( Figure 2B ), Pramlintide ( Figure 2C ) and human calcitonin (hCalcitonin) under hCTR ( Figure 2D As used herein, the term "acylated" with respect to the disclosed polypeptides means that the disclosed polypeptides are each optionally substituted with one or more lipophilic substituents via a spacer, wherein "lipophilic substituent" and "spacer" are defined herein.

[0022] Figure 3A and Figure 3B Describe the evaluation of intravenous infusion of linear or non-acylated peptides ( Figure 3A) and conjugated or acylated polypeptides ( Figure 3B ) Pharmacokinetic study data on the renal clearance (CL) of the peptide after administration. DETAILED DESCRIPTION

[0023] 1. General Description of Certain Embodiments of the Invention

[0024] The present invention relates to isolated polypeptides that are amylin analogs and pharmaceutical compositions comprising these polypeptides. The present invention also relates to methods of making and using these amylin analog polypeptides. These amylin analog polypeptides are particularly useful in treating metabolic diseases or conditions such as type 1 and type 2 diabetes, obesity, and in providing methods of weight loss.

[0025] 2. Definition

[0026] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural indicators. Thus, for example, reference to a "solvent" includes a combination of two or more such solvents, reference to a "peptide" includes one or more peptides or a mixture of peptides, reference to a "drug" includes one or more drugs, reference to an "osmotic delivery device" includes one or more osmotic delivery devices, and so on. Unless explicitly stated or obvious from the context, the term "or" as used herein should be understood to be inclusive and encompasses both "or" and "and".

[0027] Unless expressly stated or obvious from the context, the term "about" as used herein should be understood to mean within the general tolerance range in the art, for example, within 2 standard deviations of the mean. Approximately can be understood to mean 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 the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0028] Unless expressly stated or obvious from the context, as used herein, the term "substantially" is to be understood as within a narrow range of variation or otherwise within normal tolerance in the art. Substantially can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001% of the stated value.

[0029] 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 the invention belongs. Although other methods and materials similar or equivalent to those described herein can be used to practice the present invention, preferred materials and methods are described herein.

[0030] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0031] 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, for example, a hormone such as an androgen or an estrogen. The devices and methods of the present invention are well suited for the delivery of proteins, small molecules, and combinations thereof.

[0032] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and generally refer to molecules comprising a chain of two or more amino acids (e.g., most commonly L-amino acids, and also including, for example, D-amino acids, modified amino acids, amino acid analogs, and amino acid mimetics).

[0033] In some embodiments, the naturally occurring L-amino acids are represented by the conventional three-letter or uppercase single-letter amino acid names of Table 1. In other embodiments, the naturally occurring L-amino acids and D-amino acids are both represented by the conventional three-letter or uppercase single-letter amino acid names of Table 1. In other embodiments, the D-amino acids are represented by the lowercase single-letter amino acid names corresponding to the single-letter names of 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.

[0034] Table 1: Naturally occurring amino acids

[0035]

[0036] Peptides can be naturally occurring, synthetically produced, or recombinantly expressed. Peptides can also contain additional groups that alter the amino acid chain, such as functional groups added via post-translational modification. Examples of post-translational modifications include, but are not limited to, acetylation, alkylation (including methylation), biotinylation, glutamylation, glycation, glycosylation, prenylation, lipidation, phosphopantetheinylation, phosphorylation, selenoylation, and C-terminal amidation. The term peptide also includes peptides containing modifications at the amino and / or carboxyl termini. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxyl group include, but are not limited to, amide, lower alkylamide, dialkylamide, and lower alkyl ester modifications (e.g., where the lower alkyl group is C1-C4 alkyl). The term peptide also includes modifications, such as, but not limited to, those described above, of the amino acids between the amino and carboxyl termini. In one embodiment, the peptide can be modified by the addition of a small molecule drug.

[0037] The terminal amino acid at one end of the 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 carboxyl terminus). Typically, the amino acids that make up a peptide are numbered sequentially, starting from the amino terminus and increasing toward the carboxyl terminus of the peptide.

[0038] As used herein, the phrase "amino acid residue" refers to an amino acid that is incorporated into a peptide via an amide bond or an amide bond mimetic.

[0039] As used herein, the term "insulinotropic" generally 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 hormone). Such compounds generally stimulate or otherwise affect the secretion or biosynthesis of insulin in a subject. Thus, an "insulinotropic peptide" is an amino acid-containing molecule that is capable of stimulating or otherwise affecting the secretion or biosynthesis of insulin.

[0040] 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, exenatide having the amino acid sequence of SEQ ID NO: 307, and its derivatives and analogs.

[0041] As used herein, the term "acylated" with respect to the disclosed polypeptides means that the disclosed polypeptides are each optionally substituted with one or more lipophilic substituents via a spacer, wherein "lipophilic substituent" and "spacer" are defined herein. Certain lipophilic substituents, each optionally via a spacer, can bind to albumin and impart an affinity for the resulting acylated polypeptide. The extent to which the lipophilic substituents, each optionally via a spacer, bind to albumin and impart an affinity for the resulting acylated polypeptide is variable and depends on numerous factors, including the identity of the lipophilic substituent, the optional spacer, the polypeptide, and the site of covalent attachment to the polypeptide.

[0042] As used herein, the term "linear" or "linear polypeptide" refers to a "non-acylated" polypeptide, in other words, a disclosed amylin analog polypeptide that does not each have a lipophilic substituent, optionally via a spacer, wherein "lipophilic substituent" and "spacer" are defined herein.

[0043] As used herein, the term "conjugated" or "conjugated polypeptide" refers to an "acylated" polypeptide, in other words, a disclosed amylin analog polypeptide, each having one or more lipophilic substituents, optionally via a spacer, wherein "lipophilic substituent" and "spacer" are defined herein.

[0044] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. 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 are 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 amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with 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. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, 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, valerate, and the like.

[0045] Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C1-4 alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts and the like. Other pharmaceutically acceptable salts include (where appropriate) non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions (e.g., halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate).

[0046] As used herein, the phrase "incretin mimetics" 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. Examples of preferred incretin mimetics include exenatide, exenatide having the amino acid sequence of exendin-4 (a 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 referred to in the literature as "GLP-1 receptor agonists" or "GLP-1 agonists," where both terms are used interchangeably herein.

[0047] The term "exenatide" as used herein includes, but is not limited to, exenatide, exenatide having the amino acid sequence of (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2), SEQ ID NO: 307, natural incretin analog-4, exenatide peptide, exenatide peptide analogs, and exenatide peptide derivatives.

[0048] The term "GLP-1" refers to a polypeptide produced by L cells located primarily in the ileum and colon, and to a lesser extent in the duodenum and jejunum. GLP-1 is a regulatory peptide that binds to the extracellular region of the GLP-1 receptor (GLP-1R), a G-coupled protein receptor on beta cells, and stimulates the insulin response to nutrients absorbed from the intestine via adenylate cyclase activity and cAMP production [Baggio 2007, "Biology of incretins: GLP-1 and GIP", Gastroenterology, Vol. 132(6): 2131-57; Holst 2008, "The incretin system and its role in type 2 diabetes mellitus", Mol Cell Endocrinology, Vol. 297(1-2): 127-36]. The effects of GLP-1R agonism are multiple. GLP-1 maintains glucose homeostasis by enhancing endogenous glucose-dependent insulin secretion, rendering beta cells glucose competent and sensitive to GLP-1, inhibiting glucagon release, restoring first and second phase insulin secretion, slowing gastric emptying, reducing food intake, and improving 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, 52(2): 380-86; Holst 2013 “Incretin hormones and the satiation signal”, Int J Obes (Lond), 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 mode of action of GLP-1, the risk of hypoglycemia is minimal.

[0049] As described in more detail below, in some embodiments, the amylin analog polypeptides disclosed herein are provided as an adjunct to insulin therapy in methods for treating type 1 diabetes. 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:

[0050] Table 2: Exemplary insulin analogs

[0051]

[0052]

[0053]

[0054] As used herein, the term "mealtime insulin" refers to a rapid-acting insulin formulation that reaches peak blood concentrations within about 45-90 minutes after administration and peak activity within about 1 to 3 hours, and is administered at or around mealtime.

[0055] As used herein, the term "vehicle" refers to a medium for carrying a compound, such as a drug or particles containing a drug. The vehicles of the present invention typically include components such as a polymer and a solvent. The suspension vehicles of the present invention typically include a solvent and a polymer for preparing a suspension formulation that also includes a drug particle formulation.

[0056] 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, for example, when the suspension vehicle contacts an aqueous environment. In some embodiments of the present invention, the suspension vehicle is formulated to exhibit phase separation upon contact with an aqueous environment having less than about 10% water.

[0057] As used herein, the phrase "single phase" refers to a solid, semisolid, or liquid homogeneous system that is physically and chemically uniform throughout.

[0058] As used herein, the term "disperse" refers to dissolving, dispersing, suspending, or otherwise distributing a compound, such as a drug particle formulation, in a suspension vehicle.

[0059] As used herein, the phrase "chemically stable" refers to formulations that produce an acceptable percentage of degradation products over a defined period of time by chemical pathways such as deamidation (usually by hydrolysis), aggregation, or oxidation.

[0060] As used herein, the phrase "physically stable" refers to a formulation that forms an acceptable percentage of aggregates (e.g., dimers and other higher molecular weight products). Additionally, a physically stable formulation does not change its physical state, e.g., from liquid to solid or from amorphous to crystalline form.

[0061] The term "viscosity" as used herein generally refers to a value determined from the ratio of shear stress to shear rate (see, e.g., Considine, DM and Considine, GD, Encyclopedia of Chemistry, 4th ed., Van Nostrand, Reinhold, NY, 1984), essentially as follows:

[0062] F / A = μ*V / L (Equation 1)

[0063] Where F / A = shear stress (force per unit area),

[0064] μ = proportionality constant (viscosity), and

[0065] V / L = velocity per layer thickness (shear rate).

[0066] In some embodiments, the viscosity of the suspension medium of the present invention is determined by the ratio of shear stress to shear rate. The measurement of shear stress and shear rate is usually determined using the parallel plate rheology carried out under selected conditions (such as a temperature of about 37°C). Other methods for determining viscosity include using a viscometer such as a Cannon-Fenske viscometer, a Ubbelohde viscometer (Ubbelohdeviscometer) (for Cannon-Fenske opaque solutions) or an Ostwald viscometer to measure kinematic viscosity. In general, the suspension medium of the present invention has the viscosity that is enough to prevent the particle preparation suspended therein from sedimenting during storage and in a delivery method such as in an implantable drug delivery device.

[0067] As used herein, the term "non-aqueous" refers to, for example, a total moisture content of a suspension formulation that is typically less than or equal to about 10 wt %, such as less than or equal to about 7 wt %, less than or equal to about 5 wt % and / or less than about 4 wt %. In addition, the particle formulations of the present invention contain less than about 10 wt %, such as less than about 5 wt % residual moisture.

[0068] As used herein, the term "subject" refers to any member of the subphylum Chordata, including but not limited to humans and other primates, including non-human primates such as rhesus macaques and other monkey species and chimpanzees and other ape species; livestock such as cattle, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds including domesticated, wild, and game birds such as chickens, turkeys, and other quail, ducks, geese, and the like. The term does not indicate a specific age or sex. Thus, it is intended to encompass both adult and newborn individuals.

[0069] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or condition as described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have appeared. In other embodiments, treatment may be administered when symptoms are not present. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0070] The term "osmotic delivery device" as used herein generally refers to a device for delivering a drug (e.g., a disclosed amylin analog polypeptide) to a subject, wherein the device includes, for example, a reservoir (e.g., made of a titanium alloy) having an inner cavity containing a suspension formulation comprising a drug (e.g., a disclosed amylin analog polypeptide) and an osmotic agent formulation. A piston assembly disposed in the inner cavity separates the suspension formulation from the osmotic agent formulation. A semipermeable membrane is disposed at a first distal end of the reservoir adjacent to the osmotic agent formulation, and a diffusion regulator (which defines a delivery orifice through which the suspension formulation exits the device) is disposed at a second distal end of the reservoir adjacent to the suspension formulation. Typically, the osmotic delivery device is implanted within a subject, e.g., subdermally or subcutaneously (e.g., in the inner, outer, or posterior portion of the upper arm and in the abdominal region). An exemplary osmotic delivery device is (ALZA Corporation, Mountain View, Calif.) delivery device. Examples of synonymous terms 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.

[0071] As used herein, the term "continuous delivery" generally refers to the substantially continuous release of a drug from an osmotic delivery device and entry into tissues near the implant site, such as the subdermal and subcutaneous tissues. For example, an osmotic delivery device releases a drug substantially at a predetermined rate based on the principle of osmosis. Extracellular fluid enters the osmotic delivery device through a semipermeable membrane and directly into an osmotic engine, which expands to drive a piston at a slow and consistent rate of travel. The movement of the piston causes the drug formulation to be released through the orifice of a diffusion regulator. Therefore, the release of the drug from the osmotic delivery device is at a slow, controlled, consistent rate.

[0072] As used herein, the term "substantially steady-state delivery" generally refers to the delivery of a drug at or near a target concentration over a defined period of time, wherein the amount of drug delivered from an osmotic delivery device is substantially zero-order. Substantially zero-order delivery of an active agent (e.g., a disclosed amylin analog polypeptide) means that the rate of drug delivery is constant and independent of the drug available in the delivery system; for example, for zero-order delivery, if the drug delivery rate is plotted against time and a line is fit to the data, the line has a slope of about zero, as determined by standard methods (e.g., linear regression).

[0073] As used herein, the phrase "drug half-life" refers to the time it takes for a drug to eliminate half of its concentration from the plasma. When a drug is administered via injection or intravenously, the half-life of a drug is typically measured by monitoring how the drug degrades. Drugs are typically detected using, for example, radioimmunoassays (RIA), chromatographic methods, electrochemiluminescence (ECL) assays, enzyme-linked immunosorbent assays (ELISAs), or immunoenzymatic sandwich assays (IEMAs).

[0074] The terms "μg" and "mcg" and "ug" are to be understood to mean "microgram." Similarly, the terms "μl" and "uL" are to be understood to mean "microliter," and the terms "μM" and "uM" are to be understood to mean "micromolar."

[0075] The term "serum" is intended to refer to any 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 a subject's serum" would encompass "the amount of [substance] in a subject's plasma."

[0076] Baseline was defined as the last assessment on the day of initial placement of the osmotic delivery device (containing drug or placebo) or the day before.

[0077] 3. Endogenous amylin, related peptides, and amylin receptors

[0078] 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 late in type 2 diabetes, leads to insufficient secretion of both insulin and amylin. Amylin is thought to play a role in blood glucose regulation by slowing gastric emptying and promoting satiety, thereby preventing post-meal spikes in blood glucose levels. The overall effect is to slow the rate at which glucose appears in the blood after a meal.

[0079] The amino acid sequence of amylin is most closely related to that of calcitonin gene-related peptide (CGRP). CGRP also shares similarly positioned disulfide bonds and an amidated C-terminus. This is also true for calcitonin, adrenomedullin, and adrenomedullin 2. Collectively, these peptides form a small family united by these characteristic features. Consequently, there is some overlap in the cognate receptors bound by each peptide and in their pharmacological activities. Figure 1 The table illustrates a comparative sequence alignment of amylin and certain related reference polypeptides.

[0080] Peptides commonly referred to as members of the calcitonin (CT) peptide family include calcitonin gene-related peptide (CGRP), calcitonin (CT), amylin (AMY), adrenomedullin 1, and adrenomedullin 2 / mesotropin (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 the pharmacologically distinct receptors of the entire peptide family (CTR, AMY1, AMY2, AMY3, CGRPR, AM1, AM2). There appear to be at least five different receptors (AMY1, AMY2, AMY3, CTR, CGRPR) to which amylin binds with significant affinity. CTR dimerizes with RAMP 1, 2, or 3 to reconstitute AMY1, AMY2, or AMY3 receptors that are pharmacologically selective for amylin relative to calcitonin. In the absence of RAMPs, CTR pharmacology becomes selective for calcitonin relative to amylin. Dimerization of CALCRL with RAMP1 generates 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.

[0081] Amylin analog polypeptides with binding affinity for the amylin receptor complex have been developed. For example, pramlintide was developed by Amylin Pharmaceuticals and approved by the U.S. Food and Drug Administration (FDA) as a synthetic analog of human amylin for treating patients with type 1 and type 2 diabetes who use mealtime insulin but, despite optimal insulin therapy, are unable to achieve desired glycemic control. Pramlintide is an amylin mimetic that is at least as effective as human amylin. It is also a 37-amino acid polypeptide and differs from the amino acid sequence of human amylin in the amino acid substitution of proline at positions 25 (alanine), 28 (serine), and 29 (serine). Due to these substitutions, pramlintide is soluble, non-adhesive, and non-aggregating, thereby overcoming the various physicochemical tendencies of natural human amylin. The half-life of pramlintide in humans is approximately 48 minutes, which is longer than natural human amylin (approximately 13 minutes). Pramlintide requires frequent and inconvenient administration.

[0082] For the treatment of patients with type 1 diabetes, pramlintide is administered up to four times daily via subcutaneous injection in the thigh or abdomen before meals as an adjunct to insulin therapy administered after meals. Pramlintide cannot be mixed with insulin; a separate syringe is used. Pramlintide is administered with or before each meal or snack consisting of at least 250 calories or 30 grams of carbohydrates. The typical starting dose for patients with type 1 diabetes is 15 μg of subcutaneous pramlintide before each meal, subsequently titrated to a target dose of 60 μg before each meal. Reported side effects of pramlintide include nausea and vomiting. Particularly for patients with type 1 diabetes, adverse reactions can include severe hypoglycemia. Therefore, for diabetic patients starting pramlintide, the dose of mealtime insulin is reduced.

[0083] For the treatment of patients with type 2 diabetes, pramlintide is administered via subcutaneous injection at a recommended starting dose of 60 μg, with a target maintenance dose of 120 μg before each meal.

[0084] Davalintide (AC2307) is another analog of human amylin. Davalintide is an investigational compound with a half-life of approximately 26 minutes. Similar to pramlintide, davalintide will similarly require frequent administration via injection.

[0085] Certain disclosed amylin analog polypeptides, including those in Table 3 below, exhibit one or more of the following: superior solubility, stability, biological activity, and specificity, and a longer half-life compared to endogenous human amylin and known synthetic amylin analog polypeptides. Certain disclosed amylin analog polypeptides are developed to accommodate less frequent administration than required for pramlintide. Certain disclosed amylin analog polypeptides are developed for administration via weekly or monthly injections. Certain disclosed amylin analog polypeptides are developed for administration via implantation of a delivery device comprising the amylin analog polypeptide, wherein the delivery device comprises a dosage of the amylin analog polypeptide for up to 3 months, 6 months, 9 months, one year, 18 months, or two years.

[0086] 4. Description of Exemplary Embodiments

[0087] In certain embodiments, the present invention relates to isolated polypeptides that are amylin analogs.

[0088] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 199:

[0089] 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) or a pharmaceutically acceptable salt thereof, wherein:

[0090] X1 is S, K, k, H or I;

[0091] X3 is N or S;

[0092] X5 is S or A;

[0093] X6 is T or S;

[0094] X8 is A or K;

[0095] X 10 It is Q or S;

[0096] X 12It is L or K;

[0097] X 13 is A, S, E or K;

[0098] X 14 is N, n, d, Y, or Q;

[0099] X 15 is E, F, f, Y, I, k, K, or α-aminoisobutyric acid (Aib);

[0100] X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l;

[0101] X 17 is H, V, Q, R, k, K, or Aib;

[0102] X 18 is K, H or R;

[0103] X 19 It is S or Aib;

[0104] X 20 It is S or Aib;

[0105] X 22 is N or E;

[0106] X 29 is P, R or K;

[0107] X 31 is k, K, N, or H;

[0108] X 35 is e, E, N, K, G, A, Y, or P; and

[0109] X 37 is Y or P;

[0110] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0111] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0112] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[0113] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[0114] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0115] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0116] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 199 include the following:

[0117] In some embodiments, the carboxyl-terminal X 37 In some embodiments, the carboxyl terminal X 37 In some embodiments, the carboxyl terminal X 37 In some embodiments, the carboxyl terminal X 37 It is P-(OH).

[0118] 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.

[0119] As used herein, k refers to D-lysine.

[0120] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0121] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0122] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0123] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0124] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0125] In some embodiments, X 12 is L. In some embodiments, X 12 It’s K.

[0126] In some embodiments, X 13 is A. In some embodiments, X 13 is S. In some embodiments, X 13 is E. In some embodiments, X13 It’s K.

[0127] In some embodiments, X 14 Is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X 14 Is Y. In some embodiments, X 14 It's Q.

[0128] As used herein, n refers to D-asparagine.

[0129] As used herein, d refers to D-aspartic acid.

[0130] 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 1. In some embodiments, X 15 Is K. In some embodiments, X 15 is k. In some embodiments, X 15 It's Aib.

[0131] As used herein, f refers to D-phenylalanine.

[0132] As used herein, Aib refers alternatively to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine.

[0133] In some embodiments, X 16 is L. In some embodiments, X 16 Is 1. In some embodiments, X 16 Is K. In some embodiments, X 16 is k. In some embodiments, X 16 is Aib. In some embodiments, X 16 It's N-MeL.

[0134] As used herein, 1 refers to D-leucine.

[0135] As used herein, N-MeL refers to N-methylleucine.

[0136] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 is Q. In some embodiments, X17 is R. In some embodiments, X 17 Is K. In some embodiments, X 17 is k. In some embodiments, X 17 It's Aib.

[0137] In some embodiments, X 18 Is K. In some embodiments, X 18 is H. In some embodiments, X 18 It’s R.

[0138] In some embodiments, X 19 is S. In some embodiments, X 19 It's Aib.

[0139] In some embodiments, X 20 is S. In some embodiments, X 20 It's Aib.

[0140] In some embodiments, X 22 Is N. In some embodiments, X 22 It’s E.

[0141] In some embodiments, X 29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 It’s K.

[0142] In some embodiments, X 31 is k. In some embodiments, X 31 Is K. In some embodiments, X 31 Is N. In some embodiments, X 31 It’s H.

[0143] In some embodiments, X 35 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 35 is A. In some embodiments, X 35 Is Y. In some embodiments, X 35 It’s P.

[0144] As used herein, e refers to D-glutamic acid.

[0145] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 199 include the following:

[0146] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X5 is S. 10 Is Q. In some embodiments, X1 is S and X 15 Is E. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 18 Is K. In some embodiments, X1 is S and X 31 Is K. In some embodiments, X1 is S and X 35 Is E. In some embodiments, X1 is S and X 37 It’s Y.

[0147] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X5 is S. 10 Is Q. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 18 Is K. In some embodiments, X1 is K and X 31 Is K. In some embodiments, X1 is K and X 35 Is E. In some embodiments, X1 is K and X 37 It’s Y.

[0148] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X5 is S. 10 Is Q. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 18 Is K. In some embodiments, X1 is k and X 31 Is K. In some embodiments, X1 is k and X35 Is E. In some embodiments, X1 is k and X 37 It’s Y.

[0149] 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, X5 is S and X 17 Is H. In some embodiments, X5 is S and X 18 Is K. In some embodiments, X5 is S and X 31 Is K. In some embodiments, X5 is S and X 35 Is E. In some embodiments, X5 is S and X 37 It’s Y.

[0150] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 18 Is K. In some embodiments, X 10 It is Q and X 31 Is K. In some embodiments, X 10 It is Q and X 35 is E. In some embodiments, X 10 It is Q and X 37 It’s Y.

[0151] 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 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, X15 is E and X 35 is E. In some embodiments, X 15 is E and X 37 It’s Y.

[0152] 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. In some embodiments, X 16 Is L and X 37 It’s Y.

[0153] 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 It’s Y.

[0154] 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 It’s Y.

[0155] 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 It’s Y.

[0156] In some embodiments, X 31 is K and X 35is E. In some embodiments, X 31 is K and X 37 It’s Y.

[0157] In some embodiments, X 35 is E and X 37 It’s Y.

[0158] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 199 include the following:

[0159] In some embodiments, X1 is S, X5 is S, and X 15 Is E. In some embodiments, X1 is S, X5 is S, and X 16 is L. In some embodiments, X1 is S, X5 is S, and X 16 In some embodiments, X1 is S, X5 is S, and X 17 Is H. In some embodiments, X1 is S, X5 is S, and X 18 Is K. In some embodiments, X1 is S, X5 is S, and X 31 Is K. In some embodiments, X1 is S and X 35 Is E. In some embodiments, X1 is S, X5 is S, and X 37 It’s Y.

[0160] In some embodiments, X1 is K, X5 is S, and X 15 Is E. In some embodiments, X1 is K, X5 is S, and X 16 is L. In some embodiments, X1 is K, X5 is S, and X 16 In some embodiments, X1 is K, X5 is S, and X 17 Is H. In some embodiments, X1 is K, X5 is S, and X 18 Is K. In some embodiments, X1 is K, X5 is S, and X 31 Is K. In some embodiments, X1 is K, X5 is S, and X 35 Is E. In some embodiments, X1 is K, X5 is S, and X 37 It’s Y.

[0161] In some embodiments, X1 is k, X5 is S, and X 15 Is E. In some embodiments, X1 is k, X5 is S, and X 16 is L. In some embodiments, X1 is k, X5 is S, and X 16 In some embodiments, X1 is k, X5 is S, and X17 Is H. In some embodiments, X1 is k, X5 is S, and X 18 Is K. In some embodiments, X1 is k, X5 is S, and X 31 Is K. In some embodiments, X1 is k, X5 is S, and X 35 Is E. In some embodiments, X1 is k, X5 is S, and X 37 It’s Y.

[0162] In some embodiments, X5 is S, X 15 is E, and X 16 is L. In some embodiments, X5 is S, X 15 is E, and X 16 In some embodiments, X5 is S, X 15 is E, and X 17 Is H. In some embodiments, X5 is S, X 15 is E, and X 18 In some embodiments, X5 is S, X 15 is E, and X 31 In some embodiments, X5 is S, X 15 is E, and X 35 Is E. In some embodiments, X5 is S, X 15 is E, and X 37 It’s Y.

[0163] In some embodiments, X 10 It's Q, 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 It's Q, X 18 is K, and X 31 Is K. In some embodiments, X 10 It's Q, X 31 is K and X 35 is E. In some embodiments, X 10 It's Q, X 31 is K and X 37 It’s Y.

[0164] In some embodiments, X 15 It's E, X 16 is L, and X 17 is H. In some embodiments, X 15 It's E, X16 is L, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 35 is E. In some embodiments, X 15 It's E, X 16 is L, and X 37 It’s Y.

[0165] In some embodiments, X 15 It's E, X 16 is k, and X 17 is H. In some embodiments, X 15 It's E, X 16 is k, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 35 is E. In some embodiments, X 15 It's E, X 16 is k, and X 37 It’s Y.

[0166] In some embodiments, X 16 It's L, X 17 is H, and X 18 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 31 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 35 is E. In some embodiments, X 16 It's L, X 17 is H, and X 37 It’s Y.

[0167] In some embodiments, X 16 is k, X 17 is H, and X 18 Is K. In some embodiments, X 16 is k, X 17 is H, and X 31 Is K. In some embodiments, X 16is k, X 17 is H, and X 35 is E. In some embodiments, X 16 is k, X 17 is H, and X 37 It’s Y.

[0168] In some embodiments, X 17 It is H, X 18 is K, and X 31 Is K. In some embodiments, X 17 It's H, X 18 is K, and X 35 is E. In some embodiments, X 17 It's H, X 18 is K, and X 37 It’s Y.

[0169] In some embodiments, X 18 It's K, X 31 is K, and X 35 is E. In some embodiments, X 18 It's K, X 31 is K, and X 37 It’s Y.

[0170] In some embodiments, X 31 It's K, X 35 is E, and X 37 It’s Y.

[0171] 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).

[0172] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 200:

[0173] X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FGPILPX 29 TX31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200) or a pharmaceutically acceptable salt thereof, wherein:

[0174] X1 is S, K, k, H or I;

[0175] X3 is N or S;

[0176] X5 is S or A;

[0177] X6 is T or S;

[0178] X8 is A or K;

[0179] X 10 It is Q or S;

[0180] X 12 It is L or K;

[0181] X 13 is A, S, E or K;

[0182] X 14 is N, n, d, Y, or Q;

[0183] X 15 is E, F, f, Y, I, k, K, or α-aminoisobutyric acid (Aib);

[0184] X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l;

[0185] X 17 is H, V, Q, R, k, K, or Aib;

[0186] X 18 is K, H or R;

[0187] X 19 It is S or Aib;

[0188] X 20 It is S or Aib;

[0189] X 22 is N or E;

[0190] X 29 is P, R or K;

[0191] X 31 is k, K, or N; and

[0192] X 35 is e, E, or N;

[0193] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0194] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0195] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[0196] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[0197] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0198] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0199] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 201:

[0200] 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), wherein:

[0201] X1 is S, K, k, H or I;

[0202] X3 is N or S;

[0203] X5 is S or A;

[0204] X6 is T or S;

[0205] X8 is A or K;

[0206] X 10 It is Q or S;

[0207] X 12 It is L or K;

[0208] X 13 is A, S, E or K;

[0209] X 14 is N, n, d, Y, or Q;

[0210] X 15 is E, F, f, Y, I, k, K, or Aib;

[0211] X 16 is k, K, L, Aib, N-MeL, or l;

[0212] X 17 is H, V, Q, R, k, K, or Aib;

[0213] X 18 is K, H or R;

[0214] X 19 It is S or Aib;

[0215] X 20 It is S or Aib;

[0216] X 22 is N or E; and

[0217] X 29 is P, R or K;

[0218] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0219] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0220] The two cysteine ​​residues of X1CX3TX5X6C are optionally further linked via a disulfide bridge.

[0221] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 200 or SEQ ID NO: 201 include the following:

[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. In some embodiments, X1 is H. In some embodiments, X1 is I.

[0224] As used herein, k refers to D-lysine.

[0225] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0226] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0227] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0228] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0229] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0230] In some embodiments, X 12 is L. In some embodiments, X 12 It’s K.

[0231] 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 It’s K.

[0232] In some embodiments, X 14 Is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X 14 Is Y. In some embodiments, X 14 It's Q.

[0233] As used herein, n refers to D-asparagine.

[0234] As used herein, d refers to D-aspartic acid.

[0235] 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 1. In some embodiments, X 15 Is K. In some embodiments, X 15 is k. In some embodiments, X15 It's Aib.

[0236] As used herein, f refers to D-phenylalanine.

[0237] As used herein, Aib refers alternatively to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine.

[0238] In some embodiments, X 16 is L. In some embodiments, X 16 Is 1. In some embodiments, X 16 Is K. In some embodiments, X 16 is k. In some embodiments, X 16 is Aib. In some embodiments, X 16 It's N-MeL.

[0239] As used herein, 1 refers to D-leucine.

[0240] As used herein, N-MeL refers to N-methylleucine.

[0241] 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 is k. In some embodiments, X 17 It's Aib.

[0242] In some embodiments, X 18 Is K. In some embodiments, X 18 is H. In some embodiments, X 18 It’s R.

[0243] In some embodiments, X 19 is S. In some embodiments, X 19 It's Aib.

[0244] In some embodiments, X 20 is S. In some embodiments, X 20 It's Aib.

[0245] In some embodiments, X 22 Is N. In some embodiments, X 22 It’s E.

[0246] In some embodiments, X29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 It’s K.

[0247] In some embodiments, X 31 is k. In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0248] In some embodiments, X 35 In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0249] As used herein, e refers to D-glutamic acid.

[0250] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 200 or SEQ ID NO: 201 include the following:

[0251] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 Is Q. In some embodiments, X1 is S and X 15 Is E. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 18 Is K. In some embodiments, X1 is S and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0252] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X5 is S. 10 Is Q. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 18 Is K. In some embodiments, X1 is K and X 31 Is K. In some embodiments, X1 is K and X 35 It’s E.

[0253] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X5 is S. 10 Is Q. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 18 Is K. In some embodiments, X1 is k and X 31 Is K. In some embodiments, X1 is k and X 35 It’s E.

[0254] 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, X5 is S and X 17 Is H. In some embodiments, X5 is S and X 18 Is K. In some embodiments, X5 is S and X 31 Is K. In some embodiments, X5 is S and X 35 It’s E.

[0255] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 18 Is K. In some embodiments, X 10 It is Q and X 31 Is K. In some embodiments, X 10 It is Q and X 35 It’s E.

[0256] 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 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 It’s E.

[0257] 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 It’s E.

[0258] 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 It’s E.

[0259] 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 It’s E.

[0260] In some embodiments, X 18 is K and X 31 Is K. In some embodiments, X 18 is K and X 35 It’s E.

[0261] In some embodiments, X 31 is K and X 35 It’s E.

[0262] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 200 or SEQ ID NO: 201 include the following:

[0263] In some embodiments, X1 is S, X5 is S, and X 15 Is E. In some embodiments, X1 is S, X5 is S, and X 16 is L. In some embodiments, X1 is S, X5 is S, and X 16 In some embodiments, X1 is S, X5 is S, and X 17 Is H. In some embodiments, X1 is S, X5 is S, and X 18 Is K. In some embodiments, X1 is S, X5 is S, and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0264] In some embodiments, X1 is K, X5 is S, and X 15 Is E. In some embodiments, X1 is K, X5 is S, and X 16 is L. In some embodiments, X1 is K, X5 is S, and X 16 In some embodiments, X1 is K, X5 is S, and X 17 Is H. In some embodiments, X1 is K, X5 is S, and X 18 Is K. In some embodiments, X1 is K, X5 is S, and X 31 Is K. In some embodiments, X1 is K, X5 is S, and X 35 It’s E.

[0265] In some embodiments, X1 is k, X5 is S, and X 15 Is E. In some embodiments, X1 is k, X5 is S, and X 16 is L. In some embodiments, X1 is k, X5 is S, and X 16 In some embodiments, X1 is k, X5 is S, and X 17 Is H. In some embodiments, X1 is k, X5 is S, and X 18 Is K. In some embodiments, X1 is k, X5 is S, and X 31 Is K. In some embodiments, X1 is k, X5 is S, and X 35 It’s E.

[0266] In some embodiments, X5 is S, X 15 is E, and X 16is L. In some embodiments, X5 is S, X 15 is E, and X 16 In some embodiments, X5 is S, X 15 is E, and X 17 Is H. In some embodiments, X5 is S, X 15 is E, and X 18 In some embodiments, X5 is S, X 15 is E, and X 31 In some embodiments, X5 is S, X 15 is E, and X 35 It’s E.

[0267] In some embodiments, X 10 It's Q, 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 It's Q, X 18 is K, and X 31 Is K. In some embodiments, X 10 It's Q, X 31 is K and X 35 It’s E.

[0268] In some embodiments, X 15 It's E, X 16 is L, and X 17 is H. In some embodiments, X 15 It's E, X 16 is L, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 35 It’s E.

[0269] In some embodiments, X 15 It's E, X 16 is k, and X 17 is H. In some embodiments, X 15 It's E, X 16 is k, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is k, and X31 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 35 It’s E.

[0270] In some embodiments, X 16 It's L, X 17 is H, and X 18 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 31 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 35 It’s E.

[0271] In some embodiments, X 16 is k, X 17 is H, and X 18 Is K. In some embodiments, X 16 is k, X 17 is H, and X 31 Is K. In some embodiments, X 16 is k, X 17 is H, and X 35 It’s E.

[0272] In some embodiments, X 17 It's H, X 18 is K, and X 31 Is K. In some embodiments, X 17 It is H, X 18 is K, and X 35 It’s E.

[0273] In some embodiments, X 18 It's K, X 31 is K, and X 35 It’s E.

[0274] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 202: X1CNTX5TCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X 16 X 17 X 18 SSNNFGPILPPTX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 202), wherein:

[0275] X1 is S, k or K;

[0276] X5 is S or A;

[0277] X 10 It is Q or S;

[0278] X 15 is E or F;

[0279] X 16 is k, K or L;

[0280] X 17 It is H, V and Q;

[0281] X 18 is K, H or R;

[0282] X 31 is K or N; and

[0283] X 35 is E or N;

[0284] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0285] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0286] wherein the two cysteine ​​residues of X1CNTX5TC (SEQ ID NO: 308) are optionally further bound via a disulfide bridge; and

[0287] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[0288] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0289] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0290] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 203: X1CNTX5TCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X16 X 17 X 18 SSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 203), wherein:

[0291] X1 is S, k or K;

[0292] X5 is S or A;

[0293] X 10 It is Q or S;

[0294] X 15 is E or F;

[0295] X 16 is k, K or L;

[0296] X 17 are H, V, and Q; and

[0297] X 18 is K, H or R;

[0298] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0299] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0300] The two cysteine ​​residues of X1CNTX5TC (SEQ ID NO: 308) are optionally further linked via a disulfide bridge.

[0301] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 202 or SEQ ID NO: 203 include the following:

[0302] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0303] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0304] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0305] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0306] In some embodiments, X 15is E. In some embodiments, X 15 It's F.

[0307] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0308] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0309] In some embodiments, X 18 Is K. In some embodiments, X 18 is H. In some embodiments, X 18 It’s R.

[0310] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0311] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0312] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 202 or SEQ ID NO: 203 include the following:

[0313] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X5 is S. 10 Is E. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 18 Is K. In some embodiments, X1 is S and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0314] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X5 is S. 15 Is E. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 18 Is K. In some embodiments, X1 is K and X 31 Is K. In some embodiments, X1 is K and X 35 It’s E.

[0315] 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, X5 is S and X 17 Is H. In some embodiments, X5 is S and X 18 Is K. In some embodiments, X5 is S and X 31 Is K. In some embodiments, X5 is S and X 35 It’s E.

[0316] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 18 Is K. In some embodiments, X 10 It is Q and X 31 Is K. In some embodiments, X 10 It is Q and X 35 It’s E.

[0317] 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 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 35It’s E.

[0318] 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 It’s E.

[0319] 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 It’s E.

[0320] 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 It’s E.

[0321] In some embodiments, X 18 is K and X 31 Is K. In some embodiments, X 18 is K and X 35 It’s E.

[0322] In some embodiments, X 31 is K and X 35 It’s E.

[0323] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 202 or SEQ ID NO: 203 include the following:

[0324] In some embodiments, X1 is S, X5 is S, and X 10 Is Q. In some embodiments, X1 is S, X5 is S, and X 15 Is E. In some embodiments, X1 is S, X5 is S, and X 16 is L. In some embodiments, X1 is S, X5 is S, and X16 In some embodiments, X1 is S, X5 is S, and X 17 Is H. In some embodiments, X1 is S, X5 is S, and X 18 Is K. In some embodiments, X1 is S, X5 is S, and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0325] In some embodiments, X1 is K, X5 is S, and X 10 Is Q. In some embodiments, X1 is K, X5 is S, and X 15 Is E. In some embodiments, X1 is K, X5 is S, and X 16 is L. In some embodiments, X1 is K, X5 is S, and X 16 In some embodiments, X1 is K, X5 is S, and X 17 Is H. In some embodiments, X1 is K, X5 is S, and X 18 Is K. In some embodiments, X1 is K, X5 is S, and X 31 Is K. In some embodiments, X1 is K, X5 is S, and X 35 It’s E.

[0326] In some embodiments, X5 is S, X 10 is Q, and X 15 Is E. In some embodiments, X5 is S, X 10 is Q, and X 16 is L. In some embodiments, X5 is S, X 10 is Q, and X 16 In some embodiments, X5 is S, X 10 is Q, and X 17 Is H. In some embodiments, X5 is S, X 10 is Q, and X 18 In some embodiments, X5 is S, X 10 is Q, and X 31 In some embodiments, X5 is S, X 10 is Q, and X 35 It’s E.

[0327] In some embodiments, X 10 It's Q, X 15 is E, and X 16 is L. In some embodiments, X 10 It's Q, X 15 is E, and X16 is k. In some embodiments, X 10 It's Q, X 15 is E, and X 17 is H. In some embodiments, X 10 It's Q, X 15 is E, and X 18 Is K. In some embodiments, X 10 It's Q, X 15 is E, and X 31 Is K. In some embodiments, X 10 It's Q, X 15 is E, and X 35 It’s E.

[0328] In some embodiments, X 15 It's E, X 16 Is L and X 17 is H. In some embodiments, X 15 It's E, X 16 is L, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 35 It’s E.

[0329] In some embodiments, X 15 It's E, X 16 is k and X 17 is H. In some embodiments, X 15 It's E, X 16 is k, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 35 It’s E.

[0330] In some embodiments, X 16 It's L, X 17 is H, and X 18 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 31 Is K. In some embodiments, X 16 It's L, X 17is H, and X 35 It’s E.

[0331] In some embodiments, X 16 is k, X 17 is H, and X 18 Is K. In some embodiments, X 16 is k, X 17 is H, and X 31 Is K. In some embodiments, X 16 is k, X 17 is H, and X 35 It’s E.

[0332] In some embodiments, X 17 It is H, X 18 is K, and X 31 Is K. In some embodiments, X 17 It is H, X 18 is K, and X 35 It’s E.

[0333] In some embodiments, X 18 It's K, X 31 is K, and X 35 It’s E.

[0334] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 204: X1CNTSTCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 204), wherein:

[0335] X1 is S, K or k;

[0336] X 10 It is Q or S;

[0337] X 15 is E or F;

[0338] X 16 is L, K or k;

[0339] X 17 is H, V, or Q; and

[0340] X 35 is E or N;

[0341] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0342] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0343] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked via a disulfide bridge.

[0344] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0345] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 205: X1CNTSTCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 205), wherein:

[0346] X1 is S, K or k;

[0347] X 10 It is Q or S;

[0348] X 15 is E or F;

[0349] X 16 is L, K or k; and

[0350] X 17 is H, V or Q;

[0351] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0352] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[0353] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked via a disulfide bridge.

[0354] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 204 or SEQ ID NO: 205 include the following:

[0355] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0356] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0357] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0358] In some embodiments, X 15 is E. In some embodiments, X 15 It's F.

[0359] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0360] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0361] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0362] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 204 or SEQ ID NO: 205 include the following:

[0363] In some embodiments, X1 is S and X 10 Is Q. In some embodiments, X1 is S and X 10 Is S. In some embodiments, X1 is S and X 15 Is E. In some embodiments, X1 is S and X 15 Is F. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 Is K. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 17 Is V. In some embodiments, X1 is S and X 17Is Q. In some embodiments, X1 is S and X 35 Is E. In some embodiments, X1 is S and X 35 It's N.

[0364] In some embodiments, X1 is K and X 10 Is Q. In some embodiments, X1 is K and X 10 Is S. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 15 Is F. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 Is K. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 17 Is V. In some embodiments, X1 is K and X 17 Is Q. In some embodiments, X1 is K and X 35 Is E. In some embodiments, X1 is K and X 35 It's N.

[0365] In some embodiments, X1 is k and X 10 Is Q. In some embodiments, X1 is k and X 10 Is S. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 15 Is F. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 Is K. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 17 Is V. In some embodiments, X1 is k and X 17 Is Q. In some embodiments, X1 is k and X 35 Is E. In some embodiments, X1 is k and X 35 It's N.

[0366] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 15is F. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 Is K. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 17 Is V. In some embodiments, X 10 It is Q and X 17 is Q. In some embodiments, X 10 It is Q and X 35 is E. In some embodiments, X 10 It is Q and X 35 It's N.

[0367] 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 is k. In some embodiments, X 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 It's N.

[0368] 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 is k. In some embodiments, X 15is 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 It's N.

[0369] 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 is k. In some embodiments, X 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 It's N.

[0370] 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 It's N.

[0371] 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 16is K and X 35 is E. In some embodiments, X 16 is K and X 35 It's N.

[0372] 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 It's N.

[0373] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 It's N.

[0374] In some embodiments, X 17 Is V and X 35 is E. In some embodiments, X 17 Is V and X 35 It's N.

[0375] In some embodiments, X 17 It is Q and X 35 is E. In some embodiments, X 17 It is Q and X 35 It's N.

[0376] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 206: SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 206), wherein:

[0377] X 15 is E or F;

[0378] X 16 is L, K or k;

[0379] X 17is H, V, or Q; and

[0380] X 35 is E or N;

[0381] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0382] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[0383] The two cysteine ​​residues of SCNTSTC (SEQ ID NO: 310) are optionally further linked via a disulfide bridge.

[0384] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0385] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 207: SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 207), wherein:

[0386] X 15 is E or F;

[0387] X 16 is L, K or k; and

[0388] X 17 is H, V or Q;

[0389] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0390] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[0391] The two cysteine ​​residues of SC*NTSTC* are optionally further linked via a disulfide bridge.

[0392] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 206 or SEQ ID NO: 207 include the following:

[0393] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0394] In some embodiments, X 15 is E. In some embodiments, X 15 It's F.

[0395] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0396] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0397] 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 is k. In some embodiments, X 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 It's N.

[0398] 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 is k. In some embodiments, X 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 X17 is Q. In some embodiments, X 15 Is F and X 35 is E. In some embodiments, X 15 Is F and X 35 It's N.

[0399] 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 It's N.

[0400] 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 It's N.

[0401] 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 It's N.

[0402] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 It's N.

[0403] In some embodiments, X 17Is V and X 35 is E. In some embodiments, X 17 Is V and X 35 It's N.

[0404] In some embodiments, X 17 It is Q and X 35 is E. In some embodiments, X 17 It is Q and X 35 It's N.

[0405] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of:

[0406] SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 127), which is also referred to herein as Compound A127;

[0407] SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 57), which is also referred to herein as Compound A57;

[0408] SC*NTSTC*ATQRLANEKHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 128), which is also referred to herein as Compound A128;

[0409] SC*NTSTC*ATQRLANEkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 129), also referred to herein as Compound A129; and

[0410] SC*NTSTC*ATQRLANFLVKSSNEFGPILPPTKVGSETY-(NH2) (SEQ ID NO:43), which is also referred to herein as Compound A43.

[0411] In some embodiments, the isolated polypeptide of the present disclosure comprises the amino acid sequence: SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 57), which is also referred to herein as Compound A57.

[0412] In some embodiments, the isolated polypeptide of the present disclosure comprises the amino acid sequence: SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 127), which is also referred to herein as Compound A127.

[0413] In some embodiments, the isolated polypeptide of the present disclosure comprises 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.

[0414] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 208: X1CNTSTCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 208), wherein:

[0415] X1 is K or k;

[0416] X 10 It is Q or S;

[0417] X 15 is E or F;

[0418] X 16 is L, K or k;

[0419] X 17 is H, V, or Q; and

[0420] X 35 is E or N;

[0421] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0422] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[0423] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 318) are optionally further linked via a disulfide bridge.

[0424] In some embodiments, X35 is E. In some embodiments, X 35 It's N.

[0425] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of: an amino acid sequence represented by the consensus sequence of SEQ ID NO: 209: X1CNTSTCATX or a pharmaceutically acceptable salt thereof. 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 209), wherein:

[0426] X1 is K or k;

[0427] X 10 It is Q or S;

[0428] X 15 is E or F;

[0429] X 16 is L, K or k; and

[0430] X 17 is H, V or Q;

[0431] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0432] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[0433] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 318) are optionally further linked via a disulfide bridge.

[0434] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 208 or SEQ ID NO: 209 include the following:

[0435] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0436] In some embodiments, X1 is K. In some embodiments, X1 is k.

[0437] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0438] In some embodiments, X 15 is E. In some embodiments, X 15 It's F.

[0439] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0440] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0441] In some embodiments, X1 is K and X 10 Is Q. In some embodiments, X1 is K and X 10 Is S. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 15 Is F. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 Is K. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 17 Is V. In some embodiments, X1 is K and X 17 Is Q. In some embodiments, X1 is K and X 35 Is E. In some embodiments, X1 is K and X 35 It's N.

[0442] In some embodiments, X1 is k and X 10 Is Q. In some embodiments, X1 is k and X 10 Is S. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 15 Is F. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 Is K. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 17 Is V. In some embodiments, X1 is k and X17 Is Q. In some embodiments, X1 is k and X 35 Is E. In some embodiments, X1 is k and X 35 It's N.

[0443] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 15 is F. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 Is K. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 17 Is V. In some embodiments, X 10 It is Q and X 17 is Q. In some embodiments, X 10 It is Q and X 35 is E. In some embodiments, X 10 It is Q and X 35 It's N.

[0444] 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 is k. In some embodiments, X 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 X35 It's N.

[0445] 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 is k. In some embodiments, X 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 It's N.

[0446] 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 is k. In some embodiments, X 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 It's N.

[0447] 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 X35 It's N.

[0448] 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 It's N.

[0449] 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 It's N.

[0450] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 It's N.

[0451] In some embodiments, X 17 Is V and X 35 is E. In some embodiments, X 17 Is V and X 35 It's N.

[0452] In some embodiments, X 17 It is Q and X 35 is E. In some embodiments, X 17 It is Q and X 35 It's N.

[0453] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of:

[0454] KC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 130), also referred to herein as Compound A130; and

[0455] KC*NTSTC*ATQRLANFLQKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 131), which is also referred to herein as Compound A131.

[0456] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0457] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0458] 5. Conjugation of a lipophilic substituent to either peptide, optionally via a spacer

[0459] In some embodiments, any one of the disclosed polypeptides is each optionally substituted with one or more lipophilic substituents via a spacer, wherein "lipophilic substituent" and "spacer" are defined herein. In some embodiments, any one of the disclosed polypeptides (comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by any one of the common sequences of SEQ ID NO: 1 to SEQ ID NO: 143) each optionally comprises one or more lipophilic substituents via a spacer, or can be modified or further modified by each optionally covalently linking one or more lipophilic substituents via a spacer. In some embodiments, the lipophilic substituent can be linked to an amino group of the polypeptide (e.g., the ε-amino group of a lysine residue) by means of a carboxyl group of the lipophilic substituent or, optionally, an amino group of the spacer, wherein the carboxyl group of the spacer forms an amide bond with the ε-amino group of the lysine residue.

[0460] Lipophilic substituents

[0461] Each optionally via a "spacer", one or more "lipophilic substituents" are conjugated to any of the disclosed polypeptides of the present invention in order to prolong the effect of the polypeptide by promoting binding to serum albumin and delaying the renal clearance of the conjugated polypeptide. As used herein, "lipophilic substituents" include: substituents comprising 4 to 40 carbon atoms, 8 to 25 carbon atoms, 12 to 22 carbon atoms, or 6 to 20 carbon atoms. The lipophilic substituent can be linked to an amino group of the polypeptide (e.g., the ε-amino group of a lysine residue) via a carboxyl group of the lipophilic substituent or, optionally, an amino group of a spacer, which in turn forms an amide bond with the amino group of the amino acid (e.g., lysine) residue to which it is attached. In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with or without an optional spacer, as defined in more detail below.

[0462] In some embodiments, the lipophilic substituent comprises a straight or branched alkyl group. In some embodiments, the lipophilic substituent is an acyl group of a straight or branched fatty acid. In some embodiments, the lipophilic substituent is an acyl group of a straight or branched fatty acid further substituted with one or more carboxylic acid and / or hydroxamic acid groups.

[0463] 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 a monovalent group of formula I:

[0464] -CO-(CH2) m -Z

[0465] Formula I

[0466] in

[0467] Z is -CH3 or -CO2H; and

[0468] m is 4 to 24,

[0469] The lipophilic substituent forms an amide bond between an amino group of the disclosed polypeptide (eg, the epsilon-amino group of lysine) and the CO-group of the lipophilic substituent.

[0470] 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.

[0471] In some embodiments, Z is -CO2H and the lipophilic substituent has the formula -CO-(CH2) m -CO2H. In some embodiments, -CO-(CH2) m-Z is selected from the group consisting of: -CO-(CH2)4-CO2H, -CO-(CH2)5-CO2H, -CO-(CH2)6-CO2H, -CO-(CH2)7-CO2H, -CO-(CH2)8-CO2H, -CO-(CH2)9-CO2H, -CO-(CH2) 10 -CO2H, -CO-(CH2) 11 -CO2H, -CO-(CH2) 12 -CO2H, -CO-(CH2) 13 -CO2H, -CO-(CH2) 14 -CO2H, -CO-(CH2) 15 -CO2H, -CO-(CH2) 16 -CO2H, -CO-(CH2) 17 -CO2H, -CO-(CH2) 18 -CO2H, -CO-(CH2) 19 -CO2H, -CO-(CH2) 20 -CO2H.

[0472] In some embodiments, the lipophilic substituent is -CO-(CH2) 18 -CO2H.

[0473] In some embodiments, Z is -CH3, and the lipophilic substituent has the formula -CO-(CH2) m -CH3. In some embodiments, -CO-(CH2) m -Z is selected from the group consisting of: -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.

[0474] Spacer

[0475] In some embodiments, the lipophilic substituent is linked to the parent peptide with the aid of a "spacer". In some embodiments, provided herein is any one of the disclosed polypeptides comprising an amino acid sequence selected from the group consisting of the amino acid sequences represented by any one of the common sequences of SEQ ID NO: 1 to SEQ ID NO: 143 comprising a lipophilic substituent, wherein the lipophilic substituent is linked to the ε-amino group of lysine via a spacer that forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[0476] In some embodiments, the spacer comprises one or more amino acids, such as a single amino acid, such as Glu, Asp, Gly, or Lys; a dipeptide, such as 2(Glu), Glu-Gly; or a polypeptide, such as 3(Glu), 4(Glu) (SEQ ID NO: 317), 2(Glu)-Gly, etc. In some embodiments, when the spacer comprises one or more amino acids, such as 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 the amino group of the spacer can form an amide bond with the carboxyl group of the lipophilic substituent.

[0477] In some embodiments, when the spacer comprises Glu or Asp, it further comprises a carboxylic acid-terminated side chain, the terminal carboxyl group of the side chain of the spacer containing Glu or Asp can form an amide bond with the amino group of the disclosed polypeptide, and the amino group of the spacer containing Glu or Asp can form an amide bond with the carboxyl group of the lipophilic substituent, i.e., γGlu or βAsp. In some embodiments, the spacer is γGlu. In some embodiments, the spacer is 2(γGlu). In some embodiments, the spacer is 3(γGlu).

[0478] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula II:

[0479] -(Y) n -CO-(CH2) m -Z

[0480] Formula II

[0481] in

[0482] Y is selected from the group consisting of γGlu, Asp, Lys and Gly;

[0483] Z is -CH3 or -CO2H;

[0484] m is 4 to 24; and

[0485] n is 1 to 10.

[0486] In some embodiments, Y is selected from the group consisting of γGlu and Gly. In some embodiments, Y is γGlu. In some embodiments, Y is Gly.

[0487] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula III:

[0488] -(γGlu) n -CO-(CH2) m -Z (disclosed as "(γGlu)" in SEQ ID NO: 311) n ”)

[0489] Formula III

[0490] in

[0491] Z is -CH3 or -CO2H;

[0492] m is 4 to 24; and

[0493] n is 1 to 10.

[0494] In some embodiments, Z is -CH3. In some embodiments, Z is -CO2H.

[0495] 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.

[0496] 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.

[0497] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula IV:

[0498] -(γGlu) n -(Gly)-CO-(CH2) m-Z (disclosed as "(γGlu)" in SEQ ID NO: 312) n -(Gly)”)

[0499] Formula IV

[0500] in

[0501] Z is -CH3 or -CO2H;

[0502] m is 4 to 24; and

[0503] n is 1 to 10.

[0504] In some embodiments, (γGlu) n 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) disclosed as SEQ ID NO: 312) n -(Gly)") is selected from the group consisting of: 2(γGlu), Gly; and 3(γGlu), Gly (SEQ ID NO: 315).

[0505] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula V:

[0506] -(Gly)-(γGlu) n -(CO-(CH2) m -Z (disclosed as "(Gly)-(γGlu)" in SEQ ID NO: 316) n ”)

[0507] Formula V

[0508] in

[0509] Z is -CH3 or -CO2H;

[0510] m is 4 to 24; and

[0511] n is 1 to 10.

[0512] In some embodiments, certain variables represented in Formula II, Formula III, Formula IV, or Formula V include the following:

[0513] In some embodiments, Z is -CH3. In some embodiments, Z is -CO2H.

[0514] 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.

[0515] 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.

[0516] 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.

[0517] In some embodiments, n is 1, Z is -CO2H, and m is 14-20. 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.

[0518] In some embodiments, n is 1, Z is -CH3, and m is 14-20. 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.

[0519] In some embodiments, n is 2, Z is -CO2H, and m is 14-20. 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.

[0520] In some embodiments, n is 2, Z is -CH3, and m is 14-20. 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.

[0521] In some embodiments, n is 3, Z is -CO2H, and m is 14-20. 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.

[0522] In some embodiments, n is 3, Z is -CH3, and m is 14-20. 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.

[0523] In some embodiments, n is 4, Z is -CO2H, and m is 14-20. 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.

[0524] In some embodiments, n is 4, Z is -CH3, and m is 14-20. 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.

[0525] In some embodiments, n is 5, Z is -CO2H, and m is 14-20. 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.

[0526] In some embodiments, n is 5, Z is -CH3, and m is 14-20. 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.

[0527] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula VI:

[0528] -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2)m -Z

[0529] Formula VI

[0530] in

[0531] Z is -CH3 or -CO2H;

[0532] m is 4 to 24;

[0533] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0534] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0535] V is -[COCH2(O(CH2)2) t OCH2NH]-, and t is 1 to 8;

[0536] r is 1 to 8;

[0537] n1 is 0 to 10; and

[0538] n2 is 0 to 10.

[0539] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula VII:

[0540] -(Y1) n1 -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z

[0541] Formula VII

[0542] in

[0543] Z is -CH3 or -CO2H;

[0544] m is 4 to 24;

[0545] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0546] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0547] DPEG is -[CO(CH2)O(CH2)2O(CH2)NH]-;

[0548] r is 1 to 8;

[0549] n1 is 0 to 10; and

[0550] n2 is 0 to 10.

[0551] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - 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.

[0552] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula VIII:

[0553] -(V) r -(Y2) n2 -CO-(CH2) m -Z

[0554] Formula VIII

[0555] in

[0556] Z is -CH3 or -CO2H;

[0557] m is 4 to 24;

[0558] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0559] V is -[COCH2(O(CH2)2) t OCH2NH]-, and t is 1 to 8;

[0560] r is 1 to 8; and

[0561] n2 is 0 to 10.

[0562] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula IX:

[0563] -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z

[0564] Formula IX

[0565] in

[0566] Z is -CH3 or -CO2H;

[0567] m is 4 to 24;

[0568] DPEG is -[CO(CH2)O(CH2)2O(CH2)NH]-;

[0569] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0570] r is 1 to 8; and

[0571] n2 is 0 to 10.

[0572] In some embodiments, -(dpeg) r -(Y2) n2 - selected from the group consisting of: dpeg, γGlu; ​​and dpeg, dpeg, γGlu.

[0573] In some embodiments, the polypeptide comprises three, two, or preferably one lipophilic substituent, each with a spacer. In some embodiments, the lipophilic substituent and the spacer are monovalent groups of formula X:

[0574] -(Y1) n1 -(dpeg) r -CO-(CH2) m -Z

[0575] Formula X

[0576] in

[0577] Z is -CH3 or -CO2H;

[0578] m is 4 to 24;

[0579] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0580] DPEG is -[CO(CH2)O(CH2)2O(CH2)NH]-;

[0581] r is 1 to 8; and

[0582] n1 is 0 to 10.

[0583] In some embodiments, -(Y1) n1 -(dpeg) r - is 2(γGlu), dpeg.

[0584] In some embodiments, the spacer comprises a divalent group of formula XI:

[0585] -N(R1)(CHR2) p CO—[N(R3)((CH2)2O(CH2)2O) q (CH2)CO—] r

[0586] Formula XI

[0587] in

[0588] Each of R1 and R3 is hydrogen or C1-C4 alkyl;

[0589] Each R2 is H or CO2H;

[0590] p is 1, 2, 3, 4, 5, or 6;

[0591] q is 1, 2, or 3;

[0592] r is 0 or 1.

[0593] The spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[0594] In some embodiments, the spacer comprises a divalent group of Formula XII:

[0595] [-N(R3)((CH2)2O(CH2)2O) q (CH2)CO—] r

[0596] Formula XII

[0597] in

[0598] Each R3 is hydrogen or C1-C4 alkyl;

[0599] q is 1, 2, or 3;

[0600] r is 0 or 1.

[0601] The spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[0602] In some embodiments, certain variables represented in Formula XI or Formula XII include the following:

[0603] In some embodiments, each R1 is hydrogen. In some embodiments, each R3 is hydrogen. In some embodiments, each R1 and each R3 is hydrogen.

[0604] In some embodiments, at least one R2 is CO2H. In some embodiments, one R2 is CO2H.

[0605] 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.

[0606] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[0607] In some embodiments, r is 0. In some embodiments, r is 1.

[0608] In some embodiments, the spacer is a γ-glutamyl group, i.e., -NH(CHCO2H)(CH2)2CO--. In some embodiments, the spacer is a γ-aminobutyryl group, i.e., -NH(CH2)3CO--. In some embodiments, the spacer is a β-asparaginyl group, i.e., -NH(CHCO2H)(CH2)CO--. In some embodiments, the spacer is a -NH(CH2)2CO--. In some embodiments, the spacer is a glycyl group. In some embodiments, the spacer is a β-alanyl group.

[0609] In some embodiments, the spacer is -NHCH(CO2H)(CH2)2CO--[N(R3)((CH2)2O(CH2)2O) 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(CO2H)(CH2)2CO-NH((CH2)2O(CH2)2O)2(CH2)CO-. In some embodiments, the spacer is -NH(CH2)3CO-NH((CH2)2O(CH2)2O)2(CH2)CO-. In some embodiments, the spacer is -NHCH(CO2H)CH2CO--[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r In some embodiments, the spacer is -NH(CH2)2CO--[N(R3)((CH2)2O(CH2)2O) q (CH2)CO-] r .

[0610] In some embodiments, the spacer comprises a divalent group of Formula XIII:

[0611] -(Y) n -

[0612] Formula XIII

[0613] in

[0614] Y is selected from the group consisting of γGlu, Asp, Lys and Gly;

[0615] n is 1 to 10.

[0616] In some embodiments, Y is selected from the group consisting of γGlu and Gly. In some embodiments, Y is γGlu. In some embodiments, Y is Gly.

[0617] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0618] In some embodiments, the spacer comprises a divalent group of Formula XIV:

[0619] -(γGlu) n -(disclosed as "(γGlu)" in SEQ ID NO: 311) n ”)

[0620] Formula XIV

[0621] in

[0622] n is 1 to 10.

[0623] 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.

[0624] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0625] In some embodiments, the spacer comprises a divalent group of Formula XV:

[0626] -(γGlu) n -(Gly)-("(γGlu) disclosed as SEQ ID NO: 312) n -(Gly)”)

[0627] Formula XV

[0628] in

[0629] n is 1 to 10.

[0630] In some embodiments, (γGlu) n 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) disclosed as SEQ ID NO: 312) n -(Gly)") is selected from the group consisting of: 2(γGlu), Gly; and 3(γGlu), Gly (SEQ ID NO: 315).

[0631] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0632] In some embodiments, the spacer comprises a divalent group of Formula XVI:

[0633] -(Gly)-(γGlu) n -(disclosed as "(Gly)-(γGlu)" in SEQ ID NO: 316 n ”)

[0634] Formula XVI

[0635] in

[0636] n is 1 to 10.

[0637] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0638] 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.

[0639] In some embodiments, the spacer comprises a divalent group of Formula XVII:

[0640] -(Y1) n1 -(V) r -(Y2) n2 -

[0641] Formula XVII

[0642] in

[0643] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0644] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0645] V is -[COCH2(O(CH2)2) t OCH2NH]-, and t is 1 to 8;

[0646] r is 1 to 8;

[0647] n1 is 0 to 10; and

[0648] n2 is 0 to 10.

[0649] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0650] In some embodiments, the spacer comprises a divalent group of Formula XVIII:

[0651] -(Y1) n1 -(dpeg) r -(Y2) n2 -

[0652] Formula XVIII

[0653] in

[0654] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0655] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0656] DPEG is -[CO(CH2)O(CH2)2O(CH2)NH]-;

[0657] r is 1 to 8;

[0658] n1 is 0 to 10; and

[0659] n2 is 0 to 10.

[0660] In some embodiments, -(Y1) n1 -(dpeg) r -(Y2) n2 - 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.

[0661] In some embodiments, the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent. In some embodiments, one end of the spacer forms a covalent bond with the 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.

[0662] Thus, in some embodiments, the isolated polypeptide provided herein comprises an amino acid 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 pharmaceutically acceptable salt thereof, wherein the isolated peptide further comprises a lipophilic substituent and optionally comprises a spacer.

[0663] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199 or a pharmaceutically acceptable salt thereof, which 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 pharmaceutically acceptable salt thereof, which further comprises a lipophilic substituent and a 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 pharmaceutically acceptable salt thereof, which further comprises a lipophilic substituent and a spacer of Formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199 or a pharmaceutically acceptable salt thereof, which further comprises a lipophilic substituent and a spacer of Formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 199 or a pharmaceutically acceptable salt thereof, which further comprises a lipophilic substituent and a spacer of Formula VII.

[0664] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 200, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent of Formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 200, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a 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 pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 200, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 200, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VII.

[0665] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 204, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent of Formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 204, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a 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 pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 204, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 204, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VII.

[0666] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 206, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent of Formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 206, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a 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 pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 206, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 206, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VII.

[0667] In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 208, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent of Formula I. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 208, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a 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 pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula III. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 208, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VI. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 208, or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VII.

[0668] 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 the consensus sequence of SEQ ID NO: 200:

[0669] X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22 FG PILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 200) or a pharmaceutically acceptable salt thereof, wherein:

[0670] 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 17is 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 L-lysine optionally covalently bound to a lipophilic substituent via a spacer; each k independently represents D-lysine optionally covalently bound to a lipophilic substituent via a spacer; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound by a disulfide bridge; and with the proviso that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 is K; and wherein the isolated peptide further comprises a lipophilic substituent of Formula I:

[0671] -CO-(CH2) m -Z

[0672] Formula I

[0673] in

[0674] Z is -CH3 or -CO2H; and

[0675] m is 4 to 24.

[0676] 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 the consensus sequence of SEQ ID NO: 200:

[0677] 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) or a pharmaceutically acceptable salt thereof, wherein:

[0678] 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 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 L-lysine optionally covalently bound to a lipophilic substituent via a spacer; each k independently represents D-lysine optionally covalently bound to a lipophilic substituent via a spacer; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound by a disulfide bridge; and with the proviso that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 is K; and wherein the isolated peptide further comprises a lipophilic substituent and a spacer of Formula III:

[0679] -(γGlu) n -CO-(CH2) m -Z (disclosed as "(γGlu)" in SEQ ID NO: 311) n ”)

[0680] Formula III

[0681] in

[0682] Z is -CH3 or -CO2H;

[0683] m is 4 to 24; and

[0684] n is 1 to 10.

[0685] 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 the consensus sequence of SEQ ID NO: 200:

[0686] 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) or a pharmaceutically acceptable salt thereof, wherein:

[0687] 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 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 L-lysine optionally covalently bound to a lipophilic substituent via a spacer; each k independently represents D-lysine optionally covalently bound to a lipophilic substituent via a spacer; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound by a disulfide bridge; and with the proviso that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 is K; and wherein the isolated peptide further comprises a lipophilic substituent and a spacer of Formula VI:

[0688] -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2) m -Z

[0689] Formula VI

[0690] in

[0691] Z is -CH3 or -CO2H;

[0692] m is 4 to 24;

[0693] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0694] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0695] V is -[COCH2(O(CH2)2) t OCH2NH]-, and t is 1 to 8;

[0696] r is 1 to 8;

[0697] n1 is 0 to 10; and

[0698] n2 is 0 to 10.

[0699] 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 the consensus sequence of SEQ ID NO: 200:

[0700] 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) or a pharmaceutically acceptable salt thereof, wherein:

[0701] 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 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 L-lysine optionally covalently bound to a lipophilic substituent via a spacer; each k independently represents D-lysine optionally covalently bound to a lipophilic substituent via a spacer; wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound by a disulfide bridge; and with the proviso that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 is K; and wherein the isolated peptide further comprises a lipophilic substituent and a spacer of Formula VII:

[0702] -(Y1) n1 -(dpeg) r -(Y2) n2 -CO-(CH2) m -Z

[0703] Formula VII

[0704] in

[0705] Z is -CH3 or -CO2H;

[0706] m is 4 to 24;

[0707] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[0708] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[0709] DPEG is -[CO(CH2)O(CH2)2O(CH2)NH]-;

[0710] r is 1 to 8;

[0711] n1 is 0 to 10; and

[0712] n2 is 0 to 10.

[0713] In some embodiments, the isolated polypeptide comprises positions X1, X 15 or X 16 A lipophilic substituent at the position.

[0714] In some embodiments, comprising positions X1, X 15 or X 16 The isolated polypeptide containing a lipophilic substituent at said position is conjugated via the lysine at said position.

[0715] In some embodiments, the isolated polypeptide comprises a lipophilic substituent at position X1.

[0716] In some embodiments, the isolated polypeptide comprises position X 15 A lipophilic substituent at the position.

[0717] In some embodiments, the isolated polypeptide comprises position X 16 A lipophilic substituent at the position.

[0718] In some embodiments, the isolated polypeptide comprising a lipophilic substituent at position X1 is conjugated via the lysine at said position.

[0719] In some embodiments, comprising position X 15 The isolated polypeptide containing a lipophilic substituent at said position is conjugated via the lysine at said position.

[0720] In some embodiments, comprising position X 16 The isolated polypeptide containing a lipophilic substituent at said position is conjugated via the lysine at said position.

[0721] In some embodiments, the isolated polypeptide of the present disclosure comprises 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.

[0722] In some embodiments, the isolated polypeptide of the present disclosure comprises 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.

[0723] In some embodiments, the isolated polypeptide of the present disclosure comprises the amino acid sequence: K*((γGlu)2(CO(CH2) 16CO2H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:65), which is also referred to herein as Compound A65.

[0724] In some embodiments, the isolated polypeptide of the present disclosure comprises 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.

[0725] 6. Exemplary Compounds: Amylin Analog Peptides

[0726] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Table 3:

[0727] Table 3: Exemplary Compounds: Amylin Analog Peptides

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735] Notes:

[0736] • each K* independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0737] • each k* independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[0738] • two cysteine ​​residues (C*) at positions 2 and 7, optionally further bound via a disulfide bridge;

[0739] ● As used herein, (γGlu)2 and 2(γGlu) both mean -(γGlu)-(γGlu)-; (γGlu)3 and 3(γGlu) both mean -(γGlu)-(γGlu)-(γGlu)-; etc.; and

[0740] Where a variable occurs more than once in a given formula, the variable is independently determined at each occurrence. For example, for the group -(Y)3-, 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)-.

[0741] In some embodiments, the present invention provides a compound described in Table 3 above or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is an acetate salt. In some embodiments, the pharmaceutically acceptable salt is a trifluoroacetic acid (TFA) salt. In some embodiments, the pharmaceutically acceptable salt is a hydrochloric acid (HCl) salt. In one embodiment, the compound is A27. In some embodiments, the compound is the acetate salt of compound A27. In some embodiments, the compound is the trifluoroacetic acid salt of compound A27. In some embodiments, the compound is the hydrochloride salt of compound A27. In one embodiment, the compound is A57. In some embodiments, the compound is the acetate salt of compound A57. In some embodiments, the compound is the trifluoroacetic acid salt of compound A57. In some embodiments, the compound is the hydrochloride salt of compound A57. In one embodiment, the compound is A64. In some embodiments, the compound is the acetate salt of compound A64. In some embodiments, the compound is the trifluoroacetic acid salt of compound A64. In some embodiments, the compound is the hydrochloride salt of compound A64.

[0742] 7. Peptide intermediates

[0743] In certain embodiments, the present invention also relates to a synthetic intermediate of an isolated polypeptide that is an amylin analog. In some embodiments, the polypeptide intermediate of the present disclosure is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by the consensus sequence of SEQ ID NO: 210:

[0744] X1CX3TX5X6CX8TX 10 RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 NX 22FGPILPX 29 TX 31 VGSX 35 TY-(OH / NH2) (SEQ ID NO: 210), wherein:

[0745] X1 is S, K, k, H or I;

[0746] X3 is N or S;

[0747] X5 is S or A;

[0748] X6 is T or S;

[0749] X8 is A or K;

[0750] X 10 It is Q or S;

[0751] X 12 It is L or K;

[0752] X 13 is A, S, E or K;

[0753] X 14 is N, n, d, Y, or Q;

[0754] X 15 is E, F, f, Y, I, k, K, or α-aminoisobutyric acid (Aib);

[0755] X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l;

[0756] X 17 is H, V, Q, R, k, K, or Aib;

[0757] X 18 is K, H or R;

[0758] X 19 It is S or Aib;

[0759] X 20 It is S or Aib;

[0760] X 22 is N or E;

[0761] X 29 is P, R or K;

[0762] X 31 is k, K, or N; and

[0763] X 35 is e, E, or N;

[0764] Each K independently represents L-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[0765] Each k independently represents D-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[0766] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[0767] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[0768] In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0769] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0770] In some embodiments, X 31 is K and X 35 It’s E.

[0771] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include the following:

[0772] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0773] 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.

[0774] In some embodiments, X3 is N. In some embodiments, X3 is S.

[0775] In some embodiments, X5 is S. In some embodiments, X5 is A.

[0776] In some embodiments, X6 is T. In some embodiments, X6 is S.

[0777] In some embodiments, X8 is A. In some embodiments, X8 is K.

[0778] In some embodiments, X10 is Q. In some embodiments, X 10 It’s S.

[0779] In some embodiments, X 12 is L. In some embodiments, X 12 It’s K.

[0780] 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 It’s K.

[0781] In some embodiments, X 14 Is N. In some embodiments, X 14 is n. In some embodiments, X 14 In some embodiments, X 14 Is Y. In some embodiments, X 14 It's Q.

[0782] 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 1. In some embodiments, X 15 Is K. In some embodiments, X 15 is k. In some embodiments, X 15 It's Aib.

[0783] In some embodiments, X 16 is L. In some embodiments, X 16 Is 1. In some embodiments, X 16 Is K. In some embodiments, X 16 is k. In some embodiments, X 16 is Aib. In some embodiments, X 16 It's N-MeL.

[0784] 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, X17 is k. In some embodiments, X 17 It's Aib.

[0785] In some embodiments, X 18 Is K. In some embodiments, X 18 is H. In some embodiments, X 18 It’s R.

[0786] In some embodiments, X 19 is S. In some embodiments, X 19 It's Aib.

[0787] In some embodiments, X 20 is S. In some embodiments, X 20 It's Aib.

[0788] In some embodiments, X 22 Is N. In some embodiments, X 22 It’s E.

[0789] In some embodiments, X 29 is P. In some embodiments, X 29 is R. In some embodiments, X 29 It’s K.

[0790] In some embodiments, X 31 is k. In some embodiments, X 31 Is K. In some embodiments, X 31 It's N.

[0791] In some embodiments, X 35 In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0792] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include the following:

[0793] In some embodiments, X1 is S and X5 is S. In some embodiments, X1 is S and X 10 Is Q. In some embodiments, X1 is S and X 15 Is E. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 18Is K. In some embodiments, X1 is S and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0794] In some embodiments, X1 is K and X5 is S. In some embodiments, X1 is K and X5 is S. 10 Is Q. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 18 Is K. In some embodiments, X1 is K and X 31 Is K. In some embodiments, X1 is K and X 35 It’s E.

[0795] In some embodiments, X1 is k and X5 is S. In some embodiments, X1 is k and X5 is S. 10 Is Q. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 18 Is K. In some embodiments, X1 is k and X 31 Is K. In some embodiments, X1 is k and X 35 It’s E.

[0796] 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, X5 is S and X 17 Is H. In some embodiments, X5 is S and X 18 Is K. In some embodiments, X5 is S and X 31 Is K. In some embodiments, X5 is S and X 35 It’s E.

[0797] In some embodiments, X 10It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 18 Is K. In some embodiments, X 10 It is Q and X 31 Is K. In some embodiments, X 10 It is Q and X 35 It’s E.

[0798] 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 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 It’s E.

[0799] 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 It’s E.

[0800] 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 It’s E.

[0801] 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 It’s E.

[0802] In some embodiments, X 18 is K and X 31 Is K. In some embodiments, X 18 is K and X 35 It’s E.

[0803] In some embodiments, X 31 is K and X 35 It’s E.

[0804] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include the following:

[0805] In some embodiments, X1 is S, X5 is S, and X 15 Is E. In some embodiments, X1 is S, X5 is S, and X 16 is L. In some embodiments, X1 is S, X5 is S, and X 16 In some embodiments, X1 is S, X5 is S, and X 17 Is H. In some embodiments, X1 is S, X5 is S, and X 18 Is K. In some embodiments, X1 is S, X5 is S, and X 31 Is K. In some embodiments, X1 is S and X 35 It’s E.

[0806] In some embodiments, X1 is K, X5 is S, and X 15 Is E. In some embodiments, X1 is K, X5 is S, and X 16 is L. In some embodiments, X1 is K, X5 is S, and X 16 In some embodiments, X1 is K, X5 is S, and X 17 Is H. In some embodiments, X1 is K, X5 is S, and X 18 Is K. In some embodiments, X1 is K, X5 is S, and X 31 Is K. In some embodiments, X1 is K, X5 is S, and X 35 It’s E.

[0807] In some embodiments, X1 is k, X5 is S, and X 15 Is E. In some embodiments, X1 is k, X5 is S, and X 16 is L. In some embodiments, X1 is k, X5 is S, and X 16 In some embodiments, X1 is k, X5 is S, and X 17 Is H. In some embodiments, X1 is k, X5 is S, and X 18 Is K. In some embodiments, X1 is k, X5 is S, and X 31 Is K. In some embodiments, X1 is k, X5 is S, and X 35 It’s E.

[0808] In some embodiments, X5 is S, X 15 is E, and X 16 is L. In some embodiments, X5 is S, X 15 is E, and X 16 In some embodiments, X5 is S, X 15 is E, and X 17 Is H. In some embodiments, X5 is S, X 15 is E, and X 18 In some embodiments, X5 is S, X 15 is E, and X 31 In some embodiments, X5 is S, X 15 is E, and X 35 It’s E.

[0809] In some embodiments, X 10 It's Q, 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 It's Q, X 18 is K, and X 31 Is K. In some embodiments, X 10 It's Q, X 31 is K and X 35 It’s E.

[0810] In some embodiments, X 15 It's E, X 16 is L, and X 17 is H. In some embodiments, X 15 It's E, X 16 is L, and X18 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is L, and X 35 It’s E.

[0811] In some embodiments, X 15 It's E, X 16 is k, and X 17 is H. In some embodiments, X 15 It's E, X 16 is k, and X 18 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 31 Is K. In some embodiments, X 15 It's E, X 16 is k, and X 35 It’s E.

[0812] In some embodiments, X 16 It's L, X 17 is H, and X 18 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 31 Is K. In some embodiments, X 16 It's L, X 17 is H, and X 35 It’s E.

[0813] In some embodiments, X 16 is k, X 17 is H, and X 18 Is K. In some embodiments, X 16 is k, X 17 is H, and X 31 Is K. In some embodiments, X 16 is k, X 17 is H, and X 35 It’s E.

[0814] In some embodiments, X 17 It's H, X 18 is K, and X 31 Is K. In some embodiments, X 17 It is H, X 18 is K, and X 35 It’s E.

[0815] In some embodiments, X 18 It's K, X 31 is K, and X 35 It’s E.

[0816] In some embodiments, the polypeptide intermediate of the present disclosure is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by the consensus sequence of SEQ ID NO: 211:

[0817] X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 211), wherein:

[0818] X1 is S, K or k;

[0819] X 10 It is Q or S;

[0820] X 15 is E or F;

[0821] X 16 is L, K or k;

[0822] X 17 is H, V, or Q; and

[0823] X 35 is E or N;

[0824] Each K independently represents L-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[0825] each k independently represents D-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group; and

[0826] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked via a disulfide bridge.

[0827] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0828] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 211 include the following:

[0829] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0830] In some embodiments, X1 is S. In some embodiments, X1 is K. In some embodiments, X1 is k.

[0831] In some embodiments, X 10 is Q. In some embodiments, X 10 It’s S.

[0832] In some embodiments, X 15 is E. In some embodiments, X 15 It's F.

[0833] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0834] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0835] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0836] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 211 include the following:

[0837] In some embodiments, X1 is S and X 10 Is Q. In some embodiments, X1 is S and X 10 Is S. In some embodiments, X1 is S and X 15 Is E. In some embodiments, X1 is S and X 15 Is F. In some embodiments, X1 is S and X 16 is L. In some embodiments, X1 is S and X 16 Is K. In some embodiments, X1 is S and X 16 In some embodiments, X1 is S and X 17 Is H. In some embodiments, X1 is S and X 17 Is V. In some embodiments, X1 is S and X 17 Is Q. In some embodiments, X1 is S and X35 Is E. In some embodiments, X1 is S and X 35 It's N.

[0838] In some embodiments, X1 is K and X 10 Is Q. In some embodiments, X1 is K and X 10 Is S. In some embodiments, X1 is K and X 15 Is E. In some embodiments, X1 is K and X 15 Is F. In some embodiments, X1 is K and X 16 is L. In some embodiments, X1 is K and X 16 Is K. In some embodiments, X1 is K and X 16 In some embodiments, X1 is K and X 17 Is H. In some embodiments, X1 is K and X 17 Is V. In some embodiments, X1 is K and X 17 Is Q. In some embodiments, X1 is K and X 35 Is E. In some embodiments, X1 is K and X 35 It's N.

[0839] In some embodiments, X1 is k and X 10 Is Q. In some embodiments, X1 is k and X 10 Is S. In some embodiments, X1 is k and X 15 Is E. In some embodiments, X1 is k and X 15 Is F. In some embodiments, X1 is k and X 16 is L. In some embodiments, X1 is k and X 16 Is K. In some embodiments, X1 is k and X 16 In some embodiments, X1 is k and X 17 Is H. In some embodiments, X1 is k and X 17 Is V. In some embodiments, X1 is k and X 17 Is Q. In some embodiments, X1 is k and X 35 Is E. In some embodiments, X1 is k and X 35 It's N.

[0840] In some embodiments, X 10 It is Q and X 15 is E. In some embodiments, X 10 It is Q and X 15 is F. In some embodiments, X 10It is Q and X 16 is L. In some embodiments, X 10 It is Q and X 16 Is K. In some embodiments, X 10 It is Q and X 16 is k. In some embodiments, X 10 It is Q and X 17 is H. In some embodiments, X 10 It is Q and X 17 Is V. In some embodiments, X 10 It is Q and X 17 is Q. In some embodiments, X 10 It is Q and X 35 is E. In some embodiments, X 10 It is Q and X 35 It's N.

[0841] 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 is k. In some embodiments, X 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 It's N.

[0842] 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 is k. In some embodiments, X 15 is E and X 17is 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 It's N.

[0843] 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 is k. In some embodiments, X 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 It's N.

[0844] 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 It's N.

[0845] 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 35is E. In some embodiments, X 16 is K and X 35 It's N.

[0846] 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 It's N.

[0847] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 It's N.

[0848] In some embodiments, X 17 Is V and X 35 is E. In some embodiments, X 17 Is V and X 35 It's N.

[0849] In some embodiments, X 17 It is Q and X 35 is E. In some embodiments, X 17 It is Q and X 35 It's N.

[0850] In some embodiments, the polypeptide intermediate of the present disclosure is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by the consensus sequence of SEQ ID NO: 212:

[0851] SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 212), wherein:

[0852] X 15 is E or F;

[0853] X 16 is L, K or k;

[0854] X 17is H, V, or Q; and

[0855] X 35 is E or N;

[0856] Each K independently represents L-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[0857] each k independently represents D-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group; and

[0858] The two cysteine ​​residues of SCNTSTC (SEQ ID NO: 310) are optionally further linked via a disulfide bridge.

[0859] In some embodiments, X 35 is E. In some embodiments, X 35 It's N.

[0860] In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 212 include the following:

[0861] In some embodiments, the carboxy-terminal amino acid 37 is Y-(NH2). In some embodiments, the carboxy-terminal amino acid 37 is Y-(OH).

[0862] In some embodiments, X 15 is E. In some embodiments, X 15 It's F.

[0863] In some embodiments, X 16 is L. In some embodiments, X 16 Is K. In some embodiments, X 16 It's K.

[0864] In some embodiments, X 17 is H. In some embodiments, X 17 Is V. In some embodiments, X 17 It's Q.

[0865] 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 is k. In some embodiments, X 15 is E and X 17 is H. In some embodiments, X 15 is E and X17 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 It's N.

[0866] 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 is k. In some embodiments, X 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 It's N.

[0867] 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 It's N.

[0868] 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 X35 It's N.

[0869] 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 It's N.

[0870] In some embodiments, X 17 is H and X 35 is E. In some embodiments, X 17 is H and X 35 It's N.

[0871] In some embodiments, X 17 Is V and X 35 is E. In some embodiments, X 17 Is V and X 35 It's N.

[0872] In some embodiments, X 17 It is Q and X 35 is E. In some embodiments, X 17 It is Q and X 35 It's N.

[0873] 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 bound 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 bound to a protecting group.

[0874] In some embodiments, the isolated polypeptide comprises a protecting group or is optionally attached to positions X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X30, X31, X32 15 or X 16 A spacer for the protecting group at .

[0875] In some embodiments, a protecting group is included or optionally attached to positions X1, X 15 or X 16 The isolated polypeptide containing a spacer containing a protecting group at the position is conjugated via the lysine at the position.

[0876] In some embodiments, the isolated polypeptide comprises a protecting group or a spacer optionally bound to the protecting group at position X1.

[0877] In some embodiments, the isolated polypeptide comprises a protecting group or is optionally attached to position X. 15 A spacer for the protecting group at .

[0878] In some embodiments, the isolated polypeptide comprises a protecting group or is optionally attached to position X. 16 A spacer for the protecting group at .

[0879] In some embodiments, the isolated polypeptide comprising a protecting group or a spacer optionally bound to a protecting group at position X1 is conjugated via the lysine at said position.

[0880] In some embodiments, a protecting group is included or optionally attached to position X 15 The isolated polypeptide containing a spacer containing a protecting group at the position is conjugated via the lysine at the position.

[0881] In some embodiments, a protecting group is included or optionally attached to position X 16 The isolated polypeptide containing a spacer containing a protecting group at the position is conjugated via the lysine at the position.

[0882] 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.

[0883] 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 polypeptide further comprises a spacer of Formula XIV. In some embodiments, the isolated polypeptide further comprises a spacer of Formula XVII. In some embodiments, the isolated polypeptide 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 the protecting group.

[0884] Exemplary polypeptide intermediates

[0885] In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of the following peptides listed in Table 4:

[0886] Table 4: Exemplary polypeptide intermediates

[0887]

[0888]

[0889] In some embodiments, the present invention provides peptide intermediates as described 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.

[0890] 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.

[0891] 8. Use, preparation and administration

[0892] How to use

[0893] According to another embodiment, the present invention relates to a method for treating a metabolic disease or condition in a subject in need of treatment, 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 conditions include type 1 diabetes, type 2 diabetes, and obesity. Additionally, the present invention relates to a method for achieving 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.

[0894] The amylin analog polypeptides disclosed herein are particularly useful for treating diabetes, the method comprising providing an effective amount of the amylin analog polypeptide to a diabetic subject. In some embodiments, the amylin analog polypeptides disclosed herein are used to treat a subject with type 1 or type 2 diabetes to control or lower blood glucose concentration in the subject, wherein the blood glucose level can be monitored or estimated based on the measured blood concentration of glycated hemoglobin (hemoglobin A1c, HbA1c).

[0895] (i) In some embodiments, the amylin analog polypeptides of the present disclosure are used to treat a subject with type 1 diabetes;

[0896] (ii) In some embodiments, the amylin analog polypeptides of the present disclosure are used to treat a subject suffering from type 2 diabetes;

[0897] (iii) In some embodiments, the amylin analog polypeptides of the present disclosure are used to treat obesity; and

[0898] (iv) In some embodiments, the amylin analog polypeptides of the present disclosure are used to provide weight loss to a subject, such as a diabetic subject,

[0899] wherein 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 NO: 199, 200, 204, 206, 127, 57, 128, 129, 43, 209, 130, 64, 65, 131, 109 and 27.

[0900] In some embodiments, the 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, the 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, the 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, the 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.

[0901] In some embodiments, the amylin analog polypeptide is used to treat a subject with type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 27. In some embodiments, the amylin analog polypeptide is used to treat a subject with type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 64. In some embodiments, the amylin analog polypeptide is used to treat a subject with type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 65. In some embodiments, the amylin analog polypeptide is used to treat a subject with type 2 diabetes, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 131.

[0902] In some embodiments, the amylin analog polypeptide is used to treat a subject with obesity, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 27. In some embodiments, the amylin analog polypeptide is used to treat a subject with obesity, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 64. In some embodiments, the amylin analog polypeptide is used to treat a subject with obesity, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 65. In some embodiments, the amylin analog polypeptide is used to treat a subject with obesity, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 131.

[0903] In some embodiments, the amylin analog polypeptide is used to provide weight loss to a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 27. In some embodiments, the amylin analog polypeptide is used to provide weight loss to a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 64. In some embodiments, the amylin analog polypeptide is used to provide weight loss to a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 65. In some embodiments, the amylin analog polypeptide is used to provide weight loss to a subject, wherein the amylin analog polypeptide comprises the amino acid sequence of SEQ ID NO 131.

[0904] Amylin analog polypeptides (e.g., insulin) of the present disclosure are provided to diabetic subjects (i.e., administered) to maintain, control, or reduce blood glucose concentrations in the subject. Diabetic subjects treated with the amylin analog polypeptides of the present disclosure as an adjunct to insulin therapy are at risk of hypoglycemia (i.e., low blood sugar), particularly severe hypoglycemia. Therefore, reducing the mealtime insulin dose of a diabetic subject following treatment with the amylin analog polypeptides of the present disclosure is intended to reduce the risk of hypoglycemia, particularly severe hypoglycemia.

[0905] As used herein, severe hypoglycemia refers to an episode of hypoglycemia that requires assistance from another individual, including assistance with administration of oral carbohydrates, or requires administration of glucagon, intravenous glucose, or other medical intervention.

[0906] Thus, administration of the amylin analog polypeptides of the present disclosure as an adjunct to insulin therapy, particularly mealtime insulin therapy, generally requires a reduction in the dose of mealtime insulin necessary to properly maintain healthy blood glucose concentrations in the subject. In other words, a patient with type 1 or type 2 diabetes who self-administered a particular dose of mealtime insulin prior to initiating treatment with the amylin analog polypeptides of the present disclosure will reduce (e.g., by up to 25%, 50%, 75%, or 100%) the dose of mealtime insulin they continue to self-administer after initiating treatment with the amylin analog polypeptides of the present disclosure.

[0907] In some embodiments, the method comprises providing an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof to a subject in need of treatment via injection. In some embodiments, the method comprises providing an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof formulated for oral administration to a subject in need of treatment.

[0908] In some embodiments, the method comprises providing an amylin analog polypeptide of the present disclosure or a pharmaceutical composition thereof to a subject in need of treatment via implantation. In some embodiments, the method comprises providing continuous delivery of an amylin analog polypeptide from an osmotic delivery device to a subject in need of treatment. The delivery device, such as an osmotic delivery device, contains 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 the amylin analog polypeptide of the present disclosure via an osmotic delivery device eliminates the daily or multiple daily dosing of existing amylin analog polypeptides such as pramlintide. Diabetes treated with pramlintide must coordinate the administration of pramlintide before meals with mealtime insulin administered after meals. Conversely, diabetic patients treated with the 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 a reduced dose of mealtime insulin.

[0909] The substantially steady-state delivery of the amylin analog polypeptide from the osmotic delivery device is continuous over the administration period. In some embodiments, the subject or patient is a human subject or human patient.

[0910] In some embodiments of the invention, the administration period is, for example, at least about 3 months, at least about 3 months to about one year, at least about 4 months to about one year, at least about 5 months to about one year, at least about 6 months to about one year, at least about 8 months to about one year, at least about 9 months to about one year, at least about 10 months to about one year, at least about one year to about two years, at least about two years to about three years.

[0911] In other embodiments, the methods of treatment of the present invention provide a significant reduction in subject's fasting plasma glucose concentration (relative to the subject's fasting plasma glucose concentration before implanting the osmotic delivery device) after implanting the osmotic delivery device in the subject, which is achieved in about 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day or less after implanting the osmotic delivery device in the subject. A significant reduction in fasting plasma glucose is typically statistically significant, as indicated by applying appropriate statistical tests, or considered by medical professionals to be significant for the subject. A significant reduction in fasting plasma glucose relative to baseline before implantation is typically maintained within the administration period.

[0912] In some embodiments, the present invention relates to a method for treating a disease or condition in a subject in need of treatment. The method comprises providing continuous delivery of a drug from an osmotic delivery device, wherein substantially steady-state delivery of a therapeutic concentration of the drug 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 preferred subjects for the practice of the present invention. The present invention includes drugs that are effective in treating a disease or condition and osmotic delivery devices comprising the drugs of the present invention for 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 therapies associated with troughs.

[0913] In some embodiments, substantially steady-state delivery of therapeutic concentrations of the drug is achieved within a period of about 1 month, 7 days, 5 days, 3 days, or 1 day following implantation of the osmotic delivery device in the subject.

[0914] The present invention also provides a method for promoting weight loss in a subject in need thereof, a method for treating overweight or obesity in a subject in need thereof, and / or a method for suppressing appetite in a subject in need thereof. The method comprises providing for 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, 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 human.

[0915] The present invention includes isolated amylin analog polypeptides and osmotic delivery devices comprising the isolated amylin analog polypeptides for use in the methods of the present invention in a subject in need of treatment. The subject may have type 2 diabetes. The subject in need may have a baseline HbA1c% greater than 10.0%, i.e., a high baseline (HBL) subject. The subject may not have previously received medication for the treatment of type 2 diabetes.

[0916] In other embodiments, the treatment methods of the present invention provide a significant reduction in the subject's fasting plasma glucose concentration after implantation of the osmotic delivery device in the subject (relative to the subject's fasting plasma glucose concentration prior to implantation of the osmotic delivery device) that 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 in the subject, or within about 1 day or less after implantation of the osmotic delivery device in the subject. In a preferred embodiment of the present invention, the subject's fasting plasma glucose concentration is significantly reduced after implantation of the osmotic delivery device, relative to the subject's fasting plasma glucose concentration prior to implantation, that is achieved within about 2 days or less, preferably about 1 day or less, or more preferably about 1 day after implantation of the osmotic delivery device in the subject. A significant reduction in fasting plasma glucose is typically statistically significant, as demonstrated by application of an appropriate statistical test, or is considered significant for the subject by a medical professional.A significant reduction in fasting plasma glucose relative to pre-implantation baseline is typically maintained over the administration period.

[0917] In embodiments of all aspects of the invention relating to methods of treating a disease or condition in a subject, an exemplary osmotic delivery device comprises the following: an impermeable reservoir comprising an inner surface and an outer surface and a first open end and a second open end; a semipermeable membrane in sealing relationship with the first open end of the reservoir; an osmotic engine within the reservoir and adjacent to the semipermeable membrane; a piston adjacent to the osmotic engine, wherein the piston forms a movable seal with the inner surface of the reservoir, the piston separating the reservoir into a first chamber and a second chamber, the first chamber comprising the osmotic engine; a drug formulation or a suspension formulation comprising a drug, wherein the second chamber comprises the drug formulation or suspension formulation and the drug formulation or suspension formulation is flowable; and a diffusion regulator inserted into the second open end of the reservoir, the diffusion regulator being adjacent to the suspension formulation. In preferred embodiments, the reservoir comprises titanium or a titanium alloy.

[0918] In embodiments of all aspects of the invention relating to methods for treating a disease or illness in a subject, the pharmaceutical formulation may comprise a drug and a vehicle formulation. Alternatively, a suspension formulation is used in the method and may, for example, comprise a particle formulation containing the drug and a vehicle formulation. The vehicle formulation used to form the suspension formulation of the invention may, for example, comprise a solvent and a polymer.

[0919] The reservoir of the osmotic delivery device may, for example, comprise titanium or a titanium alloy.

[0920] In embodiments of all aspects of the invention, implantation of an osmotic delivery device may be used to provide subcutaneous delivery.

[0921] In embodiments of all aspects of the invention, the continuous delivery may for example be zero-order controlled continuous delivery.

[0922] combination

[0923] In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with a second agent combination. In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with a second agent combination, wherein the second agent is an insulinotropic peptide. In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with a second agent combination, wherein the second agent is a GLP-1 receptor agonist. In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with a second agent combination, wherein the second agent 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.

[0924] In some embodiments, an amylin analog polypeptide of the present disclosure is administered to a subject in combination with a second agent that is not co-formulated with the second agent, wherein the second agent is a (GLP-1) agonist, such as exenatide, a derivative of exenatide, an analog of exenatide, or semaglutide.

[0925] In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with insulin or an insulin derivative combination. In some embodiments, the amylin analog polypeptides of the present disclosure are co-formulated with a long-acting basal insulin or a long-acting basal insulin derivative combination.

[0926] In some embodiments, an amylin analog polypeptide of the present disclosure that is not co-formulated with insulin or an insulin derivative is administered to a subject in combination with insulin or an insulin derivative, i.e., as an adjunct to insulin therapy. In some embodiments, an amylin analog peptide of the present disclosure that is not co-formulated with insulin or an insulin derivative is administered to a subject in combination with mealtime insulin. In some embodiments, the subject suffers from type 1 diabetes. In some embodiments, the subject suffers from type 2 diabetes.

[0927] In some embodiments, the amylin analog polypeptides of the present disclosure are co-administered with insulin or an insulin derivative to a human patient to provide a so-called dual hormone "artificial pancreas" therapy. In some embodiments, the amylin analog polypeptides of the present disclosure that are not co-formulated with insulin or an insulin derivative are co-administered to a subject in combination 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-formulated with insulin or an insulin derivative and are therefore administered alone to a subject in combination with insulin or an insulin derivative to provide a dual hormone "artificial pancreas" therapy. In some embodiments, the artificial pancreas therapy comprises a fast-acting insulin or a fast-acting insulin derivative. In some embodiments, the artificial pancreas therapy comprises a long-acting or basal insulin or a long-acting or basal insulin derivative.

[0928] In some embodiments, any of the amylin analogs of the present disclosure is formulated in combination with exenatide. In some embodiments, any of the amylin analogs of the present disclosure is formulated in combination with a GLP-1 receptor agonist.

[0929] Some embodiments of the present invention include the use of an amylin analog polypeptide in combination with a second therapeutic agent, such as a second polypeptide, for example, as non-limiting examples, insulinotropic peptides, peptide hormones such as glucagon and incretin mimetics (e.g., GLP-1 receptor agonists, such as exenatide), and peptide analogs and peptide derivatives thereof; PYY (also known as peptide YY, peptide tyrosine tyrosine), and peptide analogs and peptide derivatives thereof, such as PYY (3-36); oxyntomodulin and peptide analogs and peptide derivatives thereof); and gastric inhibitory peptide (GIP), and peptide analogs and peptide derivatives thereof. 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.

[0930] GLP-1, including three forms of the peptide, GLP-1 (1-37), GLP-1 (7-37), and GLP-1 (7-36) amide, as well as peptide analogs of GLP-1, have been shown to stimulate insulin secretion (i.e., to be insulinotropic), which induces glucose uptake into cells and leads to a decrease in serum glucose concentration (see, e.g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).

[0931] Numerous GLP-1 receptor agonists (e.g., GLP-1 peptide derivatives and peptide analogs) that exhibit insulinotropic effects are known in the art (see, e.g., U.S. Patent 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, 295; 6,703,359; 6,706,689; 6,720,407; 6,821,949; 6,849,708; 6,849,714; 6,887,470; 6,887,849; 6,903,186; 7,022,674; 7,041,646; 7,084,243; 7,101,843; 7,138,486; 7,141,547; 7,144,863; and 7,199,217) and in clinical trials (e.g., tasirolid and albiglutide). An example of a GLP-1 receptor agonist in the practice of the present invention is (Novo Nordisk A / S, Bagsvaerd DK) (liraglutide; U.S. Patent Nos. 6,268,343, 6,458,924, and 7,235,627). Once-daily injectable (liraglutide) is commercially available in the United States, Europe and Japan. Another example of a GLP-1 receptor agonist is (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 analogs having insulinotropic activity are collectively referred to as "GLP-1."

[0932] The molecule exenatide has the amino acid sequence of incretin analog-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 (e.g., including incretin analog-3, incretin analog-4, and incretin analog-4-amide), exenatide peptide derivatives, and exenatide peptide analogs are collectively referred to as "exenatide."

[0933] Peptide YY (PYY) is a 36 amino acid residue peptide amide. PYY inhibits intestinal motility and blood flow (Laburthe, M., Trends Endocrinol Metab. 1(3): 168-74 (1990), regulates intestinal secretion (Cox, HM et al., Br J Pharmacol 101(2): 247-52 (1990); Playford, RJ et al., Lancet 335(8705): 1555-7 (1990)), and stimulates net absorption (MacFayden, RJ et al., Neuropeptides 7(3): 219-27 (1986)). Two major in vivo variants, PYY(1-36) and PYY(3-36), have been identified (e.g., Eberlein, GA et al., Peptides 10(4), 797-803 (1989). The sequences of PYY and its peptide analogs and peptide derivatives are known in the art (eg, US Pat. Nos. 5,574,010 and 5,552,520).

[0934] Oxyntomodulin is a naturally occurring 37-amino acid peptide hormone found in the colon that has been found to suppress appetite and promote weight loss (Wynne K et al., Int J Obes (Lond) 30(12):1729-36 (2006)). The sequences of oxyntomodulin and 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 Publication Nos. 2005 / 0070469 and 2006 / 0094652).

[0935] Gastric inhibitory peptide (GIP) is an insulinotropic peptide hormone (Efendic, S. et al., Horm Metab Res. 36: 742-6 (2004)) and is secreted by the mucosa of the duodenum and jejunum in response to absorbed fats and carbohydrates that stimulate the pancreas to secrete insulin. GIP circulates as a biologically active 42 amino acid peptide. GIP is also known as glucose-dependent insulinotropic protein. GIP is a 42 amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose (Tseng, C. et al., PNAS 90: 1992-1996 (1993)). The sequences of GIP and its peptide analogs and peptide 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)).

[0936] Glucagon is a peptide hormone produced by the alpha cells of the pancreas that increases glucose concentration in the bloodstream. Its action is opposite to that of insulin, which lowers glucose concentration. When blood glucose concentrations are too low, the pancreas releases glucagon. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. High blood glucose levels stimulate the release of insulin. Insulin allows insulin-dependent tissues to take up and use glucose. Thus, glucagon and insulin are part of a feedback system that keeps blood glucose levels stable.

[0937] Pharmaceutically acceptable compositions

[0938] According to another embodiment, the present invention provides a composition comprising a compound of the present invention, i.e., an isolated polypeptide or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle. The amount of the compound in the composition of the present invention is such that one or more amylin and / or calcitonin receptors in a biological sample or patient can be effectively and measurably activated. In certain embodiments, the amount of the compound in the composition of the present invention is such that human amylin 3 receptor (hAMY3) and / or human calcitonin receptor (hCTR) in a biological sample or patient can be effectively and measurably activated 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 to be administered to a patient by injection. 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.

[0939] As used herein, the term "patient" or "subject" refers to an animal, preferably a mammal, and most preferably a human.

[0940] "Pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester, or other derivative of a compound of the invention that is capable of providing, directly or indirectly, a compound of the invention or an inhibitory active metabolite or residue thereof when administered to a recipient.

[0941] The isolated polypeptides disclosed herein (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 comprise the isolated polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically 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 standard reference text in the field, the latest edition of Remington's Pharmaceutical Sciences, 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. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, they are contemplated for use in the composition. Supplementary active compounds can also be incorporated into the compositions.

[0942] Pharmaceutical composition of the present invention is formulated into and is compatible with its expected route of administration.The example of route of administration includes parenteral, such as intravenous, intradermal, subdermal, subcutaneous, oral (such as inhalation), transdermal (that is, local), transmucosal, rectal or their combination.In some embodiments, pharmaceutical composition of the present invention or separated polypeptide are formulated for and are used by topical application.In some embodiments, pharmaceutical composition of the present disclosure or separated polypeptide are formulated for and are used by inhalation administration.In some embodiments, pharmaceutical composition is formulated for and is used by device or other suitable delivery mechanism that is suitable for subdermal or subcutaneous implantation and subcutaneous delivery of pharmaceutical composition.In some embodiments, pharmaceutical composition is formulated for and is used by implant device that is suitable for subdermal or subcutaneous implantation and subcutaneous delivery of pharmaceutical composition.In some embodiments, pharmaceutical composition is formulated for and is used by osmotic delivery device, such as, is suitable for subdermal or subcutaneous placement or other implantation and subcutaneous delivery of pharmaceutical composition implantable osmotic delivery device uses. Solutions or suspensions for parenteral administration, intradermal administration, subdermal administration, subcutaneous administration, or combinations thereof may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; and an agent for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0943] The pharmaceutical composition that is suitable for injectable use includes sterile aqueous solution (when water-soluble) or dispersion and the sterile powder for the temporary preparation of sterile injectable solution or dispersion.About intravenous administration, suitable carrier includes physiological saline, antibacterial water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).In all cases, the composition must be sterile and should be fluid, and its mobility reaches the degree of easy injection. It must be stable under manufacturing and storage conditions, and must protect it from the contamination of microorganisms such as bacteria and fungi. Carrier can be a solvent or dispersion medium containing such as water, ethanol, polyol (such as glycerol, propylene glycol and liquid polyethylene glycol etc.) and its suitable mixture. Suitable mobility can be, for example, by using the coating of such as lecithin, by maintaining the required particle size and by using a surfactant to maintain. The prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0944] Sterile injectable solutions can be prepared by combining the desired amount of the active compound in an appropriate solvent with one or a combination of the ingredients listed above, and optionally followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and freeze drying are methods of preparation that produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0945] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be combined with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier suitable for use as a mouthwash, wherein the compound in the fluid carrier is administered orally and gargled and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant substances may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: a binder, such as microcrystalline cellulose, tragacanth gum, or gelatin; an excipient, such as starch or lactose; a disintegrant, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or sterotes; a slip agent, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring, such as peppermint, methyl salicylate, or orange flavoring.

[0946] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0947] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and, for transmucosal administration, include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel, or cream, as is generally known in the art.

[0948] In one embodiment, the active compound is prepared with a carrier that will protect 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 purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These substances can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0949] It is particularly advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for treating individuals.

[0950] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0951] Drug particle preparations

[0952] In some embodiments, a pharmaceutical composition comprising any of the disclosed polypeptides formulated as a trifluoroacetate salt, an acetate salt, or a hydrochloride salt is provided. In some embodiments, a pharmaceutical composition comprising any of the disclosed polypeptides formulated as a trifluoroacetate salt is provided. In some embodiments, a pharmaceutical composition comprising any of the disclosed polypeptides formulated as an acetate salt is provided. In some embodiments, a pharmaceutical composition comprising any of the disclosed polypeptides formulated as a hydrochloride salt is provided.

[0953] The compound used in the practice of the present invention, i.e., an isolated polypeptide or a pharmaceutically acceptable salt thereof, is typically added to a particle formulation, which is used to produce particles containing the polypeptide 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 in a particle formulation and converted (e.g., spray-dried) into particles. In some embodiments, particles containing the amylin analog polypeptide are suspended in a vehicle formulation to produce a suspension formulation of the vehicle and suspended particles containing the amylin analog polypeptide.

[0954] Preferably, the particle formulation is formed into particles using a process such as spray drying, freeze drying, dehumidification, freeze drying, grinding, granulation, ultrasonic droplet formation, crystallization, precipitation, or other techniques available for forming particles from a mixture of components. In one embodiment of the invention, the particles are spray dried. The particles are preferably substantially uniform in shape and size.

[0955] In some embodiments, the present invention provides pharmaceutical particle formulations for pharmaceutical use. The particle formulations typically contain a drug and include 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 experimentally based on the activity of the stabilizer and the desired characteristics of the formulation, in view of the teachings of this specification.

[0956] In any of the embodiments, the particle formulation can comprise about 50 wt% to about 90 wt% drug, about 50 wt% to about 85 wt% drug, about 55 wt% to about 90 wt% drug, about 60 wt% to about 90 wt% drug, about 65 wt% to about 85 wt% drug, about 65 wt% to about 90 wt% drug, about 70 wt% to about 90 wt% drug, about 70 wt% to about 85 wt% drug, about 70 wt% to about 80 wt% drug, or about 70 wt% to about 75 wt% drug.

[0957] Typically, the amount of carbohydrate in the particle formulation is determined by aggregation issues. Generally, the amount of carbohydrate should not be too high to avoid promoting crystal growth in the presence of water due to excess carbohydrate not bound to the drug.

[0958] Typically, the amount of antioxidant in the particle formulation is determined by oxidation issues, while the amount of amino acid in the formulation is determined by oxidation issues and / or formability of the particles during spray drying.

[0959] Typically, the amount of buffer in the particle formulation is determined by pre-processing issues, stability issues, and formability of the particles during spray drying. When all stabilizers are dissolved, a buffer may be required to stabilize the drug during processing, such as solution preparation and spray drying.

[0960] Examples of carbohydrates that may be included in particle formulations include, but are not limited to, monosaccharides (e.g., fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (e.g., lactose, sucrose, trehalose, and cellobiose), polysaccharides (e.g., raffinose, melezitose, maltodextrin, polydextrose, and starch), and alditols (non-cyclic polyols; e.g., mannitol, xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol, and inositol). Suitable carbohydrates include disaccharides and / or non-reducing sugars, such as sucrose, trehalose, and raffinose.

[0961] Examples of antioxidants that may be included in the particle formulation include, but are not limited to, methionine, ascorbic acid, sodium thiosulfate, catalase, platinum, ethylenediaminetetraacetic acid (EDTA), citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate. In addition, amino acids that are easily oxidized, such as cysteine, methionine, and tryptophan, can be used as antioxidants.

[0962] Examples of amino acids that may be included in the particle formulation include, but are not limited to, arginine, methionine, glycine, histidine, alanine, leucine, glutamic acid, isoleucine, L-threonine, 2-phenylindole, 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.

[0963] Examples of buffers that may be included in the particle formulation include, but are not limited to, citrate, histidine, succinate, phosphate, maleate, tris, acetate, carbohydrates, and gly-gly. Suitable buffers include citrate, histidine, succinate, and tris.

[0964] 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.

[0965] Additionally, the particle formulation may include other stabilizers / excipients such as surfactants and salts. Examples of surfactants include, but are not limited to, polysorbate 20, polysorbate 80, (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.

[0966] The particles are typically sized so that they can be delivered via an implantable osmotic delivery device. The uniform shape and size of the particles typically contribute to providing a consistent and uniform release rate from such a delivery device; however, particle preparations with an abnormal particle size distribution profile may also be used. For example, in a typical implantable osmotic delivery device with a delivery orifice, the size of the particles is about 30% smaller than the delivery orifice diameter, more preferably about 20% smaller, more preferably about 10% smaller. In one embodiment of a particle preparation for an osmotic delivery system, wherein the delivery orifice diameter of the implant is about 0.5 mm, the particle size may be, for example, less than about 150 microns to about 50 microns. In one embodiment of a particle preparation for an osmotic delivery system, wherein the delivery orifice diameter of the implant is about 0.1 mm, the particle size may 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.

[0967] Those skilled in the art will appreciate that particle populations follow the principles of particle size distribution. Widely used, art-recognized methods for describing particle size distribution include, for example, mean diameter and D values, such as D50 values, which are commonly used to represent the mean diameter of a range of particle sizes for a given sample.

[0968] The particles of the particle formulation have a diameter between about 2 microns and about 150 microns, such as 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.

[0969] 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, wherein each particle has a diameter of less than about 50 microns.

[0970] Typically, when incorporated into a suspension vehicle, at the delivery temperature, the particles of the particle formulation will not settle in less than about 3 months, preferably will not settle in less than about 6 months, more preferably will not settle in less than about 12 months, more preferably will not settle in less than about 24 months, and most preferably will not settle in less than about 36 months at the delivery temperature. The suspension vehicle typically has a viscosity of about 5,000 to about 30,000 poise, preferably about 8,000 to about 25,000 poise, more preferably about 10,000 to about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise ± about 3,000 poise. In general, smaller particles tend to have a lower sedimentation rate in a viscous suspension vehicle than larger particles. Therefore, micron to nanometer sized particles are generally required. In viscous suspension formulations, based on simulation modeling studies, particles of about 2 microns to about 7 microns of the present invention will not settle for at least 20 years at room temperature. In one embodiment of the particle formulation of the present invention for use in an implantable osmotic delivery device, particles having a size less than about 50 microns, more preferably less than about 10 microns, and more preferably in the range of about 2 microns to about 7 microns are included.

[0971] In summary, the disclosed polypeptides or pharmaceutically acceptable salts thereof are formulated into dry powders in the form of solid particles that retain maximum chemical and biological stability of the drug. The particles provide long-term storage stability at high temperatures and thus allow for the delivery of stable and biologically effective drugs to subjects for extended periods of time. The particles are suspended in a suspension vehicle for administration to a patient.

[0972] Particle suspension in vehicle

[0973] 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 comprises one or more polymers and one or more solvents that form a solution having a viscosity sufficient to uniformly suspend the drug-containing particles. The suspension vehicle may comprise other components, including but not limited to surfactants, antioxidants, and / or other compounds soluble in the vehicle.

[0974] The viscosity of the suspension vehicle is generally sufficient to prevent sedimentation of the drug particle formulation during storage and use in a delivery method, such as an implantable osmotic delivery device. The suspension vehicle is biodegradable because the suspension vehicle disintegrates or breaks down over a period of time in response to the biological environment while the drug particles dissolve in the biological environment and absorb the active pharmaceutical ingredient (i.e., drug) in the particles.

[0975] In embodiments, the suspension vehicle is a "single-phase" suspension vehicle, which is a solid, semisolid, or liquid homogeneous system that is physically and chemically uniform throughout.

[0976] The solvent in which the polymer is dissolved can affect the characteristics of the suspension formulation, such as the behavior of the drug particle formulation during storage. The solvent can be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation upon contact with an aqueous environment. In some embodiments of the present invention, the solvent can be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation upon contact with an aqueous environment having less than approximately 10% water.

[0977] The solvent may be an acceptable solvent that is not miscible with water. The solvent may also be selected so that the polymer is soluble in the solvent at high concentrations, for example, at a polymer concentration greater than about 30%. Examples of solvents that can be used in the practice of the present invention include, but are not limited to, lauryl alcohol, benzyl benzoate, benzyl alcohol, lauryl lactate, decanol (also known as decyl alcohol), ethyl hexyl lactate, and long-chain (C8 to C24) aliphatic alcohols, esters, or mixtures thereof. The solvent used in the suspension vehicle may be "dry" in that it has a low moisture content. Preferred solvents for preparing the suspension vehicle include lauryl lactate, lauryl alcohol, benzyl benzoate, and mixtures thereof.

[0978] Examples of polymers for preparing suspension vehicles of the present invention include, but are not limited to, polyesters (e.g., polylactic acid and polylactic polyglycolic acid), pyrrolidone-containing polymers (e.g., polyvinyl pyrrolidone having a molecular weight in the range of about 2,000 to about 1,000,000), esters or ethers of unsaturated alcohols (e.g., vinyl acetate), polyoxyethylene polyoxypropylene block copolymers, or mixtures thereof. Polyvinyl pyrrolidone can be characterized by its K value (e.g., K-17), which is a viscosity index. In one embodiment, the polymer is a polyvinyl pyrrolidone having a molecular weight of 2,000 to 1,000,000. In a preferred embodiment, the polymer is polyvinyl pyrrolidone K-17 (typically having an approximate average molecular weight range of 7,900-10,800). The polymer used in the suspension vehicle may include one or more different polymers or may include different grades of a single polymer. The polymer used in the suspension vehicle may also be dry or have a low moisture content.

[0979] In general, the composition of the suspension vehicle used in the present invention can vary based on the desired performance characteristics. In one embodiment, the suspension vehicle can comprise from about 40 wt% to about 80 wt% polymer and from about 20 wt% to about 60 wt% solvent. Preferred embodiments of the suspension vehicle include vehicles formed from polymer and solvent combined in the following ratios: about 25 wt% solvent and about 75 wt% polymer; about 50 wt% solvent and about 50 wt% polymer; about 75 wt% solvent and about 25 wt% polymer. Thus, in some embodiments, the suspension vehicle can comprise, and in other embodiments consist essentially of, the selected components.

[0980] Suspension vehicles can exhibit Newtonian behavior. Suspension vehicles are typically formulated to provide a viscosity that maintains a uniform dispersion of particle preparations for a predetermined time period. This helps facilitate the adjustment of suspension formulations to provide controlled delivery of the drug contained in the pharmaceutical particle preparation. The viscosity of the suspension vehicle can vary depending on the desired application, the size and type of particle preparation, and the loading of the particle preparation in the suspension vehicle. The viscosity of the suspension vehicle can be varied by changing the type or relative amount of the solvent or polymer used.

[0981] The suspension vehicle may have a viscosity ranging from about 100 poise to about 1,000,000 poise, preferably from about 1,000 poise to about 100,000 poise. In a preferred embodiment, the suspension vehicle typically has a viscosity of between about 5,000 and about 30,000 poise at 33°C, preferably between about 8,000 and about 25,000 poise, and more preferably between about 10,000 and about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise ± about 3,000 poise at 33°C. Viscosity can be measured using a parallel plate rheometer at a shear rate of 10-4 / sec at 33°C.

[0982] When in contact with an aqueous environment, the suspension vehicle may exhibit phase separation; however, typically the suspension vehicle exhibits substantially no phase separation as a function of temperature. For example, at temperatures in the range of about 0°C to about 70°C and after temperature cycling, e.g., from 4°C to 37°C to 4°C, the suspension vehicle typically exhibits no phase separation.

[0983] Suspension vehicles can be prepared by combining polymers and solvents under dry conditions, for example, in a drying oven. Polymers and solvents can be combined at high temperatures, for example, at about 40°C to about 70°C, and liquefied and formed into a single phase. The ingredients can be blended under vacuum to remove the bubbles produced by the dry ingredients. Conventional mixers, such as double helical blades or similar mixers, set at a speed of about 40rpm can be used to combine the ingredients. However, higher speeds can also be used to mix the ingredients. Once a liquid solution of the ingredients is obtained, the suspension vehicle can be cooled to room temperature. Differential scanning calorimetry (DSC) can be used to verify that the suspension vehicle is a single phase. In addition, the components of the vehicle (such as solvents and / or polymers) can be processed to substantially reduce or substantially remove peroxides (such as by treating with methionine; referring to, for example, U.S. Patent Application Publication No. 2007-0027105).

[0984] The drug particle formulation is added to the suspension vehicle to form a suspension formulation. In some embodiments, the suspension formulation may comprise, and in other embodiments consist essentially of, the drug particle formulation and the suspension vehicle.

[0985] Suspension preparations can be prepared by dispersing the particle preparations in a suspension vehicle. The suspension vehicle can be heated and the particle preparations can be added to the suspension vehicle under dry conditions. The ingredients can be mixed under vacuum at high temperatures, for example, at about 40°C to about 70°C. The ingredients can be mixed at a sufficient speed, for example, at about 40rpm to about 120rpm, and for a sufficient amount of time, for example, about 15 minutes, to obtain a uniform dispersion of the particle preparations in the suspension vehicle. The mixer can be a double helical 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 preparation, and cooled to room temperature before further use, for example, being loaded into an implantable drug delivery device, a unit dose container, or a multidose container.

[0986] Suspension formulations typically have a total moisture content of less than about 10 wt%, preferably less than about 5 wt%, and more preferably less than about 4 wt%.

[0987] In preferred embodiments, the suspension formulations of the present invention are substantially homogeneous and flowable to provide for delivery of the drug particle formulation from an osmotic delivery device to a subject.

[0988] In summary, the components of the suspension vehicle provide biocompatibility. The components of the suspension vehicle provide suitable chemical and physical properties to form a stable suspension of the drug particle formulation. These properties include (but are not limited to) the following: viscosity of the suspension; purity of the vehicle; residual moisture of the vehicle; density of the vehicle; compatibility with dry powders; compatibility with implantable devices; molecular weight of the polymer; stability of the vehicle; and hydrophobicity and hydrophilicity of the vehicle. These properties can be manipulated and controlled, for example, by varying the vehicle composition and manipulating the ratios of the components used in the suspension vehicle.

[0989] Suspension formulations described herein can be used in implantable osmotic delivery devices to provide zero-order, continuous, controlled and sustained delivery of a compound over an extended period of time, such as over several weeks, several months, or up to about a year or longer. Such implantable osmotic delivery devices are typically capable of delivering a suspension formulation comprising a drug at a desired flow rate over a desired time period. Suspension formulations can be loaded into an implantable osmotic delivery device by conventional techniques.

[0990] Implantable delivery

[0991] The dose and delivery rate can be selected to achieve a desired blood concentration of the drug, generally within less than about 6 half-lives of the drug in the subject following implantation of the device. The blood concentration of the drug is selected to obtain the optimal therapeutic effect of the drug while avoiding undesirable side effects that may be induced by excessive concentrations of the drug, while at the same time avoiding peaks and troughs that may induce side effects associated with peak or trough plasma concentrations of the drug.

[0992] An implantable osmotic delivery device generally includes a reservoir having at least one orifice through which a suspension formulation is delivered. The suspension formulation can be stored within the reservoir. In a preferred embodiment, the implantable drug delivery device is an osmotic delivery device, wherein drug delivery is driven by osmosis. Some osmotic delivery devices and their components have been described, for example delivery device or similar device (see, e.g., U.S. Pat. Nos. 5,609,885; 5,728,396; 5,985,305; 5,997,527; 6,113,938; 6,132,420; 6,156,331; 6,217,906; 6,261,584; 6,270,787; 6,287,295; 6,375,978; 6,395,292; 6,508,808; 6,544, 252; 6,635,268; 6,682,522; 6,923,800; 6,939,556; 6,976,981; 6,997,922; 7,014,636; 7,207,982; and 7,112,335; 7,163,688; U.S. Patent Publication Nos. 2005 / 0175701, 2007 / 0281024, 2008 / 0091176, and 2009 / 0202608).

[0993] Osmotic delivery devices typically consist of a cylindrical reservoir containing an osmotic engine, a piston, and a drug formulation. The reservoir is capped at one end by a rate-controlled semipermeable membrane and at the other end by a diffusion regulator that releases a suspension formulation containing the drug 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 chamber from entering the drug reservoir. The diffusion regulator is designed to be combined with the drug formulation to prevent body fluids from entering the drug reservoir through the orifice.

[0994] Osmotic devices release drugs at a predetermined rate based on the principle of osmosis. Extracellular fluid enters the osmotic delivery device through a semipermeable membrane and directly enters a salt engine that can swell and drive a piston at a slow and steady delivery rate. The piston motion causes the drug formulation to be released or leave the orifice at a predetermined shear rate. In one embodiment of the invention, the reservoir of the osmotic device is loaded with a suspension formulation, wherein the device can deliver a suspension formulation to the subject at a predetermined therapeutically effective delivery rate over a prolonged period of time (e.g., about 1, about 3, about 6, about 9, about 10, and about 12 months).

[0995] The rate of release of the drug from an osmotic delivery device generally provides a predetermined target dose of the drug to the subject, such as a therapeutically effective daily dose delivered over the course of a day; that is, the rate of release of the drug from the device provides substantially steady-state delivery of therapeutic concentrations of the drug to the subject.

[0996] 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 even more preferably between about 150 μl and about 200 μl.

[0997] Typically, for example, an osmotic delivery device is implanted subdermally or subcutaneously in a subject to provide subcutaneous drug delivery. The device can be implanted subdermally or subcutaneously in either or both arms (e.g., on the inside, outside, or back of the upper arm) or in the abdomen. A preferred location in the abdominal region is under the abdominal skin in an area extending below the ribs and above the waistline. In order to provide multiple locations for implanting one or more osmotic delivery devices in the abdomen, the abdominal wall can be divided into the following four quadrants: a right upper quadrant extending at least 2-3 cm below the ribs on the right side, e.g., at least about 5-8 cm below the ribs on the right side and at least 2-3 cm to the right of the midline, e.g., at least about 5-8 cm to the right of the midline; a right lower quadrant extending at least 2-3 cm above the waistline, e.g., at least about 5-8 cm above the waistline and at least 2-3 cm to the right of the midline, e.g., at least about 5-8 cm to the right of the midline; a left upper quadrant extending at least 2-3 cm below the ribs on the left side, e.g., at least about 5-8 cm below the ribs on the left side and at least 2-3 cm to the left of the midline, e.g., at least about 5-8 cm to the left of the midline; and a left lower quadrant extending at least 2-3 cm above the waistline, e.g., at least about 5-8 cm above the waistline and at least 2-3 cm to the left of the midline, e.g., at least about 5-8 cm to the left of the midline. This provides multiple available locations for implantation of one or more devices on one or more occasions. Implantation and removal of osmotic delivery devices are generally performed by a medical professional using local anesthesia (eg, lidocaine).

[0998] Termination of treatment by removing the osmotic delivery device from the subject is straightforward and offers the important advantage of immediately ceasing delivery of the drug to the subject.

[0999] Preferably, the osmotic delivery device has a fail-safe mechanism to prevent the unintentional excessive or high-dose delivery of medicine in the theoretical situation of blocking or obstructing the outlet (diffusion regulator) of the pharmaceutical preparation. In order to prevent the unintentional excessive or high-dose delivery of medicine, the osmotic delivery device is designed and constructed so that the pressure required for partly or completely removes or discharges the diffusion regulator from the reservoir exceeds the pressure required for partly or completely removes or discharges the semipermeable membrane, to the necessary degree of reservoir decompression. In this case, pressure will be built in the device until it will outwardly push the semipermeable membrane at the other end, thus releasing osmotic pressure. The osmotic delivery device will become static subsequently and no longer deliver pharmaceutical preparation, and its condition is that piston and reservoir are sealing relationship.

[1000] Suspension formulations can also be used in infusion pumps, e.g. (DURECT Corporation, Cupertino, Calif.) osmotic pumps, which are small infusion pumps used for continuous drug delivery in laboratory animals such as mice and rats.

[1001] Example

[1002] The following examples are set forth so as to provide one of ordinary skill in the art with a complete disclosure and description of how to practice the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, and percentage changes), but some experimental errors and deviations should be considered. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near atmospheric.

[1003] Example 1: Generation of Amylin Analog Peptides

[1004] Amylin analog polypeptides of the present invention as provided in Table 3 were synthesized in N,N-dimethylformamide (DMF) using a solid-phase method using the Fmoc strategy with N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (HATU) or 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HCTU) activation (5-fold molar excess of amino acid) on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ). 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.

[1005] The peptide was deprotected and cleaved from the solid support by treating the resin with (92.5% TFA, 2.5% phenol, 2.5% water, and 2.5% triisopropylsilane) for 2-3 hours. Cold diethyl ether was used to precipitate the cleavage peptide. The diethyl ether was decanted, and the solid was wet-ground with cold diethyl ether again and pelletized by centrifugation. The crude solid was then dissolved in a 1:1 solution of ACN / water and 0.01% TFA. Disulfide bridge formation was obtained by adding a solution of iodine / acetic acid (35 mg / ml) to the crude product until the solution color uniformly became amber. The reaction solution was stirred until the reaction was complete as indicated by LC / MS analysis. A 2% solution of ascorbic acid in H2O was added until the solution became clear. The final crude product solution was lyophilized in preparation for final purification.

[1006] The lyophilized solid was redissolved in a 1:1 solution of acetonitrile / water and 0.1% TFA (10-15 mL) and analyzed by reverse phase HPLC on a Waters XBridge TM BEH 130, CIS, 10pm, Purification was performed on a 30 x 250 mm ID column using a 30 gradient ranging from 5-75% acetonitrile / water with 0.1% TFA over 30-45 minutes at a flow rate of 30 mL / min, λ-215 nm.

[1007] Example 2: Purification and characterization of amylin analog polypeptides, i.e., linear polypeptides without any lipophilic substituents and optional spacers

[1008] The purified product was lyophilized and analyzed by ESI-LC / MS and analytical HPLC and proved to be pure (>98%).The mass results were all consistent with the calculated values.

[1009] Characterization of the peptide analogs was performed via C18 HPLC and LC / MS analysis (Acquity SQD Waters Corp, Milford, MA) and UV detection provided by dual absorbance signals at 215 nm and 280 nm using one of Method A, Method B, Method C, or Method D.

[1010] Method A, LC / MS conditions: using Phenomenex UPLC Aeris TM Peptide XB C18 35 column, 1.7 μm, 2.1×100 mm or ACQUiTY BEH300 or BEH130 CT8 column, 1.77 μm, 2.1×100 mm, using 5-65% acetonitrile / water with 0.05% TFA over 30 minutes at a flow rate of 0.5 mL / min, λ-215 nm, 280 nm.

[1011] Method B, C18 HPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm, at 25° C., 5-65% acetonitrile / water containing 0.05% TFA over 30 minutes, at a flow rate of 0.5 mL / min, at 1215 nm, 1280 nm.

[1012] Method C, UPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm, at 25° C., 5-65% acetonitrile / water containing 0.05% TFA over 20 minutes, at a flow rate of 0.5 mL / min, at 1215 nm and 1280 nm.

[1013] Method D, UPLC conditions: UPLC analysis was performed on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm, at 25° C. using 5-65% acetonitrile / water containing 0.05% TFA over 10 minutes at a flow rate of 0.5 mL / min at 1215 nm and 1280 nm. 5.0 μL of sample was injected using the PLNO (partial loop with needle overfilled) injection mode.

[1014] Table 3 provides exemplary amylin analog polypeptides of the invention.

[1015] Polypeptide analogs without lipophilic substituents and optional spacers are sometimes referred to herein as "linear polypeptides." Polypeptide analogs with at least one covalently bound lipophilic substituent and optional spacer are sometimes referred to herein as "conjugated polypeptides."

[1016] Example 3: Synthesis of amylin analog polypeptide intermediates

[1017] Synthesis of polypeptides with modifications at positions such as D-Lys16 or L-Lys16 (Compounds B2, A129, B4, and B8)

[1018] After the synthesis of the linear peptide described in Example 1 was completed, the resin was washed with dichloromethane (DCM) and dried under vacuum for 30 minutes. For analogs containing an allyloxycarbonyl protecting group, its removal was facilitated by Pd(PPh3)3 in a solution of (chloroform / acetic acid / n-methylmorpholine, 37:2:1). The deprotected resin was washed with 2% sodium diethyldisulfide dicarbamate trihydrate / DMF (6 x 30 seconds), 2% DIEA / DMF (6 x 30 seconds), and finally DMF (6 x 30 seconds). Extension of the spacer region was performed in a stepwise manner with manual addition of each building block under pre-activated conditions. To 1 ml of a 200 mmol solution of Fmoc-γGlu-(OH)-OtBu in DMF was added 0.5 ml of DIEA (800 mmol), followed by 0.5 ml of HCTU (400 mmol), and the resulting reaction solution was stirred for 5 minutes, where it was added to the deprotected residue on the linear sequence. Under nitrogen, stirred reaction mixture 30 minutes. Discharge resin and wash with DMF (6 × 30 seconds). Use 20% piperidines / DMF to promote the subsequent removal of Fmoc protecting group, then carry out final washing with DMF (6 × 30 seconds). Carry out final deprotection and the cracking of peptide from solid support by treating resin 2-3 hour with (95% TFA, 2% water, 2% thioanisole and 1% triisopropylsilane). Along with using the method described above to be incorporated to each structural unit, separate and characterize each intermediate via HPLC / MS.

[1019] For example, synthesis of polypeptides with modifications at the N-terminus (A130, B22, and B23)

[1020] The synthesis of the linear sequence was carried out as described in Example 1. The addition of the albumin binding moiety was promoted by removing 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 block was carried out in a stepwise manner under pre-activation conditions. 0.5 ml of DIEA (800 mmol) was added to a 200 mmol solution of 1 ml of Fmoc-γGlu-(OH)-OtBu in DMF, followed by 0.5 ml of HCTU (400 mmol). The resulting reaction solution was stirred for 5 minutes, wherein it was added to the deprotected linear sequence. The reaction mixture was stirred under nitrogen for 30 minutes. The resin was discharged and washed with DMF (6 × 30 seconds). The final deprotection and cleavage of the peptide from the solid support were carried out by treating the resin with (95% TFA, 2% water, 2% thioanisole and 1% triisopropylsilane) for 2-3 hours. Each intermediate was separated and characterized via HPLC / MS. The chemical data of both the internally modified polypeptide and the N-terminally modified polypeptide intermediate are recorded in Table 5 below.

[1021] Table 5: Exemplary intermediate compounds

[1022]

[1023] Example 4: Covalent attachment of a lipophilic substituent and an optional spacer to an amylin analog polypeptide, ie, conversion of a linear polypeptide into a conjugated polypeptide

[1024] The synthesis of amylin analog polypeptides conjugated to one or more albumin binding lipophilic substituents and an optional spacer was performed with modifications to the synthetic method described in Example 1 .

[1025] After the synthesis of the linear polypeptide as described in Example 1 is completed, the resin is washed with dichloromethane (DCM) and dried under vacuum for 30 minutes. For analogs containing allyloxycarbonyl protecting groups, their removal is achieved via a solution of Pd(PPh3)3 in (chloroform / acetic acid / n-methyl-morpholine, 37:2:1). For analogs containing BOC-Lys(Fmoc)-OH, the Fmoc protecting group is removed using 20% ​​piperidine / DMF. The resulting deprotected resin is washed with DMF (6×30 seconds). Next, the extension of the spacer region is performed by manually adding each structural unit under pre-activation conditions in a stepwise manner. The addition of lipophilic substituents (also referred to as "acyl chains") is performed under normal SPPS conditions without a pre-activation step. The final deprotection and cleavage of the peptide from the solid support are performed by treating the resin with (95% TFA, 2% water, 2% thioanisole and 1% triisopropylsilane) for 2-3 hours. The cleavage peptide is precipitated using cold diethyl ether. The diethyl ether was decanted and the solid was triturated again with cold diethyl ether and pelletized by centrifugation.

[1026] 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 each crude peptide product solution until the solution color uniformly changed to amber. The reaction solution was stirred until LC / MS analysis indicated the desired disulfide bridges had formed. A 2% solution of ascorbic acid in H2O was added to the reaction solution until the solution became clear. The solution was frozen and lyophilized. Purification was achieved by the method described in Example 1.

[1027] An exemplary synthesis of conjugated peptides is described. Synthesis of A109: The synthesis of the linear sequence was performed as described in Example 1. The addition of the albumin binding moiety was facilitated by removing the N-terminal Fmoc protecting group via a 20% solution of piperidine / DMF. The resin was then washed with DMF and the incorporation of the side chain building blocks was performed in a stepwise manner under pre-activated conditions. To a 200mmol solution of 1ml Fmoc-γGlu-(OH)-OtBu in DMF was added 0.5ml DIEA (800mmol) followed by 0.5ml HCTU (400mmol). The resulting reaction solution was stirred for 5 minutes, whereupon it was added to the deprotected linear sequence. The reaction mixture was stirred under nitrogen for 30 minutes. Next, the resin was drained and washed with DMF (6×30 seconds). The Fmoc protecting group of γGlu was removed by 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 normal solid phase conditions.

[1028] 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 crude peptide product until the solution color became uniformly amber. The reaction solution was stirred until LC / MS analysis indicated that the desired disulfide bridges had formed. A 2% solution of ascorbic acid in H2O was added to the reaction solution until the solution became clear.

[1029] The crude product was lyophilized to give an off-white solid, which was purified via the method described in Example 2.

[1030] Example 5: Stability of Amylin Analog Polypeptides

[1031] Several amylin analog polypeptides described herein were tested for stability as trifluoroacetate salts in DMSO (i.e., an organosulfur solvent) or in 1 mg / ml aqueous solution (i.e., in deionized water). These analog polypeptides were incubated at 37° C., and samples were withdrawn at various time intervals and analyzed by LC / MS and HPLC to determine the purity and quality of the parent peptide and the extent of any degradation products. The purity results of these analyses are shown in Tables 6A and 6B and are considered indicative of stability.

[1032] Table 6A: Stability of Amylin Analog Peptides

[1033]

[1034]

[1035] Table 6B: Stability of Amylin Analog Peptides

[1036]

[1037] Example 6: Solubility of Amylin Analog Peptides

[1038] The solubility of the analog polypeptides described herein was tested in saline 20% DMSO in water (i.e., bioassay buffer) or in aqueous solution (deionized water) at room temperature. The samples were visually inspected for sample clarity, turbidity, or any appearance of turbidity. The results of this analysis are shown in Table 7.

[1039] Table 7. Solubility of Amylin Analog Peptides

[1040]

[1041] Example 7: Functional Assays: Human Calcitonin Receptor and Amylin 3 Receptor

[1042] Activation of the human calcitonin receptor (hCTR) or the 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 nonspecific cAMP / cGMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Therefore, it is possible to estimate the in vitro potency (pEC50) of peptides for activating each of these receptors using fitted dose-response curves of cAMP accumulation.

[1043] Cells, culture, and cAMP assays

[1044] HEK293-CNG cells stably expressing human calcitonin receptor (hCTR) or co-expressed 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 90% DMEM, 10% FBS, 250 μg / ml G418, 1 μg / ml puromycin, 150 μg / ml hygromycin B (hAMY3R cells). Cells were maintained in growth medium for no more than 10 passages before testing.

[1045] On the day of the assay, cells expressing hCTR or hAMY3R were counted and plated at 500 cells per well in white 384-well OptiPlates (PerkinElmer #6007299) 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).

[1046] Peptides were serially diluted in the same buffer as above for each given assay condition. Two assay control solutions consisting of 50 mcM forskolin (cAMP system maximum) or assay buffer alone (cAMP system minimum) were also prepared in the appropriate stimulation buffer. Five microliters of each peptide concentration or assay control were added to triplicate wells and incubated at room temperature for thirty minutes. During this incubation step, 4× europium-labeled cAMP tracer solution and 4× tau were prepared according to the manufacturer's protocol (PerkinElmer LANCE Ultra cAMP kit). - Anti-cAMP solution (made with Ulight TMThe plate was incubated in an Envision fluorescent plate reader (PerkinElmer) for 60 minutes.

[1047] Data Analysis

[1048] Test values ​​were first normalized to the mean and system minimum cAMP system values ​​induced by forskolin using the following formula in Excel: (test value - mean system minimum) / (mean system maximum - mean system minimum) × 100. Normalized test values ​​represent the baseline-corrected percentage of the maximum system cAMP response induced by forskolin. Normalized data were analyzed from triplicate assays and used to estimate the EC50 for each peptide at each receptor. Data were fit using a 4-parameter logistic curve model in GraphPad Prism software (v7.04): Y = bottom + (top - bottom) / (1 + 10^((LogEC50 - X))). Hill slope was constrained to 1.0. EC50 values ​​were converted to pEC50 values ​​using the following formula: pEC50 = -Log(EC50).

[1049] Data Description

[1050] For a given receptor, in vitro potency estimates in the absence of HSA (pEC50) were comparable across all peptides because they reflected albumin-independent binding (free peptide) values ​​(0% HSA, Table 8). However, in the presence of albumin (BSA or HSA), potency measurements across the acylated peptides were not readily comparable (Table 8). This is due to the variability in albumin-acyl peptide binding efficiency, which generally depends on the amino acid sequence, acyl binding motif, attachment site, and linker length engineered into each peptide. However, for a given conjugated polypeptide, decreased potency (decreased pEC50 values) in the presence of HSA relative to the absence of HSA qualitatively indicates an albumin-peptide interaction (Table 8, Table 8). Figure 2A and Figure 2B In contrast, linear peptides (pramlintide, human calcitonin) were not affected by the presence of HSA (Table 8, Figure 2C and Figure 2D ), reflecting its poor albumin binding efficiency.

[1051] Table 8: Potency estimates (pEC) of conjugated and linear peptides measured on human hAMY3 and hCTR in the absence and presence of human serum albumin 50 value)

[1052]

[1053]

[1054] *Non-acylated

[1055] Example 8: Intravenous Infusion of "Linear" (i.e., Non-acylated) Amylin Analog Polypeptides: Pharmacokinetic Study to Evaluate Renal Clearance (CL) of Linear Amylin Analog Polypeptides

[1056] The peptide was dissolved in sterile saline and administered to non-fasted male Sprague-Dawley rats (n=3 per group) via a femoral vein catheter at a final dose of 0.100 mg / kg as a 3-hour intravenous infusion. The formulation was administered at a rate of 1.67 mL / kg / h. Blood samples (approximately 250 μL) were collected via a jugular vein catheter 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-term method) or 1, 1.5, 2, 2.5, and 3 hours after the start of the infusion (steady-state screening method) for pharmacokinetic analysis. All samples were collected in micro-blood tubes containing K2EDTA as an anticoagulant and 25 μL of a 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 and in a few examples in Figures 3a-3b.

[1057] Example 9: Intravenous Infusion of Conjugated (i.e., Acylated) Amylin Analog Polypeptides: Pharmacokinetic Study to Evaluate Renal Clearance (CL) of Conjugated Amylin Analog Polypeptides

[1058] The peptide was dissolved in sterile saline and administered to non-fasted male Sprague Dawley rats (n=3 per group) as an intravenous infusion over 1 hour at a final dose of 0.033 mg / kg via a femoral vein catheter. The formulation was administered at a rate of 1.67 mL / kg / h. Blood samples (approximately 250 μL) were collected via a jugular vein catheter 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 into micro-blood tubes containing K2EDTA as an anticoagulant and 25 μL of a protease inhibitor cocktail for pharmacokinetic analysis. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 9 and in a few examples in Figures 3a-3b.

[1059] Table 9: Pharmacokinetic analysis

[1060]

[1061]

[1062] Example 10: Subcutaneous Infusion: Pharmacokinetic Study to Evaluate Renal Clearance (CL) of Amylin Analog Peptides

[1063] The peptide was dissolved in sterile saline and administered to non-fasted male Sprague Dawley rats (n=3 per group) at a final dose of 0.033 mg / kg as a 1-hour subcutaneous infusion via a catheter placed in the subcutaneous space between the shoulder blades. The formulation was administered at a rate of 0.145 mL / h / kg. Blood samples (approximately 250 μL) were collected via jugular vein cannula at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 24, 30, and 48 hours after the start of the infusion into micro-blood tubes containing K2EDTA as an anticoagulant and 25 μL of a protease inhibitor cocktail for pharmacokinetic analysis. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 9.

[1064] Example 11: Subcutaneous bolus injection: Pharmacokinetic study to evaluate the renal clearance (CL) of amylin analog peptides

[1065] The peptide was dissolved in sterile saline and administered to non-fasted male Sprague Dawley rats (n=3 per group) in the subcutaneous space between the shoulder blades at a dose of 0.3 mg / kg via a single bolus injection. Blood samples (approximately 250 μL) were collected via a jugular vein catheter at 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 24, 30, and 48 hours after administration into micro-blood collection tubes containing K2EDTA as an anticoagulant and 25 μL of a protease inhibitor cocktail for pharmacokinetic analysis. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 9.

[1066] Example 12: Method for preparing plasma samples for pharmacokinetic studies

[1067] Protein precipitation

[1068] A 60 μL aliquot of each plasma sample was placed in a 96-well plate. 6 μL of 0.5% Tween-20 was added to each well. The plate was then vortexed at 1200 rpm for 10 minutes, after which 180 μL of 0.1% TFA / 2:1 ethanol:acetonitrile containing the appropriate internal standard was added to each well. The plate was vortexed at 1300 rpm for 5 minutes and then centrifuged at 2844 × g for 10 minutes. The supernatant (180 μL) was placed in a clean 96-well plate and evaporated at 45° C. under a stream of nitrogen. The residue was reconstituted in 80 μL of 20% acetonitrile (aqueous solution) containing 0.1% formic acid.

[1069] Solid Phase Extraction

[1070] 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) that had been preconditioned with 200 mL of methanol and 200 mL of deionized water. The sample was washed sequentially with 200 mL of 5% ammonium hydroxide (aqueous) and 200 mL of 20% acetonitrile in water. The analyte was eluted with 200 mL of 5% formic acid / 75:25 acetonitrile:water. The eluent was dried under a stream of nitrogen. The residue was reconstituted in 80 μL of 20% acetonitrile (aqueous) containing 0.1% formic acid.

[1071] Example 13: LC / MS Quantification of Amylin Analog Peptides in Plasma

[1072] All calibration standards were prepared in control rat plasma containing K2EDTA and a protease inhibitor cocktail.

[1073] The HPLC-MS / MS was performed using a CTC HTS PAL autoinjector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with a column oven (Palo Alto, CA), a Valco switching valve (Houston, TX), and an AB Sciex API 5600 TripleTOF TM Samples and standards were analyzed by TurboIonSpray™ UPLC-MS / MS using a system consisting of a 4000QTrap mass spectrometer (Framingham, MA) or a Sciex API 4000QTrap mass spectrometer (Framingham, MA). Samples were injected onto a 2.1×50 mm reverse-phase C18 analytical column, typically a Waters CORTECS UPLC C18+, 1.6 μm (Waters Corporation, Milford, MA) or similar column. Chromatographic separations were achieved using a gradient method using water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid as the mobile phases. Initial conditions consisted of 90% A and 10% B. The organic component was increased to 95% B over a period of 3-4 minutes, depending on the peptide. A typical flow rate was 600 μL / min. The column temperature was kept constant at 40 or 50°C. Peptides were quantified by monitoring one or more product ions generated by multiple injections of the parent ion.

[1074] Example 14: In vivo efficacy of amylin analog polypeptides with food intake inhibition in rats

[1075] Acute food intake was continuously measured for a 72-hour period using the 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, approximately 8 weeks old, were obtained. Rats were individually housed and acclimated to a 45% high-fat diet for at least 2 weeks prior to dosing. After a 1-week acclimation period, all rats were individually housed in BioDAQ cages (Research Diets, New Brunswick, NJ) and maintained at a constant temperature (approximately 22° C.) and 30-70% relative humidity with a 12-hour light / dark cycle (lights on from 7:00 AM to 7:00 PM). Rats had ad libitum access to water and pelleted chow (Research Diets D12451i, 45 kcal% fat, Research Diets, New Brunswick, NJ). All procedures were performed in accordance with the USDA Animal Welfare Act and approved by the Mispro Institutional Animal Care and Use Committee. Animals were randomly assigned to treatment groups based on body weight (n=8 rats / group). Animals were dosed (SC bolus injection) with a specified concentration of amylin analog polypeptide or vehicle control (saline) and administered between 6:00 and 6:30 before lights out, with the hopper gated while the animals were being dosed. The hopper gate was opened and continuous data collection began immediately after dosing was completed. Data were analyzed using BioDAQ Viewer software (version 2.3.07) and, if necessary, round filters were set to reduce data noise associated with non-feeding behavior. All data were expressed as % inhibition compared to vehicle control and summarized as mean values. The statistical significance of the data was analyzed using Microsoft Excel (Redmond, WA) by a 2-sample t-test. P values ​​< 0.05 were considered to indicate significant differences between treatment groups. The results of % inhibition of acute food intake for amylin analog polypeptide compared to vehicle control are shown in Table 10.

[1076] Table 10: % Acute Food Intake Inhibition in Rats Following Acute SC Administration of Amylin Analog Polypeptides

[1077]

[1078]

[1079] nd = not determined; bold = P < 0.05 vs. vehicle

[1080] Example 15: In vivo efficacy and body weight changes in LE DIO rats after 13 days

[1081] A chronic (13 day) in vivo dose-response efficacy study was conducted in a rodent model of obesity (Long Evans (LE) diet-induced obesity (DIO) rats) to investigate the efficacy and durability of amylin analog peptides on weight loss. Male LE DIO rats (Envigo Laboratories, Inc., Indianapolis, IN) were used and were fed a high-fat diet (Teklad TD 95217, 40% kcal fat, Harlan Laboratories, Madison, WI) starting at weaning. Rats were 15-17 weeks of age at the start of the study. One rat was housed per cage and given ad libitum access to a high-fat diet (Harlan TD.95217, 4.3 kcal / g) and water, maintained on a 12 hour light / dark cycle from 5:00 AM to 5:00 PM at 21° C. and 50% relative humidity, and acclimated for at least 10 days prior to surgery. All procedures were performed in accordance with the USDA Animal Welfare Act and approved by the Mispro Institutional Animal Care and Use Committee. Body weight measurements were taken twice a week starting three days before surgery. Baseline fat mass and fat-free mass measurements were taken three days before starting peptide infusion using a QMR instrument (Echo Medical Systems, Houston, TX). Rats were randomly assigned to treatment groups (n=4-6 rats / group) based on their body fat mass percentage and / or body weight. Alzet mini-osmotic pumps (2 weeks; Model 2002, Durect Corporation, Cupertino, CA) were filled with vehicle or peptide under sterile conditions the day before surgery. 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 shoulder blades. Blunt dissection was used to create a 2-3 cm subcutaneous tunnel into which a sterile, filled, mini-osmotic pump was introduced. The skin opening was closed with skin staples. Depending on its treatment group, one or two osmotic pumps containing vehicle or peptide were implanted in each rat. Data were analyzed using one-way ANOVA comparing each group to the appropriate control group in Excel and / or Prism (GraphPad Software, Inc., La Jolla, CA). P values ​​< 0.05 were considered to indicate significant differences between treatment groups. The mean weight loss (%) compared to baseline and vehicle control (ΔΔ) from the 13-day study is shown in Table 9.

[1082] Example 16: Weight Loss Efficacy of Amylin Analog Peptides Combined with Exenatide in LE DIO Rats

[1083] A chronic study was conducted to determine the effects and persistence of continuous administration of an amylin analog polypeptide in combination with exenatide (a GLP-1 receptor agonist) on body weight after 27 days of treatment in LE DIO rats. 18-week-old male LE DIO rats (14 weeks on a high-fat diet) were subcutaneously (SC) implanted with two Alzet osmotic mini-pumps containing the indicated doses of an amylin analog polypeptide and / or exenatide (10 mcg / kg / d = ED50 for weight loss) or vehicle (20% DMSO in water) (n = 8 animals / treatment group). PK support for every-other-day dosing (eod) of an amylin analog polypeptide was given by SC injection every other day rather than mini-pump administration. All other procedures were the same as described for the previous example. The mean weight loss (%) results from the chronic combination study of exenatide compared to baseline and vehicle control (ΔΔ) are shown in Table 11.

[1084] Example 17: Antidiabetic Efficacy of Amylin Analog Peptides Combined with Exenatide in ZDF Rats

[1085] A chronic study was conducted to determine the antidiabetic effect of a combination of amylin analog peptide and exenatide on HbA1c (a primary antidiabetic parameter) after 27 days of treatment in Zucker diabetic fatty (ZDF) rats. Six-week-old male ZDF rats were obtained (Charles River, Raleigh, NC) and used for the study at 8 weeks of age. Upon receipt, rats were housed one animal per cage with free access to Purina 5008 chow (Lab Diet, St. Louis, MO) and water, maintained on a 12-hour light / dark cycle from 5:00 AM to 5:00 PM at 21° C. and 50% relative humidity, and acclimated for 9 days prior to the start of the study. Blood samples were obtained as prebleeds (day -3) via the tail vein to measure glucose levels and HbA1c. ZDF rats were randomly assigned to treatment groups (n=10 / group) with similar mean HbA1c and glucose. They were implanted subcutaneously (SC) with Alzet osmotic mini-pumps (2 pumps / animal) containing the indicated doses of amylin analog polypeptide and / or exenatide (10 mcg / kg / day) or vehicle (20% DMSO in water) (n=10 animals / treatment group). PK support was given by SC injection every other day rather than mini-pump administration of the amylin analog polypeptide administered every other day (eod). All other procedures were the same as described for the previous example. Blood samples were obtained again on days 14 and 27 (end of study) to measure glucose levels and HbA1c. A final whole blood sample (day 27) was collected by cardiac puncture under isoflurane anesthesia. HbA1c analysis was performed using a Carolina Chemistries CLC720i clinical chemistry analyzer (Mindray Inc., Mahwah, NY) using the protocol and method parameters described by the manufacturer. HbA1c results expressed as mean % change from baseline and vehicle control (ΔΔ) from the exenatide chronic combination study are shown in Table 11.

[1086] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[1087] Table 11: Summary of Body Weight Loss and HbA1c Changes in Rats Treated with Amylin Analog Peptides

[1088]

[1089] Other implementation plans

[1090] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[1091] Preferred embodiment:

[1092] 1. An isolated polypeptide comprising the amino acid 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 FGPILP X 29 TX 31 VGSX 35 TX 37 -(OH / NH2) (SEQ ID NO: 199) or a pharmaceutically acceptable salt thereof, wherein:

[1093] X1 is S, K, k, H or I;

[1094] X3 is N or S;

[1095] X5 is S or A;

[1096] X6 is T or S;

[1097] X8 is A or K;

[1098] X 10 It is Q or S;

[1099] X 12 It is L or K;

[1100] X 13 is A, S, E or K;

[1101] X 14 is N, n, d, Y, or Q;

[1102] X 15 is E, F, f, Y, I, k, K, or α-aminoisobutyric acid (Aib);

[1103] X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l;

[1104] X 17is H, V, Q, R, k, K, or Aib;

[1105] X 18 is K, H or R;

[1106] X 19 It is S or Aib;

[1107] X 20 It is S or Aib;

[1108] X 22 is N or E;

[1109] X 29 is P, R or K;

[1110] X 31 is k, K, N, or H;

[1111] X 35 is e, E, N, K, G, A, Y, or P; and

[1112] X 37 is Y or P;

[1113] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[1114] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[1115] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[1116] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[1117] 2. The isolated polypeptide according to item 1, comprising the amino acid sequence of SEQ ID NO: 204: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 204) or a pharmaceutically acceptable salt thereof, wherein:

[1118] X1 is S, K or k;

[1119] X 10 It is Q or S;

[1120] X 15 is E or F;

[1121] X 16 is L, K or k;

[1122] X 17 is H, V, or Q; and

[1123] X 35 is E or N;

[1124] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[1125] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[1126] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 309) are optionally further linked via a disulfide bridge.

[1127] 3. The isolated polypeptide according to item 1, comprising the amino acid sequence of SEQ ID NO: 206: SCNTSTCATQRLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-(OH / NH2) (SEQ ID NO: 206) or a pharmaceutically acceptable salt thereof, wherein:

[1128] X 15 is E or F;

[1129] X 16 is L, K or k;

[1130] X 17 is H, V, or Q; and

[1131] X 35 is E or N;

[1132] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[1133] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[1134] The two cysteine ​​residues of SCNTSTC (SEQ ID NO: 310) are optionally further linked via a disulfide bridge.

[1135] 4. The isolated polypeptide according to item 1, wherein the isolated polypeptide is selected from the group consisting of:

[1136] SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2)(SEQ ID NO:127);

[1137] SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2)(SEQ ID NO:57);

[1138] SC*NTSTC*ATQRLANEKHKSSNNFGPILPPTKVGSETY-(NH2)(SEQ ID NO:128);

[1139] SC*NTSTC*ATQRLANEkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 129); and

[1140] SC*NTSTC*ATQRLANFLVKSSNEFGPILPPTKVGSETY-(NH2) (SEQ ID NO:43).

[1141] 5. The isolated polypeptide according to item 1, comprising the following amino acid sequence:

[1142] SC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO:57).

[1143] 6. The isolated polypeptide according to item 1, comprising the following amino acid sequence:

[1144] SC*NTSTC*ATQRLANFkHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 127).

[1145] 7. The isolated polypeptide according to item 1, comprising the following amino acid sequence:

[1146] SC*NTSTC*ATQRLANEk*((γGlu)2-CO(CH2) 14 CH3)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 27).

[1147] 8. The isolated polypeptide according to item 1, comprising the amino acid sequence of SEQ ID NO: 209: X1CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH / NH2) (SEQ ID NO: 209) or a pharmaceutically acceptable salt thereof, wherein:

[1148] X1 is K or k;

[1149] X 10 It is Q or S;

[1150] X 15 is E or F;

[1151] X 16 is L, K or k; and

[1152] X 17 is H, V or Q;

[1153] each K independently represents L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

[1154] each k independently represents D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

[1155] The two cysteine ​​residues of X1CNTSTC (SEQ ID NO: 318) are optionally further linked via a disulfide bridge.

[1156] 9. The isolated polypeptide according to item 1, comprising the following amino acid sequence:

[1157] KC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 130).

[1158] 10. The isolated polypeptide according to claim 1, comprising the following amino acid sequence:

[1159] K*((γGlu)2(CO(CH2) 18 CO2H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 64).

[1160] 11. The isolated polypeptide according to claim 1, comprising the following amino acid sequence:

[1161] K*((γGlu)2(CO(CH2) 16 CO2H))C*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 65).

[1162] 12. The isolated polypeptide of item 1, wherein the isolated polypeptide is selected from the group consisting of:

[1163] KC*NTSTC*ATQRLANFLQKSSNNFGPILPPTKVGSETY-(NH2) (SEQ ID NO: 131).

[1164] 13. The isolated polypeptide of item 1, wherein the isolated polypeptide is selected from the group consisting of:

[1165] K*(γGlu-CO(CH2) 16 CO2H)C*NTSTC*ATSRLANFLQKSSNNF GPILPTKVGSETY-NH2 (SEQ ID NO: 109).

[1166] 14. The isolated polypeptide according to item 1, comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-143.

[1167] 15. The isolated polypeptide of claim 1, further comprising a lipophilic substituent and optionally a spacer.

[1168] 16. The isolated polypeptide of item 15, further comprising a lipophilic substituent and a spacer of formula VI:

[1169] -(Y1) n1 -(V) r -(Y2) n2 -CO-(CH2) m -Z

[1170] Formula VI

[1171] in

[1172] Z is -CH3 or -CO2H;

[1173] m is 4 to 24;

[1174] Y1 is selected from the group consisting of γGlu, Asp and Gly;

[1175] Y2 is selected from the group consisting of γGlu, Asp and Gly;

[1176] V is -[COCH2(O(CH2)2) t OCH2NH]-, and t is 1 to 8;

[1177] r is 1 to 8;

[1178] n1 is 0 to 10; and

[1179] n2 is 0 to 10.

[1180] 17. The isolated polypeptide of item 15, further comprising a lipophilic substituent and a spacer of formula III:

[1181] -(γGlu) n -CO-(CH2) m -Z (disclosed as "(γGlu)" in SEQ ID NO: 311) n ”)

[1182] Formula III

[1183] in

[1184] Z is -CH3 or -CO2H;

[1185] m is 4 to 24; and

[1186] n is 1 to 10.

[1187] 18. The isolated polypeptide of claim 16, wherein the lipophilic substituent -CO-(CH2) m -Z is connected via the spacer group -(Y1) n1 -(V) r -(Y2) n2 - is linked to the epsilon-amino group of lysine of the isolated polypeptide, the spacer forming a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[1188] 19. The isolated polypeptide of claim 17, wherein the lipophilic substituent -CO-(CH2) m -Z via the spacer -(γGlu) n -(disclosed as "(γGlu)" in SEQ ID NO: 311) n ”) is attached to the ε-amino group of lysine of the isolated polypeptide, and the spacer forms a bridge between the amino group of the disclosed polypeptide and the CO-group of the lipophilic substituent.

[1189] 20. The isolated polypeptide of item 18 or 19, wherein the lipophilic substituent is -CO-(CH2) m -CO2H.

[1190] 21. The isolated polypeptide of item 20, wherein m is 14 to 20.

[1191] 22. The isolated polypeptide of item 19, wherein the spacer is γGlu or 2(γGlu).

[1192] 23. A pharmaceutical composition comprising the isolated polypeptide according to any one of items 1 to 22.

[1193] 24. The pharmaceutical composition of claim 23, wherein the composition further comprises an insulinotropic polypeptide.

[1194] 25. An osmotic delivery device comprising the isolated polypeptide of any one of items 1 to 22 or the pharmaceutical composition of item 23 or 24.

[1195] 26. The osmotic delivery device of item 25, comprising

[1196] an impermeable reservoir comprising an inner surface and an outer surface and a first open end and a second open end,

[1197] a semipermeable membrane in a sealed relationship with the first open end of the reservoir,

[1198] an osmotic engine within the reservoir and adjacent the semipermeable membrane,

[1199] a piston adjacent the osmotic engine, wherein the piston forms a movable seal with the inner surface of the reservoir, the piston separating the reservoir into a first chamber and a second chamber, the first chamber containing the osmotic engine,

[1200] a suspension formulation, wherein the second chamber contains the suspension formulation and the suspension formulation is flowable and contains the isolated polypeptide, and

[1201] A diffusion regulator is inserted into the second open end of the reservoir, the diffusion regulator being adjacent to the suspension formulation.

[1202] 27. A method of treating obesity in a human subject, providing weight loss in a human subject, or suppressing appetite in a human subject, the method comprising administering to the subject a pharmaceutical composition comprising the isolated polypeptide of any one of items 1 to 22, the pharmaceutical composition of item 23 or 24, or the osmotic device of item 25 or 26.

[1203] 28. A method of treating type 1 or type 2 diabetes in a human subject, the method comprising administering to the subject a pharmaceutical composition comprising the isolated polypeptide of any one of items 1 to 22, the pharmaceutical composition of item 23 or 24, or the osmotic device of item 25 or 26.

[1204] 29. The method of claim 28, wherein the pharmaceutical composition comprising the isolated polypeptide is administered as an adjunct to insulin administration.

[1205] 30. The method of any one of items 27-29, wherein the pharmaceutical composition comprising the isolated polypeptide is administered to the subject via implantation or injection.

[1206] 31. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 210: 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: 210), wherein:

[1207] X1 is S, K, k, H or I;

[1208] X3 is N or S;

[1209] X5 is S or A;

[1210] X6 is T or S;

[1211] X8 is A or K;

[1212] X 10 It is Q or S;

[1213] X 12 It is L or K;

[1214] X 13 is A, S, E or K;

[1215] X 14 is N, n, d, Y, or Q;

[1216] X 15 is E, F, f, Y, I, k, K, or α-aminoisobutyric acid (Aib);

[1217] X 16 is k, K, L, Aib, N-methylleucine (N-MeL), or l;

[1218] X 17 is H, V, Q, R, k, K, or Aib;

[1219] X 18 is K, H or R;

[1220] X 19 It is S or Aib;

[1221] X 20 It is S or Aib;

[1222] X 22 is N or E;

[1223] X 29 is P, R or K;

[1224] X 31 is k, K, or N; and

[1225] X 35 is e, E, or N;

[1226] Each K independently represents L-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[1227] Each k independently represents D-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

[1228] wherein the two cysteine ​​residues of X1CX3TX5X6C are optionally further bound via a disulfide bridge;

[1229] The condition is that if X 31 is N, then X 35 is E, or if X 35 is N, then X 31 It’s K.

[1230] 32. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1231] SC*NTSTC*ATQRLANEkHKSSNNFGPILPPTKVGSETY-NH2(SEQ ID NO:129);

[1232] SC*NTSTC*ATQRLANEk*(acetyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 144);

[1233] SC*NTSTC*ATQRLANEk*(allyloxycarbonyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 145); and

[1234] SC*NTSTC*ATQRLANEk*((dimethyl-2,6-dioxocyclohexan-1-ylidene)ethyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 146).

[1235] 33. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1236] SC*NTSTC*ATQRLANEk*HKSSNNFGPILPPTKVGSETY-NH2(SEQ ID NO:129);

[1237] SC*NTSTC*ATQRLANEk*(γGlu)HKSSNNFGPILPPTKVGSETY-NH2(SEQ ID NO:147);

[1238] SC*NTSTC*ATQRLANEk*(γGlu-acetyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 148);

[1239] SC*NTSTC*ATQRLANEk*(γGlu-trityl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 149); and

[1240] SC*NTSTC*ATQRLANEk*(γGlu-tert-butyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 150).

[1241] 34. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1242] SC*NTSTC*ATQRLANEk*(γGlu-γGlu)HKSSNNFGPILPPTKVGSETY-NH2(SEQ ID NO:151);

[1243] SC*NTSTC*ATQRLANEk*(γGlu-γGlu-acetyl)HKSSNNFGPILPP TKVGSETY-NH2 (SEQ ID NO: 152);

[1244] SC*NTSTC*ATQRLANEk*(γGlu-γGlu-trityl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 153); and

[1245] SC*NTSTC*ATQRLANEk*(γGlu-γGlu-tert-butyl)HKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 154).

[1246] 35. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1247] KC*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 155); and

[1248] K*(Fmoc)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 156).

[1249] 36. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1250] K*(γGlu)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 157);

[1251] K*(γGlu-acetyl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 158);

[1252] K*(γGlu-trityl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 159); and

[1253] K*(γGlu-tert-butyl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 160).

[1254] 37. The isolated peptide of item 31, wherein the isolated peptide is selected from the group consisting of:

[1255] K*(γGlu-γGlu)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 161);

[1256] K*(γGlu-γGlu-acetyl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 162);

[1257] K*(γGlu-γGlu-trityl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 163); and

[1258] K*(γGlu-γGlu-tert-butyl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2 (SEQ ID NO: 164).

Claims

1. An isolated polypeptide comprising the following amino acid sequence: K*((γGlu)2(CO(CH2) 18 CO2H))C*NTSTC*ATQRLANELHKSS NNFGPILPPTKVGSETY-(NH2)(SEQ ID NO:64), or a pharmaceutically acceptable salt thereof, wherein: C* represents cysteine ​​residues that are bound to each other via disulfide bridges, and K* represents L-lysine covalently bound to the lipophilic substituent via a spacer.

2. A pharmaceutical composition comprising the isolated polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition of claim 2, wherein the composition further comprises an insulinotropic polypeptide.

4. An osmotic delivery device comprising the isolated polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 or 3.

5. The osmotic delivery device of claim 4, further comprising an impermeable reservoir comprising an inner surface and an outer surface and a first open end and a second open end, a semipermeable membrane in a sealed relationship with the first open end of the reservoir, an osmotic engine within the reservoir and adjacent the semipermeable membrane, a piston adjacent the osmotic engine, wherein the piston forms a movable seal with the inner surface of the reservoir, the piston separating the reservoir into a first chamber and a second chamber, the first chamber containing the osmotic engine, a suspension formulation, wherein the second chamber contains the suspension formulation and the suspension formulation is flowable and contains the isolated polypeptide, and A diffusion regulator is inserted into the second open end of the reservoir, the diffusion regulator being adjacent to the suspension formulation.

6. Use of the isolated polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 or 3 in the preparation of a medicament for treating obesity in a human subject, providing weight loss to a human subject, or suppressing the appetite of a human subject.

7. Use of the isolated polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 or 3 in the preparation of a medicament for treating type 1 or type 2 diabetes in a human subject.

8. The use of claim 6 or 7, wherein the isolated polypeptide or pharmaceutical composition is administered to the subject via implantation or injection.

Images