Methods and materials for using GC-a receptor activating peptides in combination with GLP-1 / GIP receptor agonists

Abstract

Methods and materials for treating mammals having cardiovascular and / or metabolic disease are provided herein. For example, methods and materials for treating mammals having cardiovascular and / or metabolic disease by administering (a) an analog of a natriuretic peptide (NP), such as atrial natriuretic peptide (ANP) or dendroaspis natriuretic peptide (DNP), and (b) a glucagon-like peptide-1 (GLP-1) receptor / glucose-dependent insulinotropic polypeptide (GIP) receptor agonist are provided herein. The NP analogs used in the methods provided herein can be less than 20 amino acids in length and, in some cases, can have one or more variations in their ring portion (as compared to wild type ANP or DNP) that affect the potency of the analog in activating the particulate guanylyl cyclase (GC-A) receptor.

Description

[0001] METHODS AND MATERIALS FOR USING GC-A RECEPTOR ACTIVATING PEPTIDES IN COMBINATION WITH GLP-l / GIP RECEPTOR AGONISTS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority from U.S. Provisional Application Serial No. 63 / 718,058, filed November 8, 2024, and U.S. Provisional Application Serial No. 63 / 637,620, filed April 23, 2024. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2330W01_SL_ST26.xml.” The XML file, created on April 21, 2025. is 40,262 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials for treating mammals having, for example, cardiovascular and / or metabolic disease. For example, this document relates to methods and materials for treating mammals having cardiovascular and / or metabolic disease by administering, to the mammals, (a) an analog of a natriuretic peptide (NP). such as atrial natriuretic peptide (ANP) or dendroaspis natriuretic peptide (DNP). and (b) a glucagon-like peptide-1 (GLP-1) receptor agonist and / or glucose-dependent insulinotropic polypeptide (GIP) receptor agonist. The NP analogs used in the methods described herein can be less than 20 amino acids in length and, in some cases, can have one or more variations in their ring portion (as compared to wild type ANP or DNP) that affect the potency of the analogs in activating the particulate guanylyl cyclase (GC-A) receptor. BACKGROUND

[0008] The NP family in humans includes the cardiac hormones ANP, B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin (URO). ANP is a 28-amino acid (AA) hormone of cardiac origin that plays a fundamental role in regulating blood pressure (BP) and fluid balance, and has therapeutic potential in hypertension (HTN). The hormonal regulation of BP by ANP is mediated through activation of the GC-A receptor and the generation of its effector molecule, cGMP. In mammals, ANP can improve blood pressure via its natriuretic, vasodilating, and aldosterone-suppressing actions. In addition, ANP has beneficial metabolic properties, including lipolytic and adipocyte browning effects, as well as enhancement of insulin sensitivity.

[0009] New generation GLP-1 receptor agonists and GIP receptor agonists such as semaglutide (commercially available as WEGOVY® and OZEMPIC®) have become widely used peptide therapeutics for type 2 diabetes mellitus (T2DM) and obesity. These drugs function to control hyperglycemia via insulin release, slowing stomach emptying, and promoting satiety. Like insulin, semaglutides are administered as injectable drugs. Although the use of GLP-l / GIP receptor agonists is growing, they have been associated w ith loss of muscle mass and increased risk for osteoporosis. Further, while hypertension is common in association with obesity and T2DM, these drugs are not approved for control of elevated blood pressure.

[0010] SUMMARY

[0011] This document is based, at least in part, on the development of methods for using NPs (e.g., "miniaturized” NPs, referred to herein as “M-NPs”) and GLP-l / GIP receptor agonists to treat mammals having cardiovascular and / or metabolic disease. The M-NPs described herein typically are about 15 to about 20 amino acids in length and have scientific and therapeutic value, as they can be used to further the understanding of hormone / receptor interactions and facilitate the development of orally deliverable designer GC-A targeted peptide agonists. This document provides compositions and articles of manufacture containing NPs (e.g., M-NPs) and GLP- l / GIP receptor agonists, as well as methods for using NPs (e.g., M-NPs) and GLP- 1 / GIP receptor agonists to, for example, treat mammals having cardiovascular and / or metabolic disease.

[0012] In a first aspect, this document features a method for treating a mammal having cardiovascular and / or metabolic disease. The method can include, or consist essentially of, administering, to the mammal (a) an effective amount of a natriuretic polypeptide; and (b) an effective amount of a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor, wherein the administering is effective to reduce one or more symptoms of the cardiovascular and / or metabolic disease in the mammal. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 8. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 8 but with one conservative amino acid substitution. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO:8 but with two conservative amino acid substitutions. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 8. The natriuretic polypeptide can be a substantially pure polypeptide. The method can include administering the GLP- 1 receptor agonist, and the agonist can be exenatide, lixisenatide. liraglutide, dulaglutide, semaglutide, or albiglutide. The method can include administering the agonist of both a GLP-1 receptor and a GIP receptor, and the agonist can be tirzepatide or retatrutide. The method can include administering the natriuretic peptide orally. The method can include administering the GLP-1 receptor agonist, the GIP receptor agonist, or the agonist of both a GLP-1 and a GIP receptor by injection. The mammal can be a human. The method can further include identifying the mammal as being in need of the treating. The mammal can have the cardiovascular disease. The cardiovascular disease can include hypertension. The mammal can have the metabolic disease.

[0013] In another aspect, this document features a method for treating a mammal having cardiovascular and / or metabolic disease, where the method can include, or consist essentially of, administering, to the mammal (a) an effective amount of a natriuretic polypeptide; and (b) an effective amount of a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor, wherein the administering is effective to reduce one or more symptoms of the cardiovascular and / or metabolic disease in the mammal. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:12. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12 but with one conservative amino acid substitution. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12 but with two conservative amino acid substitutions. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12. The natriuretic polypeptide can be a substantially pure polypeptide. The method can include administering the GLP-1 receptor agonist, and the agonist can be exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, or albiglutide. The method can include administering the agonist of both a GLP-1 receptor and a GIP receptor, and the agonist can be tirzepatide or retatrutide. The method can include administering the natriuretic peptide orally. The method can include administering the GLP-1 receptor agonist, the GIP receptor agonist, or the agonist of both GLP-1 receptor and a GIP receptor by injection. The mammal can be a human. The method can further include identifying the mammal as being in need of the treating. The mammal can have the cardiovascular disease. The cardiovascular disease can include hypertension. The mammal can have the metabolic disease.

[0014] In another aspect, this document features a method for treating a mammal having cardiovascular and / or metabolic disease. The method can include, or consist essentially of, administering, to the mammal (a) an effective amount of a natriuretic polypeptide; and (b) an effective amount of a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor, wherein the administering is effective to reduce one or more symptoms of the cardiovascular and / or metabolic disease in the mammal. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16 but with one conservative amino acid substitution. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16 but with two conservative amino acid substitutions. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16. The natriuretic polypeptide can be a substantially pure polypeptide. The method can include administering the GLP-1 receptor agonist, and the agonist can be exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, or albiglutide. The method can include administering the agonist of both a GLP-1 receptor and a GIP receptor, and the agonist can be tirzepatide or retatrutide. The method can include administering the natriuretic peptide orally. The method can include administering the GLP-1 receptor agonist, the GIP receptor agonist, or the agonist of both GLP-1 receptor and a GIP receptor by injection. The mammal can be a human. The method can further include identifying the mammal as being in need of the treating. The mammal can have the cardiovascular disease. The cardiovascular disease can include hypertension. The mammal can have the metabolic disease.

[0015] In another aspect, this document features the use of (i) a natriuretic polypeptide and (ii) a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor for treating cardiovascular and / or metabolic disease in a mammal. The natriuretic polypeptide can include (a) the amino acid sequence set forth in SEQ ID NO: 8, (b) the amino acid sequence set forth in SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:8, (c) the amino acid sequence set forth in SEQ ID NO: 12, (d) the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 12, (e) the amino acid sequence set forth in SEQ ID NO: 16, or (!) the amino acid sequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16. The mammal can have a cardiovascular disorder. The cardiovascular disorder can include hypertension. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 8. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 12. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16. or the amino acid sequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 8. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 12. The natriuretic polypeptide can include the amino acid sequence set forth in SEQ ID NO: 16. The natriuretic polypeptide can be formulated for oral administration. The GLP-1 receptor agonist can be exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, or albiglutide. The agonist of both a GLP-1 receptor and a GIP receptor can be tirzepatide or retatrutide. The GLP-1 receptor agonist, the GIP receptor agonist, or the agonist of both a GLP-1 receptor and a GIP receptor can be formulated for administration by injection.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0018] DESCRIPTION OF DRAWINGS

[0019] FIG. 1 shows representative sequences of NPs targeting the GC-A receptor: full length human ANP (SEQ ID NO: 1), human ANP ring (SEQ ID NO:2), full length human BNP (SEQ ID NO:3), human BNP ring (SEQ ID NO:4). Dendroaspis angusticeps full length DNP (SEQ ID NO:5), D. angusticeps DNP ring (SEQ ID NO:6), mini-ANP(15)-NH2(SEQ ID NO:7), mini-Dl (SEQ ID NO:8), mini-D2 (SEQ ID NO:9), mini-D3 (SEQ ID NO: 10), mmi-D4 (SEQ ID NO: 11), mini-DlO (SEQ ID NO: 12), ANP22 (SEQ ID NO: 13), ANP20 (SEQ ID NO: 14), MANP (SEQ ID NO: 15), and MANP191 (SEQ ID NO: 16). Cysteine residues involved in ring formation are underlined. Mutations in mini-Dl, mini-D2, mini-D3, mini-D4, and mini-DlO as compared to mini-ANP(15) are in bold.

[0020] FIG. 2 is a graph plotting the amount of insulin secreted into the media by INS-1 (rat insulinoma) cells treated as indicated below the graph.

[0021] FIG. 3 is a graph plotting the amount of glycerol secreted into culture media (without glucose) by adipocytes treated with the indicated concentrations of mini-Dl (also referred to as “mini-Dl”), GLP1, or ANP.

[0022] FIG. 4 is a graph plotting the amount of glycerol secreted into culture media by adipocytes treated with IO'10M mini-Dl. IO’10M GLP1. IO'10M ANP, or GLP1 in combination with mini-Dl or ANP.

[0023] FIG. 5 is a graph plotting the amount of free fatty acid (NEFA) secreted into culture media by adipocytes treated with the indicated concentrations of mini-Dl, GLP1, or ANP.

[0024] FIG. 6 is a graph plotting the amount of NEFA secreted into culture media by adipocytes treated with IO’10M mini-Dl, IO'10M GLP1, IO'10M ANP, or GLP1 in combination with mini-Dl or ANP.

[0025] FIG. 7 is a graph plotting cGMP generation by human visceral adipocytes treated with the indicated concentrations of ANP or mini-Dl.

[0026] FIG. 8 is a graph plotting the amount of glycerol secreted into culture media by human visceral adipocytes that were differentiated for 10 days and then treated with the indicated concentrations of mini-Dl, MANP 191, GLP1, ANP, or MANP for 6 hours.

[0027] FIG. 9 is a graph plotting the amount of glycerol secreted into culture media by adipocytes incubated with GLP1, the indicated peptides, and combinations thereof.

[0028] FIG. 10 is a graph plotting the amount of NEFA secreted into culture media by human visceral adipocytes that were differentiated for 10 days and then treated with the indicated concentrations of mini-Dl, MANP191. GLP1, ANP. or MANP for 6 hours.

[0029] FIG. 11 is a graph plotting the amount of NEFA secreted into culture media by adipocytes incubated with GLP1, the indicated peptides, and combinations thereof. FIG. 12 is a graph ploting cGMP production by human visceral adipocytes treated with the indicated concentrations of ANP, mini-Dl, MANP. and MANP191.

[0030] DETAILED DESCRIPTION

[0031] This disclosure provides methods and materials related to using variants of NPs (e.g., shortened variants of NPs) in combination with GLP-l / GIP receptor agonists to treat mammals having cardiovascular and / or metabolic disease. For example, this document provides methods and materials that include using (a) substantially pure, shortened variants of NPs (also referred to as “mini-NPs” or “M- NPs’") having a NP activity, or compositions containing such polypeptides, in combination with (b) GLP-l / GIP receptor agonists, for treating a disorder (e.g., a cardiovascular or metabolic disorder) in a mammal (e.g., a human, a rodent, a pig, a sheep, a dog, or a non-human primate). In some cases, this document provides methods and materials that include using (a) substantially pure variants of NPs that are not shortened and that have a NP activity. or compositions containing such polypeptides, in combination with (b) GLP-l / GIP receptor agonists, for treating a disorder (e.g., a cardiovascular or metabolic disorder) in a mammal (e.g., a human, a rodent, a pig, a sheep, a dog, or a non-human primate).

[0032] In some cases, NPs (e.g., M-NPs) can be effective to increase plasma cGMP levels, increase urinary cGMP excretion, increase net renal cGMP generation, increase urine flow, increase urinary sodium excretion, increase urinary potassium excretion, increase hematocrit, increase plasma BNP immunoreactivity, increase renal blood flow, increase plasma ANP immunoreactivity, decrease renal vascular resistance, decrease proximal and distal fractional reabsorption of sodium, decrease mean arterial pressure, decrease pulmonary wedge capillary pressure, decrease right atrial pressure, decrease pulmonary arterial pressure, decrease plasma renin activity, decrease plasma angiotensin II levels, decrease plasma aldosterone levels, decrease renal perfusion pressure, and / or decrease systemic vascular resistance.

[0033] The amino acid sequence for endogenous human mature ANP is SLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO:1). Like other mature NPs, ANP includes a 17-amino acid ring structure with a cysteine bond between the cysteine residues at positions 1 and 17 (underlined in the above sequence) of the ring. The sequences of mature human BNP, CNP, and urodilatin are as follows:

[0034] BNP: SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO:3) CNP: GLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 17)

[0035] URO: TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 18) Dendroaspis natriuretic peptide (DNP), from the venom of Dendroaspis angusticeps (green mamba snake), has sequence and structural similarity to ANP, BNP, and CNP: EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA (SEQ ID NO:5).

[0036] The cysteine residues at the ends of the ring structures formed by the above sequences are underlined.

[0037] In some cases, aNP described herein (e.g., a M-NP) can include an amino acid sequence similar to one or more sequences present in a human polypeptide having NP activity’ (e.g., ANP, BNP, CNP, or urodilatin), and / or to an amino acid sequence present in DNP. In some cases, NPs (e.g., M-NPs) that can be used in the methods provided herein can have a non-naturally occurring sequence or can include a sequence present in any species (e.g., human, horse, pig, goat, cow, dog, cat, rat, or mouse).

[0038] The term “isolated” as used herein with reference to a polypeptide means that the polypeptide (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source (e.g., free of human proteins), (3) is expressed by a cell from a different species, or (4) does not occur in nature. An isolated polypeptide can be, for example, encoded by DNA or RNA, including synthetic DNA or RNA, or some combination thereof.

[0039] The term “substantially pure” as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. A substantially pure polypeptide can be any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65. 70. 75, 80, 85, 90, 95, or 99 percent pure, or about 65 to 75. 75 to 80. 80 to 85, 85 to 90, 90 to 95, or 95 to 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacry lamide gel. In some embodiments, a substantially pure polypeptide can be a chemically synthesized polypeptide.

[0040] Any method can be used to obtain a substantially pure polypeptide. For example, polypeptide purification techniques, such as affinity chromatography and HPLC, as well as polypeptide synthesis techniques can be used. In addition, any material can be used as a source to obtain a substantially pure polypeptide. For example, tissue from wild-type or transgenic animals can be used as a source material. In addition, tissue culture cells engineered to over-express a particular polypeptide can be used to obtain substantially pure polypeptide. Further, a polypeptide can be engineered to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini, or in between. Other fusions that can be used include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.

[0041] NPs (e.g., M-NPs) that can be used in the methods provided herein can be variants of one or more wild type NPs (e.g., ANP or DNP). For example, M-NPs that can be used in the methods provided herein can have lengths that are less than the length of the corresponding wild type NP. In some cases, a M-NP described herein can have a length of about 15 amino acids to about 22 amino acids (e.g., 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, about 15 to 20 amino acids, about 16 to 19 amino acids, or about 17 to 18 amino acids). In some cases, a M-NP described herein can have a length of 20 or less amino acids (e.g., 19 or less, 18 or less, 17 or less. 16 or less, or 15 or less amino acids). In some cases, a M-NP described herein can have an amide group at its C-terminus. In some cases, aNP described herein can have a length of about 44 amino acids to about 52 amino acids (e.g., 44 amino acids, 45 amino acids, 46 amino acids. 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids. 52 amino acids, about 45 to 51 amino acids, about 46 to 50 amino acids, or about 47 to 49 amino acids).

[0042] Non-limiting examples of polypeptides that are variants (e.g., shortened variants) of wild type NPs are shown in FIG. 1. In some cases, a M-NP described herein that can be used in the methods provided herein can have the amino acid sequence of the “mini-Dl” polypeptide set forth in SEQ ID NO: 8. In some cases, a M-NP described herein can have the amino acid sequence of the ‘'mini -DIO” polypeptide set forth in SEQ ID NO: 12. In some cases, a NP described herein can have the amino acid sequence of the “MANP191” polypeptide set forth in SEQ ID NO: 16. As shown in FIG. 1. SEQ ID NOS:8 and 12 have an amino group at their C- terminus.

[0043] In some cases, aNP (e.g., a M-NP) described herein that can be used in the methods provided herein can contain the entire amino acid sequence set forth in SEQ ID NO:8. SEQ ID NO: 12, or SEQ ID NO: 16, except that the amino acid sequence can contain one, two. or three amino acid additions, subtractions, or substitutions. For example, a M-NP described herein can contain the amino acid sequence set forth in SEQ ID NO: 8 with one or two single amino acid residue additions, subtractions, or substitutions. For example, a M-NP described herein can contain the amino acid sequence set forth in SEQ ID NO: 12 with one, two, or three single amino acid residue additions, subtractions, or substitutions. For example, a NP described herein can contain the amino acid sequence set forth in SEQ ID NO: 16 with one, two, or three single amino acid residue additions, subtractions, or substitutions.

[0044] Any amino acid residue set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO: 16 can be subtracted, and any amino acid residue (e.g., any of the 20 conventional amino acid residues or any other type of amino acid such as ornithine or citrulline) can be added to or substituted within the sequence set forth in SEQ ID NO: 8, SEQ ID NO:12, or SEQ ID NO:16. The majority of naturally occurring amino acids are L- amino acids, and naturally occurring polypeptides are largely comprised of L-amino acids. D-amino acids are the enantiomers of L-amino acids. In some cases, a polypeptide described herein can contain one or more D-amino acids. In some embodiments, a polypeptide described herein can contain chemical structures such as e-aminohexanoic acid; hydroxylated amino acids such as 3 -hydroxy proline, 4- hydroxyproline, (5R)-5-hydroxy-L-lysine. allo-hydroxylysine, and 5-hydroxy-L- norvaline; or glycosylated amino acids such as amino acids containing monosaccharides (e.g., D-glucose, D-galactose, D-mannose, D-glucosamine, and D- galactosamine) or combinations of monosaccharides. NPs (e.g.. M-NPs) described herein having one or more amino acid additions, subtractions, or substitutions relative to a native NP amino acid sequence (also referred to herein as “variant” NPs) can be prepared and modified as described herein. In some cases, amino acid substitutions can be made by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side-chain properties: (1) hydrophobic amino acids (methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of useful conservative substitutions can include, without limitation, substitution of valine, leucine, or isoleucine for alanine; lysine, glutamine, or asparagine for arginine; glutamine for asparagine; glutamic acid for aspartic acid; serine for cysteine; asparagine for glutamine; aspartic acid for glutamic acid; proline for glycine; arginine, glutamine, lysine, or asparagine for histidine; leucine, valine, methionine, alanine, or phenylalanine for isoleucine; isoleucine, valine, methionine, alanine, or phenylalanine for leucine; arginine, glutamine, or asparagine for lysine; leucine, phenylalanine, or isoleucine for methionine; leucine, valine, isoleucine, or alanine for phenylalanine; glycine for proline; threonine for serine; serine for threonine; tyrosine for tryptophan; phenylalanine, tryptophan, threonine, or serine for tyrosine; and leucine, isoleucine, methionine, phenylalanine, or alanine for valine.

[0045] In some embodiments, aNP (e.g., a M-NP) described herein that can be used in the methods provided herein can include one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Such production can be desirable to provide large quantities or alternative embodiments of such compounds. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the peptide variant using, for example, methods disclosed herein.

[0046] In some embodiments, aNP (e.g., a M-NP) described herein that can be used in the methods provided herein can include an amino acid sequence with at least 85% (e.g., at least 86%, at least 90%, at least 93%, or at least 95%, or 100%) sequence identity to a reference NP sequence (e.g., SEQ ID NO: 8, SEQ ID NO: 12. or SEQ ID NO: 16). Percent sequence identity is calculated by determining the number of matched positions in aligned amino acid sequences, dividing the number of matched positions by the total number of aligned amino acids, and multiplying by 100. A matched position refers to a position in which identical amino acids occur at the same position in aligned amino acid sequences. Percent sequence identity also can be determined for any nucleic acid sequence.

[0047] In particular, the percent sequence identity7between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained online at fr.com / blast or at ncbi.nlm.nih.gov. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g.. C:\seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g.. C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt - p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0048] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID N0:8), or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleotide sequence that has 14 matches when aligned with the sequence set forth in SEQ ID NO:8 is 93.3 percent identical to the sequence set forth in SEQ ID NO:8 (i.e., 14 / 15 x 100 = 93.3). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example. 75.11, 75.12. 75. 13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 7.17, 75.18, and 7.19 are rounded up to 7.2. It also is noted that the length value will always be an integer.

[0049] Non-limiting examples of such NPs include those set forth in TABLE 1.

[0050] TABLE 1. Exemplary natriuretic peptides

[0051]

[0052] Isolated NPs (e.g., M-NPs) described herein can be produced using any appropriate methods, including solid phase synthesis, and can be generated using manual techniques or automated techniques (e.g., using an Applied BioSystems (Foster City, CA) Peptide Synthesizer or a Biosearch Inc. (San Rafael, CA) automatic peptide synthesizer). Disulfide bonds between cysteine residues can be introduced by mild oxidation of the linear polypeptides using KCN as taught, e g., in U.S. Patent No. 4,757,048. NPs (e.g., M-NPs) also can be produced recombinantly or obtained commercially. NPs (e.g.. M-NPs) described herein that can be used in the methods provided herein typically are cyclic due to disulfide bonds between the cysteine residues underlined in the sequences shown above. In some embodiments, a sulfhydryl group on a cysteine residue can be replaced with an alternative group (e.g., -CH2CH2-). To replace a sulfhydryl group with a -CH2- group, for example, a cysteine residue can be replaced by alpha-aminobutyric acid. Such cyclic analog polypeptides can be generated, for example, in accordance with the methodology of Lebl and Hruby ((1984) Tetrahedron Lett. 25:2067-2068), or by employing the procedure disclosed in U.S. Patent No. 4,161,521.

[0053] In addition, ester bridges can be formed by reacting the OH of serine or threonine with the carboxyl group of aspartic acid or glutamic acid to yield a bridge having the structure -CH2CO2CH2-. Similarly, an amide can be obtained by reacting the side chain of lysine with aspartic acid or glutamic acid to yield a bridge having the structure -CH2C(O)NH(CH)4-. Methods for synthesis of these bridges are described elsewhere (see. e.g., Schiller et al. (1985) Biochem. Biophys. Res. Comm. 127:558, and Schiller et al. (1985) Int. J. Peptide Protein Res. 25:171 ). For example, one method for preparing esters of the present polypeptides, when using the Merrifield synthesis technique, is to cleave the completed polypeptide from the resin in the presence of the desired alcohol under either basic or acidic conditions, depending upon the resin. The C-terminal end of the polypeptide then can be directly esterified when freed from the resin, without isolation of the free acid. Amides of polypeptides also can be prepared using techniques for converting a carboxylic acid group or precursor to an amide. One method for amide formation at the C-terminal carboxyl group includes cleaving the polypeptide from a solid support with an appropriate amine, or cleaving in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. Other bridge-forming amino acid residues and reactions are provided in, for example. U.S. Pat. No. 4,935,492. Preparation of peptide analogs that include non-peptidyl bonds to link amino acid residues also are described elsewhere. See. e.g., Spatola et al. (1986) Life Sci. 38: 1243; Spatola (1983) Vega Data 1(3); Morley (1980) Trends Pharm. Sci. 463-468; Hudson et al. (1979) Int. J. Pept. Prot. Res. 14:177; Spatola, in Chemistr}' and Biochemistry of Amino Acid Peptides and Proteins. B. Weinstein, ed.. Marcel Dekker, New York, p. 267 (1983); Hann (1982) J. Chem. Soc. Perkin Trans. 1 :307; Almquist et al. (1980) J. Med. Chem. 23: 1392; Jennings-White et al. (1982) Tetrahedron Lett. 23:2533; European Patent Application EP 45665; Holladay et al. (1983) Tetrahedron Lett. 24:4401; and Hruby (1982) Life Sci. 31:189. N-acyl derivatives of an amino group of a polypeptide can be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives can be prepared for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.

[0054] In some cases, aNP (e.g., a M-NP) described herein can be pegylated, acety lated, or both. In some cases, a polypeptide provided herein can be covalently attached to oligomers, such as short, amphiphilic oligomers that enable administration or improve the pharmacokinetic or pharmacodynamic profile of the conjugated polypeptide. The oligomers can comprise water soluble polyethylene glycol (PEG) and / or lipid soluble alkyls (short, medium, or long chain fatty7acid polymers, such as, without limitation, palmitic acid, myristic acid, lauric acid, capric acid, or steric acid). The fatty acid molecule can be attached to the free amino terminus or to any lysine side chain (an epsilon amino group), and a lysine residue for this attachment can be placed at either the C-terminal or N-terminal end of the peptide. Linkage to PEG or another suitable polymer, or fusion to albumin or another suitable polypeptide can result in a modified NP (e.g.. a modified M-NP) having an increased half-life as compared to an unmodified NP (e.g., an unmodified M-NP). Without being bound by a particular mechanism, an increased serum half-life can result from reduced proteolytic degradation, immune recognition, or cell scavenging of the modified NP. Methods for modifying a polypeptide by linkage to PEG (also referred to as ■'PEGylationJ or other polymers are described elsewhere, and include those set forth in U.S. Patent No. 6,884,780; PCT Publication No. WO 2004 / 047871; Cataliotti et al. (2007) Trends Cardiovcisc. Med. 17:10-14; Veronese and Mero (2008) BioDrugs 22:315-329; Miller et al. (2006) Bioconjugate Chem. 17:267-274; and Veronese and Pasut (2005) Drug Discov. Today 10:1451-1458, all of which are incorporated herein by reference in their entirety. Methods for modifying a polypeptide by fusion to albumin also are described elsewhere, and include those set forth in U.S. Patent Publication No. 20040086976, and Wang et al. (2004) Pharm. Res. 21:2105-2111, both of which are incorporated herein by reference in their entirety. In some cases, aNP (e.g., a M-NP) described herein that can be used in the methods provided herein can be fused to the Fc domain of an immunoglobulin molecule (e.g., an IgGl molecule) such that active transport of the fusion polypeptide across epithelial cell barriers occurs via the Fc receptor. In some cases, a polypeptide can be a cyclic polypeptide. A cyclic polypeptide provided herein can be obtained by bonding cysteine residues. However, the replacement of a sulfhydryl group on the cysteine residue with an alternative group (e.g., -CH2-CH2-) also is envisioned, for example, to replace sulfhydryl groups with a -CH2- group, the cysteine residues can be replaced by the analogous alpha-aminobuty ric acid. These cyclic analog peptides can be formed, for example, in accordance with the methodology of Lebl and Hruby (supra), or by employing the procedure disclosed in U.S. Patent No. 4, 161.521.

[0055] Salts of carboxyl groups of polypeptides can be prepared by contacting a polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base (e.g., sodium hydroxide), a metal carbonate or bicarbonate base (e.g., sodium carbonate or sodium bicarbonate), or an amine base (e.g., triethylamine, triethanolamine, and the like). Acid addition salts of polypeptides can be prepared by' contacting the polypeptide with one or more equivalents of an inorganic or organic acid (e.g., hydrochloric acid).

[0056] NPs (e.g.. M-NPs) described herein that can be used in the methods provided herein can function through one or more of the guanylyl cyclase receptors through which native (wild ty pe) NPs function. For example, NPs (e.g., M-NPs) described herein can bind to and function through the GC-A receptor through which ANP and BNP function, although they also may function through the GC-B receptor through which CNP functions. Further, in some cases, a NP (e.g., a M-NP) that can be used in the methods provided herein can bind to and function through more than one guanylyl cyclase receptor, including GC-A and GC-B, for example. Any appropriate method can be used to evaluate which receptor is involved in the function of a particular NP, including methods described herein. For example, glomeruli, which contain both GC- A and GC-B. can be isolated (e.g.. from a laboratory animal such as a dog or a rat) and incubated with a NP (e.g., a M-NP) provided herein, and cGMP levels can be measured. Glomeruli can be pretreated with antagonists of GC-A or GC-B to determine whether cGMP production stimulated by aNP through one or the other receptor can be attenuated. In some cases, a cell line that overexpresses a particular guanylyl cyclase receptor (e.g., GC-A or GC-B) can be incubated with a NP (e.g., a M-NP) provided herein, and cGMP produced by the cells can be measured.

[0057] In some cases, a NP (e.g., a M-NP) described herein can be encoded by a nucleic acid molecule. For example, a nucleic acid molecule can encode a NP having the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO: 16. In some cases, a nucleic acid molecule can encode aNP that contains the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO:16, except that the amino acid sequence contains one, two, or three amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:8. SEQ ID NO: 12, or SEQ ID NO: 16.

[0058] The term '‘nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0059] The term “isolated” as used herein with reference to nucleic acid refers to a naturally -occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5’ end and one on the 3’ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally -occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g.. a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence. The term “isolated"’ as used herein with reference to nucleic acid also includes any non-naturally -occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally -occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally -occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence. It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

[0060] Isolated nucleic acid molecules can be produced using standard techniques, including, without limitation, common molecular cloning, and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing nucleotide sequence that encodes aNP (e.g., a M-NP) provided herein. PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond ty pically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize complementary DNA (cDNA) strands. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254:1292.

[0061] Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3’ to 5’ direction using phosphorami dite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity7(e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.

[0062] Vectors containing nucleic acids molecules described herein also are provided. A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0063] In an expression vector, a nucleic acid (e.g., a nucleic acid encoding a NP described herein) can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 to 500 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked’' and “under the control'’ of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence. Expression vectors thus can be useful to produce antibodies as well as other multivalent molecules.

[0064] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA).

[0065] An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

[0066] In some cases, a nucleic acid molecule (e.g.. a vector) encoding a NP described herein can be contained in a host cell. The term “host cell” is intended to include prokaryotic and eukary otic cells into which a recombinant expression vector can be introduced (e.g., vector encoding a NP described herein). As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Suitable methods for transforming and transfecting host cells can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2ndedition), Cold Spring Harbor Laboratory. New York (1989). For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer can be used introduce nucleic acid into cells. In addition, naked DNA can be delivered directly to cells in vivo as described elsewhere (U.S. Patent Nos. 5,580,859 and 5,589,466). The host cells can express the encoded polypeptide, but it is noted that cells containing an isolated nucleic acid molecule provided herein are not required to express a polypeptide. The isolated nucleic acid molecule transformed into a host cell can be integrated into the genome of the cell or maintained in an episomal state. Thus, host cells can be stably or transiently transfected with a construct containing an isolated nucleic acid molecule provided herein.

[0067] Any suitable method can be used to introduce an isolated nucleic acid molecule into a cell in vivo or in vitro. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are methods that can be used to introduce an isolated nucleic acid molecule into a cell. In addition, naked DNA can be delivered directly to cells in vivo as described elsewhere (e.g.. U.S. Patent Nos. 5.580,859 and 5,589.466, and continuations thereof). Further, isolated nucleic acid molecules can be introduced into cells by generating transgenic animals.

[0068] Any suitable method can be used to identify cells containing an isolated nucleic acid molecule encoding a NP (e.g., a M-NP) described herein. Such methods include, without limitation, PCR, and nucleic acid hybridization techniques such as Northern and Southern analyses. In some cases, irrnnunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular isolated nucleic acid molecule by detecting the expression of a polypeptide encoded by that nucleic acid molecule.

[0069] NPs (e.g., M-NPs) described herein (e.g., polypeptides having the sequence set forth in SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16, or containing one, two, or three subtractions, additions, or substitutions with respect to SEQ ID NO:8. SEQ ID NO: 12. or SEQ ID NO: 16) can be screened for biological activity using any appropriate assay. For example, the activity of aNP (e.g., a M-NP) can be evaluated in vitro by testing its effect on cGMP production in cultured cells (e.g., cultured cardiac fibroblasts, aortic endothelial cells, glomerular cells, or a cell line transfected with a nucleic acid encoding a guanylyl cyclase receptor such as GC-A or GC-B). Cells can be exposed to a NP (e.g.. IO’10to 10'4M NP). and samples can be assayed to evaluate the NP’s effects on cGMP generation. cGMP generation can be detected and measured using, for example, a competitive RIA cGMP kit (Perkin-Elmer, Boston, MA). The activity of aNP (e.g., a M-NP) also can be evaluated in vivo by, for example, testing its effects on factors such as plasma cGMP levels, urinary cGMP excretion, net renal generation of cGMP, glomerular filtration rate, blood pressure, heart rate, hemodynamic function such as cardiac output, pulmonary wedge pressure, systemic vascular resistance, and renal function such as renal blood flow, urine volume, and sodium excretion rate in a mammal (e.g.. a rodent, pig, sheep, dog, or human). In some cases, such parameters can be evaluated after inducing heart failure (e.g., by rapid right ventricular pacing) or hypertension.

[0070] The NPs (e.g., M-NPs) described herein (e.g., NPs having the amino acid sequences set forth in SEQ ID NO: 8 or variants thereof. SEQ ID NO: 12 or variants thereof, and SEQ ID NO: 16 or variants thereof), or nucleic acids encoding the NPs (e.g., M-NPs) described herein, can be incorporated into compositions for administration to a mammal (e.g., a mammal having or at risk for cardiovascular and / or metabolic disease). Dosages typically are dependent on the responsiveness of the subject to the compound, with the course of treatment lasting from several days to several months, or until a suitable response is achieved. Persons of ordinary skill in the art can readily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of an antibody, and generally can be estimated based on the EC50 found to be effective in in vitro and / or in vivo animal models. Compositions containing the NPs (e.g.. M-NPs) and nucleic acids provided herein may be given once or more daily, weekly, monthly, or even less often, or can be administered continuously for a period of time (e.g., hours, days, or weeks). For example, a NP (e.g., a M-NP) or a composition containing aNP (e.g., a M-NP) can be administered to a patient at a dose of at least about 0.01 ng NP / kg to about 100 mg NP / kg of body mass, or can be administered continuously as an infusion for about one to seven days (e.g., at a dose of about 0.01 ng NP / kg / minute to about 0.5 pg NP / kg / minute).

[0071] ANP (e.g., a M-NP) described herein and nucleic acids encoding aNP (e.g., a M-NP) described herein can be admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, receptor or cell targeted molecules, or oral, topical, or other formulations for assisting in uptake, distribution and / or absorption. In some embodiments, a composition can contain a NP (e.g., a M-NP) described herein, in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable earners include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering antibodies to a subj ect. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, without limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose or dextrose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0072] Pharmaceutical compositions containing molecules described herein can be administered by a number of methods, depending upon whether local or systemic treatment is desired. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous (i.v.) drip): oral; topical (e.g., transdermal, sublingual, ophthalmic, or intranasal); or pulmonary (e.g., by inhalation or insufflation of powders or aerosols), or can occur by a combination of such methods. Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations).

[0073] Without being bound by a particular mechanism, oral administration of aNP (e.g., a M-NP) described herein may be particularly useful. Compositions and formulations for oral administration include, for example, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Such compositions also can incorporate thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders.

[0074] Pharmaceutical compositions include, without limitation, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, for example, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Emulsion formulations are particularly useful for oral delivery of therapeutic compositions due to their ease of formulation and efficacy of solubilization, absorption, and bioavailability. Liposomes can be particularly useful due to their specificity and the duration of action they offer from the standpoint of drug delivery.

[0075] Compositions provided herein can contain any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a subject, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof for the relevant compound (e.g., NP). Accordingly, for example, this document provides pharmaceutically acceptable salts of NPs, prodrugs and pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. A prodrug is a therapeutic agent that is prepared in an inactive form and is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and / or conditions. The term ■‘pharmaceutically acceptable salts’’ refers to physiologically and pharmaceutically acceptable salts of the NPs (e.g., M-NPs) useful in methods provided herein (i.e., salts that retain the desired biological activity of the parent NPs without imparting undesired toxicological effects). Examples of pharmaceutically acceptable salts include, but are not limited to, salts formed with cations (e.g.. sodium, potassium, calcium, or polyamines such as spermine); acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid); salts formed with organic acids (e.g., acetic acid, citric acid, oxalic acid, palmitic acid, or fumaric acid); and salts formed with elemental anions (e.g., bromine, iodine, or chlorine).

[0076] Compositions additionally can contain other adjunct components conventionally found in pharmaceutical compositions. Thus, the compositions also can include compatible, pharmaceutically active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or additional materials useful in physically formulating various dosage forms of the compositions, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. Furthermore, the composition can be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, penetration enhancers, and aromatic substances. When added, however, such materials should not unduly interfere with the biological activities of the other components within the compositions.

[0077] In some cases, a NP (e.g., a M-NP) described herein can be formulated as a sustained release dosage form. For example, aNP (e.g., a M-NP) described herein can be formulated into a controlled release formulation. In some cases, coatings, envelopes, or protective matrices can be formulated to contain one or more of the polypeptides provided herein. Such coatings, envelopes, and protective matrices can be used to coat indwelling devices such as stents, catheters, and peritoneal dialysis tubing. In some cases, a polypeptide provided herein can incorporated into polymeric substances, liposomes, microemulsions, microparticles, nanoparticles, or w axes.

[0078] Pharmaceutical formulations as disclosed herein, which can be presented conveniently in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients (i.e., the antibodies) w ith the desired pharmaceutical carrier(s). Typically, the formulations can be prepared by uniformly and intimately bringing the active ingredients into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations can be sterilized if desired, provided that the method of sterilization does not interfere w ith the effectiveness of the molecules(s) contained in the formulation.

[0079] In some cases, a NP (e.g., a M-NP) described herein can be formulated for subcutaneous delivery via depot polymers, drug patch, injection, pump, or microparticle / nano particle. By way of example and not limitation, PCT Publication No. WO 2008 / 061355 discloses materials and methods for formulating a polypeptide for delivery’ in a hydrogel tube. The polypeptide can be mixed with one or more excipients that are pharmaceutically acceptable and are compatible with the polypeptide in amounts suitable for use in the methods described herein. For example, a polypeptide can be combined w ith one or more excipients such as, without limitation, microcrystalline cellulose, colloidal silicon dioxide, lactose, starch, sorbitol, cyclodextrin, and combinations thereof. The excipient can be a solid, semisolid. or liquid material that acts as a vehicle, carrier, or medium for the polypeptide. In some embodiments, the polypeptide can be compressed, compacted, or extruded with one or more excipients prior to inserting it into a hydrogel tube. Such formulations can result in a pharmaceutical composition with desirable release properties, improved stability, and / or other desirable properties.

[0080] Pharmaceutical compositions also can include auxiliary agents or excipients, such as glidants, dissolution agents, surfactants, diluents, binders, disintegrants, and / or lubricants. For example, dissolution agents can increase the dissolution rate of the polypeptide from the dosage formulation, and can include, for example, organic acids and / or salts of organic acids (e.g., sodium citrate with citric acid). Other examples of excipients useful in such formulations include synthetic, semi -synthetic, modified, and natural polymers (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starches, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, PEG, cyclodextrin, alkoxy- modified cyclodextrins, hydroxyethylcellulose, hydroxypropylcellulose, microcrystalline cellulose, albumin, dextran, malitol, xylitol, kaolin, and methyl cellulose). The polypeptide also can be mixed with a lubricating agent (e.g., talc, magnesium stearate, stearic acid, or mineral oil, calcium stearate, hydrogenated vegetable oils, sodium benzoate, sodium chloride, leucine carbowax, magnesium lauryl sulfate, or glyceryl monostearate), a wetting agent, an emulsifying and suspending agent, or a preserving agent (e.g., methyl or propyl hydroxybenzoate).

[0081] Other agents that can be added to a pharmaceutical composition can alter the pH of the microenvironment on dissolution and establishment of a therapeutically effective plasma concentration profile of the polypeptide. Such agents include salts of inorganic acids and magnesium hydroxide. Other agents that can be used include surfactants and other solubilizing materials.

[0082] Useful diluents include, for example, pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, sucrose, fructose, glucose dextrose, or other sugars, dibasic calcium phosphate, calcium sulfate, cellulose, ethylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, saccharides, dextrin, maltodextrin or other polysaccharides, inositol, or combinations thereof. Water-soluble diluents can be particularly useful.

[0083] Glidants can be used to improve the flow and compressibility of composition ingredients during processing. Useful glidants include, for example, colloidal silicon dioxide (also referred to as colloidal silica, fumed silica, light anhydrous silicic acid, silicic anhydride, and silicon dioxide fumed).

[0084] Surfactants that are suitable for use in the pharmaceutical compositions provided herein include, without limitation, sodium lauryl sulphate, polyethylene stearates, polyethylene sorbitan fatty' acid esters, polyoxyethylene castor oil derivatives, polyoxyethylene alkyl ethers, benzyl benzoate, cetrimide, cetyl alcohol, docusate sodium., glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, lecithin, medium chain triglycerides, monoethanol amine, oleic acid, poloxamers, polyvinyl alcohol and sorbitan fatty' acid esters.

[0085] Suitable disintegrants include, for example, starches, sodium starch glycolate, crospovidone, croscarmellose, microcrystalline cellulose, low substituted hydroxypropyl cellulose, pectins, potassium methacrylate- divinylbenzene copolymer, polyvinyl alcohol), thylamide, sodium bicarbonate, sodium carbonate, starch derivatives, dextrin, beta cyclodextrin, dextrin derivatives, magnesium oxide, clays, bentonite, and combinations thereof.

[0086] In some embodiments, aNP (e.g., a M-NP) described herein can be incorporated into a hydrogel delivery system. For example, a polypeptide can be formulated for subcutaneous delivery' to a patient via a xerogel-hydrogel system that can release the polypeptide in a continuous sustained manner over an extended period of time. See, for example, U.S. Patent No. 5,226,325, and PCT Publication No. WO 2004 / 071736.

[0087] Liquid polymerizable materials useful in the preparation of hydrogel tubes include a wide variety7of polymerizable hydrophilic, and ethylenically unsaturated compounds. See, for example, the compounds listed in PCT Publication No. WO 2008 / 061355. Mixtures of such hydrophilic monomers typically are used in the polymerization reaction. The type and proportion of monomers are selected to yield a polymer (e.g., a crosslinked homogeneous polymer) that on hydration possesses the desired characteristics (e.g., equilibrium water content (EWC) value and / or pore size) for the contemplated application or use.

[0088] In some cases, the polymerization of hydrophilic monomeric mixtures can result in homogeneous hydrophilic copolymers which dissolve, to a varying extent, in an aqueous medium. In such cases, a small amount (e.g., up to about 3 percent) of a copolymerizable polyethylenically unsaturated crosslinking agent can be included in the monomeric mixture to obtain homogeneous crosslinked copolymers that are water-insoluble as well as water-swellable. A slightly crosslinked homopolymer of (hydroxyethyl)methacrylate (HEMA) has an EWC value of about 38%. Crosslinked copolymers of HEMA and N-(2-hydroxypropyl) methacrylamide (HP MA) have EWC values below 38%. while crosslinked copolymers of HEMA and acrylamide exhibit EWC values above 38 w / v %. Therefore, depending on the useful or effective elution rate of the polypeptide, copolymer hydrogels can be customized to elute the polypeptide at the desired rate. Typically, copolymers contain about 15 to about 70 weight % of HEMA units and from about 85 to 30 weight % of a second ethylenic monomer, and thus possess EWC values in the range of from about 20% to about 75%. In some embodiments, a mixture of copolymers can further contain a small amount of a polyethylenically unsaturated crosslinking agent (e.g., ethyleneglycol dimethacrylate (“EDMA”) or trimethylolpropane trimethacrylate C'TMPTM A ")).

[0089] In some embodiments, a pharmaceutical composition for controlled release delivery of a NP (e.g., a M-NP) described herein in a subject can include (a) a complex of the polypeptide (where the polypeptide has at least one basic functional group) and a polyanion derived from hexahydroxy cyclohexane (where the polyanion has at least two negatively charged functional groups); and (b) a pharmaceutically acceptable carrier containing a biodegradable, water-insoluble polymer. Such compositions are described in, for example, PCT Publication No. WO 2006 / 017852, and can be prepared in the form of solutions, suspensions, dispersions, emulsions, drops, aerosols, creams, semisolids, pastes, capsules, tablets, solid implants, or microparticles, for example. The term "controlled release delivery,’7as used herein, refers to continual delivery of a pharmaceutical agent in vivo over a period of time (e.g., several days to weeks or months) following administration. Sustained controlled release delivery of a NP (e.g., a M-NP) can be demonstrated by, for example. continued therapeutic effects of the polypeptide over time (e.g., continued reductions in symptoms over time). Sustained delivery of the polypeptide also can be demonstrated by detecting the presence of the polypeptide in vivo over time. The compositions can provide a low initial burst delivery, followed by stable, controlled release of the polypeptide in vivo for prolonged periods of time (e.g., from days to months).

[0090] In such embodiments, a physically and chemically stable complex can form upon appropriate combining of a NP (e.g., a M-NP) described herein and a polyanion. The complex can take the form of a precipitate that is produced upon combining an aqueous preparation of the poly peptide and the poly anion. Optionally, one or more pharmaceutically acceptable excipients can be incorporated into the complex. Such excipients can function as stabilizers for the polypeptide and / or the complex. Nonlimiting examples of suitable excipients include sodium bisulfite, p-aminobenzoic acid, thiourea, glycine, methionine, mannitol, sucrose, and PEG.

[0091] A stable complex between a NP (e.g., a M-NP) described herein and a polyanion can be incorporated into a pharmaceutically acceptable carrier containing a biodegradable water-insoluble polymer, optionally with one or more excipients. The term “biodegradable water-insoluble polymer"’ refers to biocompatible and / or biodegradable synthetic and natural polymers that can be used in vivo. The term also is meant to include polymers that are insoluble or become insoluble in water or biological fluid at 37°C. The polymers can be purified (e.g., to remove monomers and oligomers) using any appropriate technique(s). See, e.g., U.S. Patent No. 4,728,721. Examples of useful polymers include, without limitation, polylactides, polyglycolides, poly(lactide-co-glycolide)s, polycaprolactones, polydioxanones, polycarbonates, polyhydroxybutyrates, polyalkylene oxalates, polyanhydrides, polyamides, polyesteramides, polyurethanes, polyacetals, polyorthocarbonates, polyphosphazenes, polyhydroxyvalerates, polyalkylene succinates, and poly orthoesters, and copolymers, block copolymers, branched copolymers, terpolymers, and combinations thereof.

[0092] Biodegradable water-insoluble polymers also can include end capped, end uncapped, or mixtures of end capped and end uncapped polymers. An end capped polymer generally is defined as having capped carboxyl end groups, while an uncapped polymer has free carboxyl end groups. Factors to consider when determining suitable molecular weights for the polymer can include desired polymer degradation rate, mechanical strength, and rate of dissolution of polymer in solvent. Useful molecular weights for polymers can be from about 2,000 Daltons to about 150,000 Daltons, for example, with a poly dispersity of from 1.1 to 2.8, depending upon which polymer is selected for use.

[0093] The pharmaceutically acceptable carrier can be a carrier with environment responsive properties (e.g., thermosensitive, pH sensitive, or electrical sensitive), in the form of an injectable solution or suspension, particle, film, pellet, cylinder, disc, microcapsule, microsphere, nanosphere, microparticle, wafer, micelle, liposome, or any other polymeric configuration useful for drug delivery-.

[0094] Methods of forming various pharmaceutically acceptable polymer carriers include, without limitation, those that are described in U.S. Patent Nos. 6,410,044; 5,698,213; 6,312,679; 5,410,016; 5.529,914; 5,501,863; 4,938,763; 5,278,201; and 5,278,202; and PCT Publication No. WO 93 / 16687.

[0095] Compositions can be produced when a polypeptide / polyanion complex is dispersed in a polymeric matrix to form a solid implant, which can be injected or implanted into a subject. Such implants can be prepared using conventional polymer melt-processing techniques, such as extrusion, compression molding, and injection molding, for example. Preparations of such implants can be carried out under aseptic conditions, or alternatively by terminal sterilization by irradiation (e.g.. using gamma irradiation or electron beam sterilization).

[0096] In some embodiments, compositions in the form of microspheres can be produced by encapsulating a polypeptide / polyanion complex in a polymeric carrier, using various biocompatible and / or biodegradable polymers having properties that are suitable for delivery to different biological environments or for effecting specific functions. The rate of dissolution and, therefore, delivery of polypeptide is determined by factors such as the encapsulation technique, polymer composition, polymer crosslinking, polymer thickness, polymer solubility, and size and solubility of polypeptide / polyanion complex.

[0097] To prepare such microspheres, a polypeptide / polyanion complex to be encapsulated can be suspended in a polymer solution in an organic solvent, such that the polymer solution completely coats the polypeptide / polyanion complex. The suspension then can be subjected to a microencapsulation technique such as spray drying, spray congealing, emulsion, or solvent evaporation emulsion. For example, the suspended complexes or microparticles along with the polymer in an organic solvent can be transferred to a larger volume of an aqueous solution containing an emulsifier, such that the organic solvent evaporates or diffuses away from the polymer and the solidified polymer encapsulates the polypeptide / poly anion complex.

[0098] Emulsifiers useful to prepare encapsulated polypeptide / poly anion complexes include poloxamers and polyvinyl alcohol, for example. Organic solvents useful in such methods include acetic acid, acetone, methylene chloride, ethyl acetate, chloroform, and other non-toxic solvents that will depend on the properties of the polymer. Solvents typically are chosen that solubilize the polymer and are ultimately non-toxic.

[0099] In some embodiments, a polypeptide can be formulated in a depot, which can provide constantly high exposure levels and may reach high exposure levels rapidly (with a short or no lag phase). See. e.g., U.S. Publication No. 2010 / 0266704. Depot formulations can include aNP (e.g., a M-NP) provided herein or a pharmaceutically - acceptable salt thereof (e.g., an acid addition salt with an inorganic acid, polymeric acid, or organic acid). Acid addition salts can exist as mono- or divalent salts, depending on whether one or two acid equivalents are added.

[0100] As described in U.S. Publication No. 2010 / 0266704, depot formulations can contain two different linear poly(lactic-co-gly colic acid) (PLGA) polymers having a molar ratio of lactide: glycolide comonomer (L:G) from 85: 15 to 65:35, where at least one of the polymers has a low inherent viscosity. Such formulations can provide sustained high plasma levels of the polypeptide for extended periods of time. Examples of suitable polymers include the linear poly(D,L-lactide) and poly(D,L- lactide-co-glycolide) polymers sold under the trade names RESOMER®, LACTEL®, and MEDISORB® by Boehringer Ingelheim Pharma GmBH & Co. KG (Ingelheim, Germany), Absorbable Polymers International (Pelham. AL), and Alkermes, Inc. (Cambridge, MA), respectively.

[0101] High exposure depot formulations for subcutaneous administration can show immediate or at least very rapid action, such that therapeutic plasma concentrations are achieved in a short time (e.g., one, two, three, four, five, six, or seven days after subcutaneous injection), and can show constantly high exposure levels over about one month or longer.

[0102] In some embodiments, a depot formulation can contain two different PLGA polymers mixed or blended in a % wt ratio of 95:5 to 50:50 (e.g., 85:15 to 50:50, 80:20 to 60:40, 90: 10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45 or 50:50% wt). In some embodiments, the polymer with the higher inherent viscosity can have a higher % wt than the polymer with the lower inherent viscosity. In some embodiments, the polymer with the higher inherent viscosity can have an ester end- group. Depot formulations can contain further polymers, including other linear or star shaped PLGA polymers, or poly(D,L-lactide-co-glycolide) (PLG) or polylactic acid (PLA) polymers, provided that favorable PK properties are retained.

[0103] The polypeptide content of the depot formulation (the loading) can be in a range of 1% to 30% (e.g., 10% to 25%, more preferred 15% to 20%. The loading is defined as the weight ratio of polypeptide to the total mass of the PLGA formulation.

[0104] Depot compositions can be manufactured aseptically, or can be manufactured non-aseptically and terminally sterilized (e.g., using gamma irradiation). Terminal sterilization can result in a product with the highest sterility7assurance possible.

[0105] Depot compositions also can contain one or more pharmaceutical excipients that can modulate the release behavior of the polypeptide. Such excipients can be present in the composition in an amount of about 0. 1% to about 50%. Suitable excipients include, without limitation, polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose sodium, dextrin, PEG, surfactants such as poloxamers (also known as poly (oxy ethylene-block-oxy propylene), poly (oxy ethylene)-sorbitan-fatty acid esters commercially available under the trade name TWEEN®, sorbitan fatty acid esters, lecithins, inorganic salts such as zinc carbonate, magnesium hydroxide, magnesium carbonate, protamine, and natural or synthetic polymers bearing amine- residues such as polylysine.

[0106] Depot compositions can contain a mixture or blend of different polymers in terms of compositions, molecular weight and / or polymer architectures. A polymer blend is defined herein as a solid solution or suspension of tw o different linear polymers in one implant or microparticle. A mixture of depots is defined herein as a mixture of tw o depot-like implants or microparticles or semisolid formulations of different composition with one or more PLGAs in each depot. Pharmaceutical depot compositions in which two PLGAs are present as a polymer blend can be particularly useful.

[0107] Pharmaceutical depot compositions can be in the form of implants, semisolids (gels), liquid solutions, microparticles, or suspensions that solidify in situ once they are injected. The following paragraphs are focused on polymer microparticles, although the descriptions also are applicable for implants, semisolids, and liquids.

[0108] Microparticles can have a diameter from a few submicrons to a few millimeters (e.g., from about 0.01 micron to about 2 mm, about 0.1 micron to about 500 microns, about 10 to about 200 microns, about 10 to about 130 microns, or about 10 to about 90 microns).

[0109] In some cases, microparticles can be mixed or coated with an antiagglomerating agent. Suitable anti-agglomerating agents include, for example, mannitol, glucose, dextrose, sucrose, sodium chloride, and water soluble polymers such as polyvinyl alcohol, polyvinyl pyrrolidone and PEG.

[0110] Microparticles can be manufactured using processes such as, for example, coacerv ation or phase separation, spray drying, or water-in-oil (W / O), water-in-oil-in- water (W / O / W), or solids-in-oil-in-water (S / O / W) emulsion / suspension methods followed by solvent extraction or solvent evaporation. Emulsion / suspension methods can be particularly useful, and can include the following steps:

[0111] (i) preparing an internal organic phase, comprising

[0112] (a) dissolving a polymer or polymers in a suitable organic solvent (e.g., ethyl acetate, acetone, THF, acetonitrile, or a halogenated hydrocarbon such as methylene chloride, chloroform, or hexafluoroisopropanol) or solvent mixture, and optionally dissolving / dispersing suitable additives;

[0113] (b) dissolving / suspending / emulsifying a polypeptide in the polymer solution obtained in step (a);

[0114] (ii) preparing an external aqueous phase containing one or more stabilizers (e.g.. poly(vinylalcohol). hydroxyethyl cellulose, hydroxypropyl cellulose, poly(vinyl pyrolidone), or gelatin) and optionally a buffer salt;

[0115] (iii) mixing the internal organic phase with the external aqueous phase to form an emulsion; and (iv) hardening the microparticles by solvent evaporation or solvent extraction, washing the microparticles (e.g.. with water), collecting and drying the microparticles (e.g., by freeze-drying or drying under vacuum), and sieving the microparticles (e.g., through 140 pm).

[0116] A dry microparticle composition can be terminally sterilized by gamma irradiation, either in bulk or after dispensing into the final container. In some embodiments, bulk sterilized microparticles can be resuspended in a suitable vehicle and dispensed into a suitable device such as double chamber syringe with subsequent freeze drying.

[0117] In some embodiments, microparticle depot compositions can include a vehicle to facilitate reconstitution. In addition, prior to administration, microparticles can be suspended in a suitable vehicle for injection (e.g., a water-based vehicle containing one or more pharmaceutical excipients such as mannitol, sodium chloride, glucose, dextrose, sucrose, or glycerin, and / or one or more non-ionic surfactants such as a poloxamer. poly(oxyethylene)-sorbitan-fatty acid ester, carboxymethyl cellulose sodium, sorbitol, poly(vinylpyrrolidone), or aluminium monostearate).

[0118] As described herein, one or more NPs (e.g., one or more M-NPs described herein) and a GLP-l / GIP receptor agonist can be administered to a mammal having cardiovascular and / or metabolic disease. GLP-1 and GIP are components of the incretin pathway, which is a self-regulating feedback system that connects the gut with the brain, the pancreas, and the liver. GLP-1 and GIP (also referred to as “incretins”) are the predominant hormones released from the gut lining upon ingestion of food; they regulate glucose metabolism and appetite, and are responsible for postprandial insulin secretion (Rizvi and Rizzo (2022) Diabetes Metab Syndr Obes., 15: 1023-1030). The “incretin effect” is severely reduced in patients with T2DM (Nauck and Meier (2016) Lancet Diabetes Endocrinol., 4(6):525-536), but administration of GLP-1 receptor agonists can replace the action of incretins and reduce glucose levels. GIP also has a role in glucose homeostasis, and agonists that take advantage of the interplay between GIP and GLP-1 may have both glycemic and nonglycemic (e.g., cardiovascular) benefits.

[0119] Examples of GLP-1 receptor agonists include, without limitation, exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, and albiglutide. Examples of agonists of both GLP-1 receptor and GIP receptor include, without limitation, tirzepatide and retatrutide.

[0120] Any appropriate combination of NP(s) (e.g., M-NP) and GLP-l / GIP receptor agonist(s) can be administered to a mammal in the methods provided herein. Non- limiting examples of such combinations are listed in TABLE 2.

[0121] TABLE 2: Representative combinations ofNPs and GLP-1 GIP receptor agonists

[0122] This document also provides methods for treating cardiovascular and / or metabolic disease in a mammal by administering (a) one or more isolated NPs (e.g., one or more M-NPs) described herein or a composition containing one or more isolated NPs (e.g., one or more M-NPs) described herein, and (b) one or more GLP- 1 / GIP receptor agonists or a composition containing one or more GLP-l / GIP receptor agonists. For example, one of more of the polypeptides described herein can be used in combination with one or more GLP-l / GIP receptor agonists to treat cardiovascular disorders (e.g., hypertension, resistant hypertension, myocardial infarction, and heart failure) and / or metabolic disorders (e.g., type II diabetes and obesity) in mammals. As used herein, the term “GLP-l / GIP receptor agonist’" refers to an agent that is a GLP-1 receptor agonist, a GIP receptor agonist, or a GLP-1 receptor and GIP receptor agonist (that is, an agonist of both the GLP-1 receptor and the GIP receptor). The terms “treat” and “treatment” as used herein refer to prescribing, administering, or providing a medication to beneficially affect or alleviate one or more symptoms associated with a disease or disorder, or one or more underlying causes of a disease or disorder.

[0123] Any appropriate method can be used to assess the presence or extent of cardiovascular and / or metabolic disease, including, without limitation, general clinical and metabolic examination to evaluate blood pressure, heart rate, heart rhythm, arterial oxygen, hemoglobin levels, serum glucose, free fatty acids, glycerol, insulin, Hlc, HOMA-IS and HOMA-IR. cGMP. cAMP, adiponectin, body mass index, waist circumference, and / or weight; echocardiography to measure ejection fraction, LV and left atrium (LA) diameter, LV wall motion, LV filling pressure, and diastolic function by pulse and tissue Doppler; use of a Swan-Ganz catheter to measure cardiac output, pulmonary wedge capillary’ pressure, pulmonary arterial pressure, right ventricle pressure, right atrium pressure, and systemic and pulmonary' vascular resistance; assessment of kidney function by determination of glomerular filtration rate, serum creatinine, and blood urea nitrogen; and measurement of biomarkers such as ANP, amino-terminal pro ANP (NT-proANP), GLP-1, BNP, amino-terminal proBNP (NT- proBNP), troponin-T, troponin-1. C-reactive protein (CRP), and creatine-kinase, serum cystatin-C, albuminuria, neutrophil gelatinize associated lopocalin (NGAL), N- acetyl-beta-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), angiotensin-II, renin, aldosterone, and inflammatory cytokines (e.g., interleukin (IL)- 6, IL-18, etc.). In some cases, an isolated NP (e.g., an isolated M-NP) provided herein can reduce one or more symptoms of acute HF, including clinical parameters such as edema, shortness of breath, and fatigue, as well as cardiac unloading (i.e., reduced pressure in the heart), increased glomerular filtration rate (GFR), decreased PRA, decreased levels of angiotensin II, decreased proliferation of cardiac fibroblasts, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicative of reduced fibrosis and hypertrophy), decreased PWCP (an indirect measure of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased levels of aldosterone (indicative of an anti-fibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end systolic diameter. To determine whether a NP (e.g., a M-NP) is capable of inhibiting or reducing a symptom of acute HF, one or more of these parameters can be evaluated (e.g., before and after treatment with the NP), using any appropriate method.

[0124] Before administering aNP (e.g., a M-NP) described herein and a GLP-l / GIP receptor agonist to a mammal, the mammal can be assessed to determine whether or not the mammal has a need for treatment of a cardiovascular and / or a metabolic disorder. After identifying a mammal as having a need for such treatment, the mammal can be treated with a NP (e.g., a M-NP) described herein and a GLP-l / GIP receptor agonist. For example, a composition containing a NP (e.g.. a M-NP) can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce one or more symptoms of a cardiovascular and / or metabolic disease, or to prevent or delay w orsening of one or more such symptoms).

[0125] The methods provided herein can include administering (a) a NP (e.g., a M- NP) described herein or a composition containing a NP (e.g., a M-NP) described herein, and (b) a GLP-l / GIP receptor agonist or a composition containing a GLP- l / GIP receptor agonist, in any appropriate order. In some cases, the methods provided herein can include administering aNP (e.g., a M-NP) described herein simultaneously with a GLP-l / GIP receptor agonist. In some cases, the methods provided herein can include sequentially administering a NP (e.g., a M-NP) described herein and a GLP- l / GIP receptor agonist. In such cases, the NP can be administered before the GLP- l / GIP receptor agonist, or the GLP-l / GIP receptor agonist can be administered before the NP. In some cases, the NP and the GLP-l / GIP receptor agonist can be administered multiple times, either simultaneously or sequentially.

[0126] In some cases, a NP (e.g., a M-NP) described herein or a composition containing a NP (e.g., a M-NP) described herein can be administered at a dose of at least about 0.01 ng NP / kg to about 100 mg NP / kg of body mass (e.g., about 10 ng NP / kg to about 50 mg NP / kg, about 20 ng NP / kg to about 10 mg NP / kg, about 0. 1 ng NP / kg to about 20 ng NP / kg, about 3 ng NP / kg to about 10 ng NP / kg, or about 50 ng NP / kg to about 100 ug / kg) of body mass, although other dosages also may provide beneficial results. A composition can be administered at a dose of, for example, about 0.1 ng NP / kg / minute to about 500 ng NP / kg / minute (e.g., about 0.5 ng NP / kg / minute, about 1 ng NP / kg / minute, about 2 ng NP / kg / minute. about 3 ng NP / kg / minute, about 5 ng NP / kg / minute, about 7.5 ng NP / kg / minute, about 10 ng NP / kg / minute, about 12.5 ng NP / kg / minute, about 15 ng NP / kg / minute, about 20 ng NP / kg / minute, about 25 ng NP / kg / minute, about 30 ng NP / kg / minute, about 50 ng NP / kg / minute, about 100 ng NP / kg / minute, or about 300 ng NP / kg / minute).

[0127] In some cases, a NP (e.g., a M-NP) described herein or a composition containing a NP (e.g., a M-NP) described herein can be administered via a first route (e.g., intravenously) for a first period of time, and then can be administered via another route (e.g., orally, topically, or subcutaneously) for a second period of time. For example, a composition containing a NP (e.g.. a M-NP) can be intravenously administered to a mammal (e.g., a human) at a dose of about 0. 1 ng NP / kg / minute to about 300 ng NP / kg / minute (e.g., about 1 ng NP / kg / minute to about 15 ng NP / kg / minute, about 3 ng NP / kg / minute to about 10 ng NP / kg / minute, or about 10 ng NP / kg / minute to about 30 ng NP / kg / minute) for one to seven days (e.g., one, two, three, four, five, six, or seven days), and subsequently can be subcutaneously administered to the mammal at a dose of about 10 ng NP / kg / day to about 100 ng NP / kg / day (e.g., about 10 ng NP / kg / day, about 20 ng NP / kg / day, about 25 ng NP / kg / day, about 30 ng NP / kg / day, about 50 ng NP / kg / day, or about 100 ng NP / kg / day) for five to 30 days (e.g.. seven, 10, 14, 18, 21, 24. or 27 days).

[0128] In some cases, a GLP-l / GIP receptor agonist or a composition containing a GLP-l / GIP receptor agonist can be administered at a dose of at least about 0.025 mg to about 20 mg, although other dosages also may provide beneficial results. A composition can be administered at a dose of, for example, about 0.025 mg to about 20 mg (e.g., about 0.025 mg to about 0.05 mg, about 0.05 mg to about 0. 1 mg, about 0.1 mg to about 0.25 mg, about 0.25 mg to about 0.5 mg, about 0.25 mg to about 1 mg, about 0.25 mg to about 2 mg, about 0.5 mg to about 1 mg, about 0.5 mg to about 2.5 mg, about 1 mg to about 1.5 mg, about 1 mg to about 3 mg, about 1.5 mg to about 2 mg. about 2 mg to about 2.5 mg. about 2.5 mg to about 5 mg, about 5 mg to about 10 mg, or about 10 mg to about 20 mg).

[0129] In some cases, a GLP-l / GIP receptor agonist or a composition containing a GLP-l / GIP receptor agonist can be subcutaneously administered to a mammal (e.g., a human) at a dose of about 0.025 mg to about 20 mg (e.g., about 0.25 mg to about 2.5 mg) for five to 30 days (e.g.. five, seven, 10, 14. 18. 21. 24. 27, or 30 days). In some cases, a GLP-l / GIP receptor agonist can be orally administered to a mammal at a dose of about 3 mg to about 14 mg (e.g., about 3 mg to about 5 mg, about 5 mg to about 7 mg. about 7 mg to about 10 mg, about 10 mg to about 12 mg, or about 12 mg to about 14 mg) for five to 30 days (e.g., seven, 10. 14. 18. 21, 24, or 27 days).

[0130] In some cases, the methods provided herein can include administering to a mammal (a) an effective amount of a NP (e.g., a M-NP) described herein (e.g., a NP having the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO: 16, or having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16 but with one, two, or three subtractions, additions, or substitutions) or a nucleic acid encoding such a NP, or an effective amount of a composition containing such a NP, and (b) an effective amount of a GLP-l / GIP receptor agonist. As used herein, the term “effective amount” is an amount of a molecule or composition that is sufficient to alter a selected parameter by at least 10%. For example, an effective amount of aNP (e.g., a M-NP) described herein can be an amount of the NP (e.g., the M-NP) that, in combination with an effective amount of a GLP-l / GIP receptor agonist, is sufficient to reduce the occurrence of a symptom of cardiovascular and / or metabolic disease by at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%). In some cases, an effective amount of a NP (e.g., a M-NP) described herein can be an amount that, in combination with an effective amount of a GLP-l / GIP receptor agonist, reduces a symptom of cardiovascular and / or metabolic disease in a treated mammal by at least 10% as compared to the level of the symptom in the mammal prior to administration of the NP (e.g., the M-NP) and the GLP-l / GIP receptor agonist or without administration of the NP and the GLP-l / GIP receptor agonist, or as compared to the level of the symptom in a control, untreated mammal. The presence or extent of such symptoms can be evaluated using any appropriate method. In some cases, an effective amount of a NP (e.g., a M-NP) provided herein can be an amount that, in combination with an effective amount of a GLP-l / GIP receptor agonist, reduces blood pressure in a mammal identified as having hypertension by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%) as compared to the blood pressure in the mammal prior to administration of the NP and the GLP-l / GIP receptor agonist or without administration of the NP and the GLP-l / GIP receptor agonist, or as compared to the level of the symptom in a control, untreated mammal.

[0131] In some embodiments, the amount and frequency of administration of a NP (e.g., a M-NP) described herein and a GLP-l / GIP receptor agonist to a mammal can be titrated in order to, for example, identify a dosage that is most effective to treat hypertension and / or cardiovascular and / or metabolic disease while having the least number of adverse effects. For example, an effective amount of a composition can be any amount that reduces fibrillation within a mammal without having significant toxicity in the mammal. If a particular mammal fails to respond to a particular amount, then the amount can be increased by, for example, two-fold, three-fold, fivefold, or ten-fold. After receiving this higher concentration, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments in the dosage can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment.

[0132] In some cases, an effective frequency of administration of (a) aNP (e.g., a M- NP) described herein or a pharmaceutical composition containing a NP (e.g.. a M-NP) described herein, and (b) a GLP-l / GIP receptor agonist or a pharmaceutical composition containing a GLP-l / GIP receptor agonist, can be a frequency that reduces one or more symptoms associated with a disorder (e.g., cardiovascular and / or metabolic disease) in the mammal, without producing significant toxicity to the mammal. In some cases, an effective frequency of administration of (a) a NP (e.g., a M-NP) described herein or a pharmaceutical composition containing the NP (e.g., the M-NP), and (b) a GLP-l / GIP receptor agonist or a pharmaceutical composition containing a GLP-l / GIP receptor agonist, can be a frequency that reduces one or more symptoms associated with a disorder (e.g.. cardiovascular and / or metabolic disease) in a mammal as compared to a control mammal having a comparable disorder and not treated with the NP and the GLP-l / GIP receptor agonist. For example, an effective frequency of administration of aNP (e.g., a M-NP) described herein or a pharmaceutical composition containing the NP (e.g., the M-NP) can be one to four times a day to about once every other month, or from about once a day to about once a month, or from about once every other day to about once a week. For example, an effective frequency of administration of a GLP-l / GIP receptor agonist or a pharmaceutical composition containing the GLP-l / GIP receptor agonist can be one to four times a day to about once every other month, or from about once a day to about once a month, or from about once every other day to about once a week. The frequency of administration of (a) a NP (e.g., a M-NP) described herein or a pharmaceutical composition containing the NP (e.g., the M-NP), and (b) a GLP-l / GIP receptor agonist or a pharmaceutical composition containing a GLP-l / GIP receptor agonist can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application. For example, the effective amount, the severity of the disorder w hen treating a mammal, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments, and the judgment of the treating physician may require an increase or decrease in the actual effective frequency of administration.

[0133] In some cases, an effective duration of administration of (a) a NP (e.g., a M- NP) described herein or a pharmaceutical composition containing aNP (e.g., a M-NP) described herein, and (b) a GLP-l / GIP receptor agonist or a pharmaceutical composition containing a GLP-l / GIP receptor agonist, can be a duration that reduces one or more symptoms associated with a disorder (e.g., cardiovascular and / or metabolic disease) in a mammal, without producing significant toxicity to the mammal. In some cases, an effective duration of administration of (a) a NP (e.g., a NINE) described herein or a pharmaceutical composition containing the NP (e.g., the NINE), and (b) a GLP-l / GIP receptor agonist or a pharmaceutical composition containing a GLP-l / GIP receptor agonist, can be a duration that reduces one or more symptoms associated with a disorder (e.g., cardiovascular and / or metabolic disease) in a mammal as compared to a control mammal having a comparable disorder and not treated with the NP and the GLP-l / GIP receptor agonist. For example, an effective duration of administration of aNP (e.g., a M-NP) provided herein or a pharmaceutical composition containing the NP (e.g., the M-NP) can range from one to several days, to several weeks, months, or years. For example, an effective duration of administration of a GLP-l / GIP receptor agonist or a pharmaceutical composition containing the GLP-l / GIP receptor agonist can range from one to several days, to several weeks, months, or years. In general, the effective duration can range in duration from several days to several months. For example, an effective duration can range from about one to two weeks to about 36 months. Prophylactic treatments can be typically longer in duration and may last throughout an individual mammal’s lifetime. Multiple factors can influence the actual effective duration used for a particular application. For example, the severity of the disorder, the effective frequency, the effective amount, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments, and the judgment of the treating physician may require an increase or decrease in the actual effective duration of administration.

[0134] After administering a NP (e.g.. a M-NP) and a GLP-l / GIP receptor agonist to a mammal having cardiovascular and / or metabolic disease, the mammal can, in some cases, be monitored to determine whether or not the cardiovascular and / or metabolic disorder has improved. For example, a mammal can be assessed after treatment to determine whether or not one or more symptoms of the disorder have decreased. Any suitable method can be used to assess improvements in function. If a mammal fails to respond to a particular dose of the NP (e.g., the M-NP) and the GLP-l / GIP receptor agonist, then the amount of one or both can be increased by, for example, two-fold, three-fold, five-fold, or ten-fold. After receiving this higher concentration, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used. For example, the frequency of administration, duration of treatment, route(s) of administration, and severity’ of the disease may require an increase or decrease in the actual effective amount administered.

[0135] In some cases, the methods provided herein can further include monitoring the concentration of aNP (e.g., a M-NP) described herein in serum or plasma drawn from the patient. Blood can be drawn at regular intervals (e g., every 15 minutes, 30 minutes, 1 hour, 2 hours. 4 hours, 6 hours. 10 hours. 12 hours, 20 hours, 22 hours, daily, biweekly, weekly, or monthly). Alternatively, blood can be drawn at random intervals. In still another aspect, an additional step may include creating a feedback loop by increasing or decreasing the amount of polypeptide administered after measuring its concentration.

[0136] Any suitable method can be used to measure serum levels of a NP (e.g., a M- NP) provided herein including, without limitation, mass spectrometry and immunological methods such as ELISA. An antibody used in an immunological assay can be, without limitation, a polyclonal, monoclonal, human, humanized, chimeric, or single-chain antibody, or an antibody fragment having binding activity, such as a Fab fragment, F(ab’) fragment, Fd fragment, fragment produced by a Fab expression library, fragment comprising a VL or VH domain, or epitope binding fragment of any of the above. An antibody can be of any type, (e.g., IgG, IgM, IgD, IgA or IgY), class (e.g., IgGl. IgG4, or IgA2), or subclass. In addition, an antibody can be from any animal including birds and mammals. For example, an antibody can be a human, rabbit, sheep, or goat antibody. Such an antibody can be capable of binding specifically to a polypeptide provided herein.

[0137] Antibodies can be generated and purified using any suitable method. For example, monoclonal antibodies can be prepared using hybridoma, recombinant, or phage display technology, or a combination of such techniques. In some cases, antibody fragments can be produced synthetically or recombinantly from a gene encoding the partial antibody sequence. In some cases, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody. An antibody directed against a polypeptide provided herein typically can bind the polypeptide at an affinity of at least 104mol'1(e.g., at least 105, 106, 107, 108, 109, IO10, 1011, or 1012mol'1).

[0138] Also provided herein are articles of manufacture containing (a) one or more NPs (e.g., one or more M-NPs) or pharmaceutical compositions as described herein (e.g., a depot formulation containing a M-NP described herein) and (b) one or more GLP-l / GIP receptor agonists. The one or more NPs (e.g., one or more M-NPs) and the one or more GLP-l / GIP receptor agonists can be contained in bottles, vials, syringes, or other vessels, or in a combination thereof. The article of manufacture also can include a transfer set and / or a water-based vehicle in a separate vessel, or the NP / composition and vehicle can be separated in a double chamber syringe.

[0139] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0140] EXAMPLES

[0141] Example 1 - Effects of NPs and GLP-1 on Insulin Release from INS-1 Cells

[0142] INS-1 rat insulinoma cells were stimulated with high (20 mM) glucose and cultured in the presence of vehicle, GLP-1, MANP191 (SEQ ID NO: 16), mini-Dl (SEQ ID NO: 8), MANP (SEQ ID NO: 15), ANP (SEQ ID NO: 1). or GLP-1 in combination with MANP191, mini-Dl, MANP, or ANP (each at a concentration of 10'8M), and insulin secretion into the media was measured. These studies demonstrated that MANP191 and mini-Dl synergized with GLP-1 to augment insulin secretion from the INS-1 cells, while MANP and ANP did not show synergy with GLP-1 (FIG. 2).

[0143] Example 2 - Synergistic Insulinotropic and Lipolytic Actions of GLP-1 and NPs in Beta Cells and Adipocytes

[0144] Studies were conducted to investigate the metabolic properties of mini- Dl / GC-A and its interaction with GLP-1 / GLP-1R in vitro. Rat pancreatic beta (INS- 1) cells were treated with GLP-1 and / or mini-Dl at concentrations of IO'10to 10'6M for 1 hour. Supernatants were collected and insulin was measured by ELISA. Human visceral adipocytes were differentiated for 10 days and then treated with GLP-1 and / or mini-Dl ( 1 O'10to 10‘6M) for 6 hours. Supernatants were collected and then glycerol and free fatty acid (NEFA), indicators of lipolysis, were measured.

[0145] Mini-Dl (p<0.05) and GLP-1 (p<0.05) each stimulated insulin secretion in INS-1 cells in a dose-dependent manner in the presence of high glucose (20 mM). The combination of mini-Dl with GLP-1 resulted in greater enhancement (p<0.05) of insulin secretion than either peptide alone (FIG. 2). FIGS. 3-12 show cGMP production (receptor engagement for GC-A) of 4 peptides - endogenous ANP, mini- Dl, MANP, and MANP 191. In summary, mini-Dl at the highest tested dose (10‘6M) had the greatest increase in cGMP production in human adipocytes. MANP 191 had the greatest increase in cGMP at the two lower doses of 10’10M and 10’8M, compared to ANP, mini-Dl , and MANP. ANP and MANP resulted in similar increases in cGMP, but overall the increases were less than those observed for mini-Dl and MANP 191.

[0146] While high dose mini-Dl and GLP-1 (IO-6M) stimulated lipolysis in visceral adipocytes, with increases in glycerol and NEFA production, only mini-Dl at low dose (10‘10M), and not low dose GLP-1 (1O‘10M), stimulated glycerol and NEFA production (p<0.05) (FIGS. 3, 5, 8, and 10). When low dose GLP-1 (10‘10M) was added to low dose mini-Dl (10‘10M), lipolysis indicated by glycerol secretion was similar to the level observed with mini-Dl alone (FIGS. 4 and 9), while lipolysis indicated by NEFA secretion for the combination was higher than the level of lipolysis observed with mini-Dl alone (FIGS. 6 and 11). cGMP production by human visceral adipocytes treated with increasing concentrations of ANP, mini-Dl, MANP, or MANP191 also was evaluated, revealing that IO-6M mini-Dl resulted in greatest increase in cGMP production, while MANP 191 also had a greater effect than MANP (FIG. 12).

[0147] These results indicated synergy' in insulin release from beta cells with combined stimulation of GLP-1 and the potent, miniaturized GC-A designer peptide, mini-Dl. Mim-Dl had greater lipolytic properties than GLP-1 in visceral adipocytes at low doses, advancing understanding of the metabolic actions of mini-D 1 alone or in combination with GLP-1R activation. These in vitro studies of dual receptor activation of GC-A and GLP-1R also highlighted the therapeutic potential of combining mini -DI with GLP-1 -based therapies for the treatment of cardiometabolic diseases.

[0148] Example 3 - Treating Cardiovascular Disease

[0149] A human having cardiovascular disease is administered a M-NP having the sequence set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO: 16 in combination with a GLP-l / GIP receptor agonist such as semaglutide. The administered M-NP and GLP-l / GIP receptor agonist are effective to reduce the occurrence of at least one symptom of the cardiovascular disease in the human.

[0150] Example 4 - Treating Metabolic Disease

[0151] A human having metabolic disease is administered a M-NP having the sequence set forth in SEQ ID NO:8, SEQ ID NO: 12, or SEQ ID NO: 16 in combination with a GLP-l / GIP receptor agonist such as semaglutide. The administered M-NP and GLP-l / GIP receptor agonist are effective to reduce the occurrence of at least one symptom of the metabolic disease in the human.

[0152] OTHER EMBODIMENTS

[0153] It is to be understood that while the invention has been described in conjunction with the detailed description 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 following claims.

Claims

WHAT IS CLAIMED IS:

1. Use of (i) a natriuretic polypeptide and (ii) a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor for treating cardiovascular and / or metabolic disease in a mammal.

2. The use of claim 1. wherein said natriuretic polypeptide comprises:(a) the amino acid sequence set forth in SEQ ID NO: 8,(b) the amino acid sequence set forth in SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 8,(c) the amino acid sequence set forth in SEQ ID NO: 12,(d) the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 12,(e) the amino acid sequence set forth in SEQ ID NO: 16,(1) the amino acid sequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16.

3. The use of claim 1 or claim 2, wherein said mammal has a cardiovascular disorder.

4. The use of claim 3, wherein said cardiovascular disorder comprises hypertension.

5. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:8.

6. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 12.

7. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, or the amino acidsequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16.

8. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8.

9. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12.

10. The use of any one of claims 1 to 4, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

11. The use of any one of claims 1 to 10, wherein said natriuretic polypeptide is formulated for oral administration.

12. The use of any one of claims 1 to 11, wherein said GLP-1 receptor agonist is exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, or albiglutide.

13. The use of any one of claims 1 to 11, wherein said agonist of both a GLP-1 receptor and a GIP receptor is tirzepatide or retatrutide.

14. The use of any one of claims 1 to 13, wherein said GLP-1 receptor agonist, said GIP receptor agonist, or said agonist of both a GLP-1 receptor and a GIP receptor is formulated for administration by injection.

15. A method for treating a mammal having cardiovascular and / or metabolic disease, wherein said method comprises administering, to said mammal:(a) an effective amount of a natriuretic polypeptide; and(b) an effective amount of a GLP-1 receptor agonist, a GIP receptor agonist, or an agonist of both a GLP-1 receptor and a GIP receptor. wherein said administering is effective to reduce one or more symptoms of said cardiovascular and / or metabolic disease in said mammal.

16. The method of claim 15, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forthin SEQ ID NO: 8 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 8.

17. The method of claim 16, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8 but with one conservative amino acid substitution.

18. The method of claim 16, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8 but with two conservative amino acid substitutions.

19. The method of claim 16, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8.

20. The method of claim 15, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 12.

21. The method of claim 20, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12 but with one conservative amino acid substitution.

22. The method of claim 20, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12 but with two conservative amino acid substitutions.

23. The method of claim 20, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12.

24. The method of claim 15, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence set forth in SEQ ID NO: 16 but with one or two amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 16.

25. The method of claim 24, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 but with one conservative amino acid substitution.

26. The method of claim 24, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 but with two conservative amino acid substitutions.

27. The method of claim 24, wherein said natriuretic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

28. The method of any one of claims 15 to 27, wherein said natriuretic polypeptide is a substantially pure polypeptide.

29. The method of any one of claims 15 to 28, wherein said method comprises administering said GLP-1 receptor agonist, and wherein said agonist is exenatide, lixisenatide, liraglutide, dulaglutide, semaglutide, or albiglutide.

30. The method of any one of claims 15 to 28, wherein said method comprises administering said agonist of both a GLP-1 receptor and a GIP receptor, and wherein said agonist is tirzepatide or retatrutide.

31. The method of any one of claims 15 to 30, comprising administering said natriuretic peptide orally.

32. The method of any one of claims 15 to 31, comprising administering said GLP-1 receptor agonist, said GIP receptor agonist, or said agonist of both a GLP-1 and a GIP receptor by injection.

33. The method of any one of claims 15 to 32, wherein said mammal is a human.

34. The method of any one of claims 15 to 33, further comprising identifying said mammal as being in need of said treating.

35. The method of any one of claims 15 to 34. wherein said mammal has said cardiovascular disease.

36. The method of claim 35, wherein said cardiovascular disease comprises hypertension.

37. The method of any one of claims 15 to 34, wherein said mammal has said metabolic disease.

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