Polypeptides having natriuretic and insulin-secreting actions, and methods for their use

Abstract

Chimeric polypeptides that have the ability to stimulate insulin secretion and that have natriuretic actions are provided herein. For example, provided herein are chimeric polypeptides containing a GLP-1-like region that can stimulate insulin secretion, and a NP-like region having natriuretic activity. Methods for using the chimeric polypeptides described herein to treat mammals having, for example, diabetes mellitus, cardiovascular disease, metabolic syndrome, nonalcoholic fatty liver disease, and / or obesity also are provided herein.

Description

[0001] POLYPEPTIDES HAVING NATRIURETIC AND INSULIN-SECRETING ACTIONS, AND METHODS FOR THEIR USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority7from U.S. Provisional Application No. 63 / 643.113, filed on May 6, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under HL 136440 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named ‘L07039-2328W01.xml.‘’ The XML file, created on April 28. 2025. is 9,059 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] This document relates to chimeric polypeptides that have the ability to stimulate insulin secretion via glucagon-like peptide-1 (GLP-1) receptors and also have natriuretic polypeptide (NP) actions via NP receptors. For example, this document provides chimeric polypeptides containing a GLP-1 -like region that can stimulate insulin secretion, and a NP-like region that has natriuretic activity. This document also provides methods for using the chimeric polypeptides described herein to treat mammals having, for example, fibrosis, nonalcoholic fatty7liver disease, cardiovascular disease, metabolic syndrome, obesity7and / or diabetes mellitus.

[0010] BACKGROUND

[0011] Diabetes (DM) is associated with cardiovascular, renal, humoral, and metabolic dysfunction. Insulin and insulin-like peptides such as glucagon-like- peptide (GLP-1) can be used to manage DM. Despite the efficacy of insulin for lowering blood glucose, however, DM patients treated with insulin still develop cardiovascular, renal, humoral, and metabolic complications. In contrast, patients treated with GLP-1 can have improved CV outcomes.

[0012] SUMMARY

[0013] As described herein, polypeptides having the combined actions of GLP-1 and NPs can enhance cardiovascular, renal, humoral, and metabolic protective actions in DM (e.g., diabetic cardiomyopathy), metabolic syndrome, fibrosis, Metabolic Dysfunction- Associated Steatotic Liver Disease (MASLD, previously referred to as nonalcoholic fatty liver disease or NAFLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH, previously referred to as nonalcoholic steatohepatitis or NASH), obesity, and other disorders. For example, as demonstrated herein, ISP-NP (specifically. CRRL-101) treatment of rat beta (INS-1) cells resulted in a dose-dependent increase in insulin secretion from the cells, and treatment of cardiomyocyte cells with the same ISP-NP led to a decrease in insulin-induced apoptosis. CRRL-101 also reduced blood glucose levels and increased plasma insulin levels in diabetic rats, while another ISP-NP (CRRL-094) reduced blood glucose levels, increased blood insulin levels, increased blood cGMP levels, reduced left ventricular interstitial fibrosis, increased glomerular filtration rate (GFR), and reduced proteinuria in diabetic rats. ISP-NP (CRRL094) also reduced left ventricular interstitial fibrosis, reduced glomerular hyperfiltration, and reduced proteinuria in diabetic rats treated with insulin.

[0014] This document provides methods and materials related to a novel class of dualacting chimeric polypeptides (referred to herein as ISP-NPs) that have pGC-activating and GLP-1 insulin-secreting properties. For example, this document provides ISP-NPs that have pGC-activating and GLP-1 insulin-secreting properties when administered to mammals (e.g., humans). This document also provides compositions containing one or more of the ISP-NPs disclosed herein, as well as methods for using the ISP-NPs and compositions containing one or more ISP-NPs provided herein to treat mammals (e.g., humans) in need thereof. In some cases, the ISP-NPs and the compositions provided herein can be used to treat mammals (e.g.. humans) having DM, diabetic cardiomyopathy, diabetic nephropathy, metabolic syndrome, heart failure (e g., heart failure with preserved ejection fraction), MASLD, MASH, fibrosis, obesity, and other disorders.

[0015] In a first aspect, this document features a method for treating a mammal having fibrosis, MASLD, MASH, cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus. The method can include, consist of, or consist essentially of, administering, to the mammal, an effective amount of a composition containing a polypeptide that includes (a) the amino acid sequence set forth in SEQ ID NO:2, (b) the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2, (c) the amino acid sequence set forth in SEQ ID NO: 1, or (d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1. The mammal can have fibrosis, and wherein the composition can be administered to the mammal in an amount effective to reduce the fibrosis in the mammal. The fibrosis can include one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and Peyronie’s disease. The mammal can have MASH, and the composition can be administered in an amount effective to reduce a symptom of the MASH in the mammal. The mammal can have diabetes mellitus, and the composition can be administered to the mammal in an amount effective to reduce blood glucose levels and / or increase blood insulin levels in the mammal. The composition can be administered in an amount effective to increase blood cGMP levels in the mammal. The mammal can have cardiovascular disease, metabolic syndrome, and / or obesity. The polypeptide can include the amino acid sequence set forth in SEQ ID NO:2, or the amino acid sequence set forth in SEQ ID NO: 2 but with one. two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NOT. The polypeptide can include the amino acid sequence set forth in SEQ ID NO:2. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1. The method can include administering the composition subcutaneously, intravenously, or orally. The mammal can be a human. The method can further include identifying the mammal as being in need of the treating.

[0016] In another aspect, this document features a method for increasing natriuretic activity within a mammal having fibrosis, MASLD, MASH, cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus. The method can include, consist of, or consist essentially of, administering, to the mammal, an effective amount of a composition containing a polypeptide that includes (a) the amino acid sequence set forth in SEQ ID N0:2. (b) the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2, (c) the amino acid sequence set forth in SEQ ID NO: 1, or (d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:1. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 2, or the amino acid sequence set forth in SEQ ID NO: 2 but with one, two. three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence set forth in SEQ ID NO: 1 but with one, two. three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:1. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1. The method can include administering the composition subcutaneously, intravenously, or orally. The mammal can be a human. The method can further include identifying the mammal as being in need of increased natriuretic activity. The mammal can have fibrosis. The fibrosis can include one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and Peyronie’s disease. The mammal can have MASLD.

[0017] In another aspect, this document features the use of a polypeptide in the manufacture of a medicament for treating fibrosis, MASLD, MASH, cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus, where the polypeptide includes, consists of, or consists essentially of (a) the amino acid sequence set forth in SEQ ID NO:2, (b) the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2, (c) the amino acid sequence set forth in SEQ ID NO: 1, or (d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1. The medicament can be for treating fibrosis. The fibrosis can include one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiati on- induced lung injury', gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and Peyronie’s disease. The medicament can be for treating MASLD. The medicament can be for treating cardiovascular disease. The cardiovascular disease can include hypertension. The medicament can be for treating diabetes mellitus, metabolic syndrome, nonalcoholic fatty’ liver disease, and / or obesity. The polypeptide can include the amino acid sequence set forth in SEQ ID NO:2, or the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2. The poly peptide can include the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1. The polypeptide can include the amino acid sequence set forth in SEQ ID NO:2. The polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1. The medicament can be formulated for subcutaneous, intravenous, or oral administration.

[0018] 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.

[0019] 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 w ill be apparent from the description and drawings, and from the claims.

[0020] DESCRIPTION OF DRAWINGS

[0021] FIG. 1 shows the sequences of CRRL-101 (SEQ ID NO: 1), CRRL-094 (SEQ ID NO:2), and CRRL-090 (SEQ ID NO:3). Boxes indicate GLP-l-based amino acid sequences. Underlining indicates cysteine residues that can form disulfide bonds to impart a ring structure to CRRL-101 and CRRL-094.

[0022] FIGS. 2A-2B are graphs plotting insulin secretion from INS-1 (rat beta) cells treated with the indicated concentrations of GLP-1 or CRRL-101.

[0023] FIG. 3 is a graph plotting the effect of CRRL-101 on insulin induced apoptosis in AC 16 cells. FIG. 4 is a graph plotting blood glucose levels in diabetic male Wistar rats treated with placebo or the indicated concentrations of CRRL-101 over a 120 minute time course.

[0024] FIGS. 5A and 5B are graphs plotting plasma insulin (FIG. 5A) and plasma glucose (FIG. 5B) levels in four groups of male Wistar rats: Control, untreated nondiabetic (n=17); UDM, untreated diabetic (n=17); CDM, CRRL-094-treated diabetic (n=ll); IDM, insulin-treated diabetic (n=9). *p<0.0001; **p=0.0064; ***p=0.0008.

[0025] FIG. 6 is a graph plotting plasma cGMP levels in four groups of male Wistar rats (Control, UDM, CDM, and IDM as in FIGS. 5A and 5B). *p=0.0025; **p<0.0001.

[0026] FIG. 7A includes representative images of left ventricle sections obtained from male Wistar rats: Control, UDM, CDM, and IDM as in FIGS. 5A and 5B. FIG. 7B is a graph plotting quantified levels of left ventricle interstitial fibrosis in the male Wistar rats.

[0027] FIGS. 8A and 8B are graphs plotting glomerular filtration rate (GFR) (FIG. 8A) and proteinuria (FIG. 8B) in male Wistar rats: Control, UDM, CDM, and IDM as in FIGS. 5A and 5B. For FIG. 8A, *p=0.0022; **p=0.0337; ***p=0.0090, #p<0.0001; ##p=0.0002. For FIG. 8B, *p<0.0001; ns, p=0.0722.

[0028] FIGS. 9A-9D are graphs plotting levels of glycerol (FIGS. 9A-9B) and non- esterified fatty acids (NEFA; FIGS. 9C-9D) released from adipocytes incubated for 6 hours with the indicated concentrations of GLP-1, CRRL-90, CRRL-101, or CRRL-094.

[0029] FIG. 10A includes representative images of adipocytes that were untreated or were treated with CRRL-101, CRRL-094. GLP-1. or CRRL-090 (ISP), and then stained for UCP1 (red) to indicate browning, lipid (green), and nuclei DAPI (blue). FIGS. 10B- 10C are graphs plotting the UCPl / beta- actin ratio determined from Western blotting of adipocyte lysates from cells treated with the indicated concentrations of GLP-1, CRRL- 094, CRRL-101, and CRRL-090 (ISP).

[0030] FIGS. 11A and 11B are graphs plotting the level of oleic acid (OA) uptake into HepG2 cells treated with (FIG. 11A) OA alone or OA in combination with the indicated concentrations of CNP, glucagon, GLP-1, or CRRL-094, or treated with (FIG. 11B) OA along or OA in combination with the indicated concentrations of ANP, glucagon, GLP-1, or CRRL-101.

[0031] FIG. 12 is a graph plotting the change in body weight of control rats, rats that were induced to have Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) by feeding a high-fat, high-glucose / fructose diet for 8 weeks, without subsequent treatment (“UM”), and rats with induced MASLD that were treated with CRRL-094 for 4 weeks (“MASLD+CRRL094”). FIGS. 13 A and 13B are graphs ploting ratios of heart weight to body weight (FIG. 13A) and left ventricle weight to body weight (FIG. 13B) in the control, UM, and MASLD+CRRL094 rats.

[0032] FIGS. 14A and 14B are graphs ploting left ventricle interstitial fibrosis (FIG. 14A) and apoptosis (FIG. 14B) in the control, UM and MASLD+CRRL094 rats, as assessed by histological analysis.

[0033] FIG. 15 is a graph ploting left ventricular ejection fraction (LVEF) in the control, UM, and MASLD+CRRL094 rats.

[0034] FIG. 16 is a graph ploting kidney weight to body weight ratios in the control, UM, and MASLD+CRRL094 rats.

[0035] FIG. 17 is a graph ploting kidney cortex fibrosis in the control. UM, and MASLD+CRRL094 rats, as determined by histological analysis.

[0036] FIG. 18 is a graph ploting plasma creatinine levels in the control, UM, and MASLD+CRRL094 rats.

[0037] FIG. 19 is a graph plotting liver-to-body weight ratio in the control. UM, and MASLD+CRRL094 rats.

[0038] FIG. 20 is a graph ploting plasma insulin levels in the control, UM, and MASLD+CRRL094 rats.

[0039] FIGS. 21A-21D are graphs ploting total cholesterol (FIG. 21A), plasma HDL (FIG. 21B), plasma LDL (FIG. 21C) and triglyceride (FIG. 21D) levels in the control, UM, and MASLD+CRRL094 rats.

[0040] DETAILED DESCRIPTION

[0041] This document provides methods and materials related to chimeric, dual-acting polypeptides that have pGC-activating and GLP-1 insulin-secreting properties. For example, this document provides substantially pure chimeric polypeptides that are referred to herein as ISP-NPs. The ISP-NPs provided herein have GLP-l-like activity7that results in insulin secretion, as well as NP-like activity that activates the pGC receptor. This document also provides compositions containing chimeric ISP-NPs having insulinsecreting and natriuretic polypeptide activities, nucleic acid molecules encoding ISP-NPs having insulin-secreting and natriuretic polypeptide activities, and host cells containing isolated nucleic acid molecules that encode chimeric ISP-NPs having insulin-secreting and natriuretic polypeptide activities. In addition, this document provides methods for treating disorders such as DM and its associated complications (e.g., diabetic cardiomyopathy), metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, and obesity in a mammal (e.g., a human, a rodent, a pig, a sheep, a dog, or a non-human primate).

[0042] The ISP-NPs provided herein are effective to increase insulin secretion, and also have natriuretic effects such that they can 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.

[0043] Exemplary natriuretic peptides include ANP, BNP, CNP, urodilatin, and DNP. Natriuretic peptides typically have a 17-amino acid ring structure resulting from a disulfide bond between cysteine residues. The amino acid sequence for mature human ANP, BNP, CNP, and urodilatin (URO) are shown below, with underlining to indicate the cysteine residues involved in the disulfide bond:

[0044] ANP: SLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO:4).

[0045] BNP: SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO:5)

[0046] CNP: GLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:6)

[0047] URO: TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO:7) Dendroaspis natriuretic peptide (DNP), from the venom of Dendroaspis angusticeps (green mamba snake), has sequence and structural similarity to ANP, BNP, and CNP:

[0048] DNP: EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA (SEQ ID NO: 8).

[0049] An ISP-NP provided herein can include an amino acid sequence similar to one or more sequences present in a human polypeptide having natriuretic polypeptide activity (e.g., ANP, BNP, CNP, urodilatin, and / or DNP), but due to their chimeric nature, the ISP- NPs provided herein have non-naturally occurring sequences. It is to be noted that the ISP-NPs provided herein can include a sequence present in any species (e.g., human, horse, pig, goat, cow, dog, cat, rat, or mouse).

[0050] GLP-1 is a peptide hormone that typically is 30 or 31 amino acids in length and is derived from tissue-specific posttranslational processing of proglucagon. GLP-1 is produced and secreted by intestinal enteroendocrine L-cells and certain neurons within the nucleus of the solitary tract in the brainstem upon food consumption. The initial GLP- 1 product (amino acids 1-37 of proglucagon) is susceptible to amidation and proteolytic cleavage, resulting in two truncated and equipotent biologically active products: GLP-1 amide (amino acids 7-36 of proglucagon), and GLP-1 (amino acid 7-37 of proglucagon). GLP-1 can decrease blood sugar levels in a glucose-dependent manner by enhancing insulin secretion. A representative 31-amino acid human GLP-1 amino acid sequence is set forth in SEQ ID NO:9: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0051] 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.

[0052] 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 polyacrylamide gel. In some embodiments, a substantially pure polypeptide can be a chemically synthesized polypeptide.

[0053] 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.

[0054] The ISP-NPs provided herein are chimeric polypeptides that contain an NP-like region and a GLP-l-like region. An ISP-NP can have any appropriate length. In some cases, the polypeptides provided herein can have a length of about 45 amino acids to about 62 amino acids (e.g.. 45 amino acids, 46 amino acids. 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, 61 amino acids, 62 amino acids, about 45 to about 50 amino acids, about 48 to about 54 amino acids, about 53 to about 59 amino acids, or about 55 to about 62 amino acids).

[0055] Non-limiting examples of ISP-NPs are shown in FIG. 1. In some cases, an ISP- NP provided herein can have the amino acid sequence of the “CRRL-101” polypeptide set forth in SEQ ID NO: 1. In some cases, an ISP-NP provided herein can have the amino acid sequence of the “CRRL-094” polypeptide set forth in SEQ ID NO:2. The boxed portions of SEQ ID NOS: 1 and 2 shown in FIG. 1 represent the GLP-l-like region, and the unboxed portions of SEQ ID NOS: 1 and 2 shown in FIG. 1 represent the NP-like region.

[0056] In some cases, an ISP-NP provided herein can contain the entire amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, except that the amino acid sequence can contain one, two, three, four, or five amino acid additions, subtractions, or substitutions. For example, a polypeptide can contain the amino acid sequence set forth in SEQ ID NO: 1 with one, tw o. three, four, or five single amino acid residue additions, subtractions, or substitutions. For example, a polypeptide can contain the amino acid sequence set forth in SEQ ID NO:2 with one, two, three, four, or five single amino acid residue additions, subtractions, or substitutions.

[0057] Any amino acid residue set forth in SEQ ID NO: 1 or SEQ ID NO:2 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: 1 or SEQ ID NO:2. 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 provided herein can contain one or more D-amino acids. In some embodiments, a polypeptide can contain chemical structures such as 8-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.

[0058] ISP-NPs having one or more amino acid additions, subtractions, or substitutions relative to the amino acid sequences set forth in SEQ ID NOS: 1 and 2 (also referred to herein as “variant” ISP-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. In some embodiments, an ISP-NP provided herein can include one or more nonconservative substitutions as compared to SEQ ID NO: 1 or SEQ ID NO:2. Nonconservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Such substitutions can be desirable to provide large quantities or alternative embodiments of such polypeptides. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide variant using, for example, methods disclosed herein (e.g., in the Examples section).

[0059] In some embodiments, an ISP-NP provided herein can include an amino acid sequence with at least 85% (e.g., at least 88%, at least 89%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100%) sequence identity to a reference ISP-NP sequence (e.g., SEQ ID NO: 1 or SEQ ID NO:2). 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.

[0060] In particular, the percent sequence identity between 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 tw o compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0061] 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 NO: 1), 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 53 matches when aligned with the sequence set forth in SEQ ID NO: 1 is 94.6 percent identical to the sequence set forth in SEQ ID NO: 1 (i.e.. 53 / 56 x 100 = 94.6). 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.

[0062] Isolated polypeptides can be produced using any suitable 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. Polypeptides also can be produced recombinantly or obtained commercially.

[0063] The ISP-NP provided herein can be cyclic due to disulfide bonds between the cysteine residues underlined in the sequences shown in FIG. 1. 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.

[0064] 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 P arm. Sci. 463-468; Hudson et al. (1979) Int. J. Pept. Prot. Res. 14: 177; Spatola, in Chemistry7and Biochemistry of Amino Acid Peptides and Proteins. B. Weinstein, ed., Marcel Dekker, New7York, 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.

[0065] 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.

[0066] In some cases, a polypeptide provided herein can be pegylated, acetylated, 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 fatty acid 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 polypeptide having an increased half-life as compared to an unmodified polypeptide. 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 polypeptide. Methods for modifying a polypeptide by linkage to PEG (also referred to as ■’PEGylation") 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 Cardiovasc. 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.

[0067] In some cases, an ISP-NP 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, an ISP-NP provided herein 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-aminobutyric 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.

[0068] 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).

[0069] The ISP-NPs provided herein can function through one or more of the guanylyl cyclase receptors through which native (wild type) natriuretic polypeptides function. For example, the ISP-NPs provided 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, an ISP-NP 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 funebon of a particular ISP-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 an ISP-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 an ISP-NP through one or the other receptor can be atenuated. In some cases, a cell line that overexpresses a particular guanylyl cyclase receptor (e.g., GC-A or GC-B) can be incubated with an ISP-NP provided herein, and cGMP produced by the cells can be measured.

[0070] In some cases, an isolated ISP-NP provided herein can be used to treat mammals (e.g., humans) having DM, cardiovascular disorders (e.g., diabetic cardiomyopathy), metabolic syndrome, MASLD, MASH, obesity, and other disorders. For example, the ISP-NPs provided herein can be used to treat diabetic cardiomyopathy or metabolic syndrome. Any suitable method can be used to assess the presence or extent of disease, including, without limitation, general clinical examination to evaluate blood (e.g., plasma) glucose levels (e.g., fasting blood glucose levels), hemoglobin Ale, lipid panel, blood pressure, heart rate, heart rhythm, arterial oxygen, and hemoglobin levels; 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; and measurement of biomarkers such as BNP, aminoterminal proBNP (NT-proBNP), troponin-T, troponin-I, C-reactive protein (CRP), and creatine-kinase, serum cystatin-C, albuminuria, neutrophil gelatinize associated lopocalin (NGAL). N-acetyl-beta-D-glucosaminidase (NAG). angiotensin-II. renin, aldosterone, and inflammatory cytokines (e.g., interleukin (IL)-6, IL-18, etc.). In some cases, an isolated ISP-NP provided herein can reduce one or more symptoms of DM, cardiovascular disorders (e.g., diabetic cardiomyopathy and / or diabetic nephropathy), metabolic syndrome, fibrosis (e.g.. hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, and / or obesity, including clinical parameters such as edema, shortness of breath, fatigue, weakness, abdominal pain, and / or unexplained weight loss, as well as cardiac unloading (i.e., reduced pressure in the heart), decreased 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 an ISP-NP is capable of inhibiting or reducing a symptom of DM, a cardiovascular disorder (e.g., diabetic cardiomyopathy and / or diabetic nephropathy), metabolic syndrome, fibrosis, MASLD, MASH, and / or obesity, one or more of these parameters can be evaluated (e.g., before and after treatment with the ISP-NP), using any appropriate method.

[0071] ISP-NPs provided herein (e.g., polypeptides having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. or containing one, two, three, four, or five subtractions, additions, or substitutions with respect to SEQ ID NO: 1 or SEQ ID NO:2) can be screened for biological activity using any appropriate assay, including those described in the Examples herein. For example, the activity of an ISP-NP can be evaluated in vitro by testing its effect on cGMP production, in cultured cells (e.g., cultured cardiac fibroblasts, aortic endothelial cells, or a cell line transfected with a nucleic acid encoding a guanylyl cyclase receptor such as GC-A or GC-B). In some cases, the activity of an ISP-NP can be evaluated in vitro by testing its effect on insulin secretion and / or insulin-induced apoptosis in cultured cells (e.g., rat beta cells). In some cases, the activity of an ISP-NP can be evaluated in vitro by testing its effect on glycerol release and / or release of fatty acids from cultured cells (e.g., adipocytes). In some cases, the activity of an ISP-NP can be evaluated in vitro by testing its effect on fatty acid (e.g., oleic acid) uptake into cultured liver cells (e.g., HepG2 cells). Cells can be exposed to an ISP-NP (e.g., 10‘10to 10'4M ISP-NP), and samples can be assayed to evaluate the effects of the ISP-NP on cGMP generation, insulin release, insulin-induced apoptosis, glycerol release, fatty acid release, and / or fatty acid uptake.

[0072] The activity of an ISP-NP also can be evaluated in vivo by, for example, testing its effects on factors such as plasma cGMP levels, urinary cGMP excretion, blood (e.g., plasma) insulin levels, blood (e.g., plasma) glucose levels, 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, GFR, 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 DM (e.g., by streptozotocin administration), heart failure (e.g., by rapid right ventricular pacing), obesity (e.g., using a high fat diet), and / or hypertension.

[0073] This document also provides nucleic acid molecules encoding the ISP-NPs provided herein. For example, this document provides nucleic acid molecules encoding ISP-NPs having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. In some cases, a nucleic acid molecule provided herein can encode an ISP-NP that contains the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, except that the amino acid sequence contains one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:1 or SEQ ID NO:2.

[0074] 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.

[0075] 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.

[0076] 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 prokary ote 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.

[0077] 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 an ISP-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 typically 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 complementary7DNA (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.

[0078] 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 phosphoramidite 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 complementarity (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.

[0079] 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.

[0080] In an expression vector, a nucleic acid (e.g., a nucleic acid encoding an ISP-NP) 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’7and “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.

[0081] 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).

[0082] 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.

[0083] Host cells containing vectors also are provided. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced (e.g., vector encoding an ISP-NP provided 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.

[0084] 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.

[0085] Any suitable method can be used to identify cells containing an isolated nucleic acid molecule provided herein. Such methods include, without limitation, PCR and nucleic acid hybridization techniques such as Northern and Southern analyses. In some cases, immunohistochemistry' 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.

[0086] The ISP-NPs described herein (e.g., ISP-NPs having the amino acid sequences set forth in SEQ ID NOT and SEQ ID NO:2, or variants thereof), or nucleic acids encoding the ISP-NPs described herein, can be incorporated into compositions for administration to a mammal (e.g., a human having or at risk for DM, metabolic syndrome, heart failure, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury', gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, and / or obesity). Dosages typically are dependent on the responsiveness of the subj ect to the polypeptide, 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 ECso found to be effective in in vitro and / or in vivo animal models. Compositions containing one or more ISP-NPs or one or more 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, an ISP-NP or a composition containing an ISP-NP can be administered to a mammal (e.g., a human) at a dose of at least about 0.01 ng ISP-NP / kg to about 100 mg ISP-NP per 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 ISP-NP / kg / minute to about 0.5 pg ISP-NP / kg / minute).

[0087] One or more ISP-NPs provided herein, or nucleic acids encoding one or more ISP- NPs provided 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.

[0088] In some embodiments, a composition can contain an ISP-NP provided herein in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering antibodies to a subject. 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).

[0089] Pharmaceutical compositions containing ISP-NPs 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.) infusion); 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).

[0090] In some cases, an ISP-NP provided herein can be administered orally to a mammal (e.g., a human). 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.

[0091] 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 can be particularly useful for oral deliver}' of therapeutic compositions due to their ease of formulation and efficacy of solubilization, absorption, and bioavailabilify. Liposomes can be particularly useful due to their specificity and the duration of action they offer from the standpoint of drug delivery.

[0092] 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., an ISP-NP provided herein). Accordingly, for example, this document provides pharmaceutically acceptable salts of ISP-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 ISP-NPs useful in methods provided herein (salts that retain the desired biological activity of the parent ISP-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). 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.

[0093] In some cases, an ISP-NP provided herein can be formulated as a sustained release dosage form. For example, an ISP-NP 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 waxes.

[0094] 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., ISP-NPs) with 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 with the effectiveness of the molecules(s) contained in the formulation.

[0095] In some cases, an ISP-NP provided 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 ISP-NP 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, an ISP-NP can be combined with 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, semi-solid, 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.

[0096] 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 ISP-NP 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, microcrystal line cellulose, polyvinylpyrrolidone, cellulose, water, syrup. PEG. cyclodextrin, alkoxy- modified cyclodextrins, hydroxyethylcellulose, hydroxypropylcellulose, microciystalline 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).

[0097] 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 an ISP-NP. Such agents include salts of inorganic acids and magnesium hydroxide. Other agents that can be used include surfactants and other solubilizing materials.

[0098] 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. 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).

[0099] 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, glycery l monostearate, glycery l palmitostearate, lecithin, medium chain triglycerides, monoethanolamine, oleic acid, poloxamers, polyvinyl alcohol and sorbitan fatty’ acid esters.

[0100] 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.

[0101] In some embodiments, an ISP-NP provided herein can be incorporated into a hydrogel delivery' system. For example, an ISP-NP 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.

[0102] Liquid polymerizable materials useful in the preparation of hydrogel tubes include a wide variety of 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.

[0103] In some cases, the polymerization of hydrophilic monomeric mixtures can result in homogeneous hydrophilic copolymers which dissolve, to a vary ing 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 waterinsoluble 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., ethylene glycol dimethacrylate (“EDMA”) or trimethylolpropane trimethacrylate ('‘TMPTMA”)].

[0104] In some embodiments, a pharmaceutical composition for controlled release delivery’ of an ISP-NP in a subject can include (a) a complex of the ISP-NP (where the ISP-NP 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 ,'’ as used herein, refers to continual delivery7of 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 an ISP-NP can be demonstrated by, for example, continued therapeutic effects of the ISP-NP over time (e.g., continued reductions in symptoms over time). Sustained delivery7of an ISP-NP polypeptide also can be demonstrated by detecting the presence of the ISP-NP 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).

[0105] In such embodiments, a physically and chemically stable complex can form upon appropriate combining of an ISP-NP and a polyanion. The complex can take the form of a precipitate that is produced upon combining an aqueous preparation of the polypeptide and the polyanion. 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. Non-limiting examples of suitable excipients include sodium bisulfite, p-aminobenzoic acid, thiourea, glycine, methionine, mannitol, sucrose, and PEG.

[0106] A stable complex between an ISP-NP 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 polyorthoesters, and copolymers, block copolymers, branched copolymers, terpolymers, and combinations thereof.

[0107] 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.

[0108] 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 polydispersity7of from 1.1 to 2.8, depending upon which polymer is selected for use.

[0109] 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. 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.

[0110] 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).

[0111] 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 / poly anion complex.

[0112] 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 / polyanion complex.

[0113] Emulsifiers useful to prepare encapsulated polypeptide / polyanion 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.

[0114] In some embodiments, an ISP-NP 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 an ISP-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.

[0115] 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 an ISP-NP 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.

[0116] High exposure depot formulations for subcutaneous administration of ISP-NPs 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.

[0117] In some embodiments, depot formulations 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 viscosity7can 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.

[0118] The ISP-NP content of a 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.

[0119] 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 sterility assurance possible. Depot compositions also can contain one or more pharmaceutical excipients that can modulate the release behavior of an ISP-NP. 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(oxyethylene- block-oxypropylene), poly(oxyethylene)-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.

[0120] 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 two different linear polymers in one implant or microparticle. A mixture of depots is defined herein as a mixture of two depotlike implants or microparticles or semisolid formulations of different composition with one or more PLGAs in each depot. Pharmaceutical depot compositions in which tw o PLGAs are present as a polymer blend can be particularly useful.

[0121] 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.

[0122] 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).

[0123] In some embodiments, 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.

[0124] Microparticles can be manufactured using processes such as, for example, coacervation or phase separation, spray drying, or w ater-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:

[0125] (i) preparing an internal organic phase, comprising (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;

[0126] (b) dissolving / suspending / emulsifying a polypeptide in the polymer solution obtained in step (a);

[0127] (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;

[0128] (iii) mixing the internal organic phase with the external aqueous phase to form an emulsion; and

[0129] (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).

[0130] 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.

[0131] 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).

[0132] Also provided herein are articles of manufacture containing one or more ISP-NPs or pharmaceutical compositions as described herein (e.g., a depot formulation containing an ISP-NP provided herein) in a bottle, vial, syringe, or other vessel. The article of manufacture also can include a transfer set and / or a water-based vehicle in a separate vessel, or the polypeptide / composition and vehicle can be separated in a double chamber syringe. Methods for increasing natriuresis in a mammal also are provided herein. The methods can include administering (e.g.. orally) to a mammal, in an amount effective to increase natriuresis in the mammal, an ISP-NP provided herein. As used herein, an “increase” in natriuresis is an increase of at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 50%, or at least 100%) in natriuresis of the mammal, as compared to a reference level of natriuresis. A “reference level” can be a control level of natriuresis in the mammal prior to administration of the ISP-NP, for example.

[0133] This document also provides methods for treating clinical conditions, disorders, and / or diseases, such as DM, cardiovascular disease (e.g., diabetic cardiomyopathy and / or heart failure, such as heart failure with preserved ejection fraction), metabolic syndrome, fibrosis (e.g.. hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, obesity, and other disorders in mammals. Any appropriate mammal can be treated as described herein. For example, humans or other primates, dogs, cats, horses, cows. pigs, sheep, mice, and rats can be treated as described herein. 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.

[0134] In some cases, before administering a polypeptide or composition provided herein to a mammal, the mammal can be assessed to determine whether or not the mammal has a need for treatment of DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity. After identifying a mammal as having a need for such treatment, the mammal can be treated w ith a composition containing one or more ISP-NPs provided herein. For example, a composition containing an ISP-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 DM, a cardiovascular disorder such as diabetic cardiomyopathy and / or heart failure, metabolic syndrome, fibrosis, MASLD, MASH, or obesity, or to prevent or delay worsening of one or more such symptoms).

[0135] In some cases, an ISP-NP or a composition containing an ISP-NP can be administered at a dose of at least about 0.01 ng ISP-NP / kg to about 100 mg ISP-NP / kg of body mass (e.g., about 10 ng ISP-NP / kg to about 50 mg ISP-NP / kg, about 20 ng ISP- NP / kg to about 10 mg ISP-NP / kg, about 0. 1 ng ISP-NP / kg to about 20 ng ISP-NP / kg, about 3 ng ISP-NP / kg to about 10 ng ISP-NP / kg, or about 50 ng ISP-NP / kg to about 100 .g / 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 ISP- NP / kg / minute to about 500 ng ISP-NP / kg / minute (e g., about 0.5 ng ISP-NP / kg / minute, about 1 ng ISP-NP / kg / minute, about 2 ng ISP-NP / kg / minute, about 3 ng ISP- NP / kg / minute, about 5 ng ISP-NP / kg / minute, about 7.5 ng ISP-NP / kg / minute, about 10 ng ISP-NP / kg / minute, about 12.5 ng ISP-NP / kg / minute, about 15 ng ISP-NP / kg / minute, about 20 ng ISP-NP / kg / minute, about 25 ng ISP-NP / kg / minute, about 30 ng ISP- NP / kg / minute, about 50 ng ISP-NP / kg / minute, about 100 ng ISP-NP / kg / minute, or about 300 ng ISP-NP / kg / minute).

[0136] In some embodiments, an ISP-NP or a composition containing an ISP-NP 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) for a second period of time. For example, a composition containing an ISP-NP can be intravenously administered to a mammal (e.g.. a human) at a dose of about 0. 1 ng ISP-NP / kg / minute to about 300 ng ISP-NP / kg / minute (e.g., about 1 ng ISP-NP / kg / minute to about 15 ng ISP-NP / kg / minute, about 3 ng ISP- NP / kg / minute to about 10 ng ISP-NP / kg / minute, or about 10 ng ISP-NP / kg / minute to about 30 ng ISP-NP / kg / minute) for one to seven days (e.g., one, two, three, four, five, six, or seven days), and subsequently can be orally administered to the mammal at a dose of about 10 ng ISP-NP / kg / day to about 100 ng ISP-NP / kg / day (e.g., about 10 ng ISP- NP / kg / day, about 20 ng ISP-NP / kg / day, about 25 ng ISP-NP / kg / day, about 30 ng ISP- NP / kg / day, about 50 ng ISP-NP / kg / day, or about 100 ng ISP-NP / kg / day) for five to 30 days (e.g., seven, 10, 14, 18, 21, 24, or 27 days).

[0137] Methods provided herein can include administering to a mammal an effective amount of an ISP-NP provided herein (e.g., an ISP-NP having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 but with one, two, three, four, or five subtractions, additions, or substitutions) or a nucleic acid encoding such an ISP-NP, or an effective amount of a composition containing such an ISP-NP. 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, in some embodiments, an “effective amount” of an ISP-NP is an amount of the ISP-NP that is sufficient to reduce blood (e.g., plasma) glucose levels, to increase insulin secretion, to reduce left ventricle interstitial fibrosis, and / or to increase natriuresis and / or diuresis (or to increase or decrease a characteristic of natriuresis and / or diuresis such as plasma cGMP levels, urinary cGMP excretion, net renal cGMP generation, urine flow, urinary sodium excretion, urinary potassium excretion, hematocrit, plasma BNP immunoreactivity, renal blood flow, plasma ANP immunoreactivity7, renal vascular resistance, proximal and distal fractional reabsorption of sodium, mean arterial pressure, pulmonary wedge capillary7pressure, right atrial pressure, pulmonary arterial pressure, plasma renin activity, plasma angiotensin II levels, plasma aldosterone levels, renal perfusion pressure, and systemic vascular resistance) 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%), as compared to the level of the same parameter prior to treatment, or as compared to the level of the parameter in a control, untreated mammal. For example, an “effective amount” of an ISP-NP can be an amount that increases sodium excretion in a treated mammal by at least 10% as compared to the level of sodium excretion in the mammal prior to administration of the ISP-NP, or as compared to the level of sodium excretion in a control, untreated mammal. In some cases, an “effective amount” of an ISP-NP can be an amount that reduces blood (e.g., plasma) glucose levels in a treated mammal by at least 10% as compared to the blood glucose level in the mammal prior to administration of the ISP-NP, or as compared to the blood glucose level in a control, untreated mammal.

[0138] In some embodiments, an “effective amount” of an ISP-NP provided herein can be an amount of the ISP-NP that is sufficient to reduce the occurrence of a symptom of DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation- induced lung injury', gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity7by 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, for example, an “effective amount” of an ISP-NP provided herein can be an amount that reduces a symptom of DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury7, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity in a treated mammal by at least 10% as compared to the level of the symptom in the mammal prior to administration of the ISP- NP or without administration of the ISP-NP, 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 an ISP- NP provided herein can be an amount that reduces blood (e.g., plasma) glucose and / or increases natriuresis in a mammal identified as having DM 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 level of blood (e.g., plasma) glucose in the mammal prior to administration of the ISP-NP or without administration of the ISP-NP, or as compared to the level of the symptom in a control, untreated mammal. In some cases, an “effective amount” of an ISP-NP can be an amount that reduces fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation- induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie's disease) in a treated mammal by at least 10% as compared to the amount of fibrosis in the mammal prior to administration of the ISP-NP, or as compared to the level of fibrosis in a control, untreated mammal. In some cases, an “effective amount” of an ISP-NP can be an amount that reduces a symptom of MASH (e.g., fatigue, weakness, abdominal pain, and / or unexplained weight loss) in a treated mammal by at least 10% as compared to the amount of the symptom in the mammal prior to administration of the ISP-NP, or as compared to the level of the symptom in a control, untreated mammal.

[0139] In some embodiments, the amount and frequency of ISP-NP administration to a mammal can be titrated in order to, for example, identify a dosage that is most effective to treat DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiati on- induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity while having the lowest amount of adverse effects. For example, an effective amount of a composition can be any amount that reduces blood (e.g., plasma) glucose and / or increases natriuresis 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, 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 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.

[0140] In some cases, an effective frequency of administration of an ISP-NP provided herein or a pharmaceutical composition containing the ISP-NP can be a frequency that reduces one or more symptoms associated with a disorder (e.g., DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury', gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity ) in the mammal, without producing significant toxicity to the mammal. In some cases, an effective frequency of administration of an ISP-NP provided herein or a pharmaceutical composition containing the ISP-NP can be a frequency7that reduces one or more symptoms associated with a disorder (e.g., DM, cardiovascular disease, metabolic syndrome, fibrosis, MASLD, MASH, or obesity) in a mammal as compared to a control mammal having a comparable disorder and not treated with the ISP-NP or the composition. For example, an effective frequency of administration of an ISP-NP provided herein or a pharmaceutical composition containing the ISP-NP can be four rimes 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 an ISP-NP provided herein or a pharmaceutical composition containing the ISP-NP 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 when 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 clinician may require an increase or decrease in the actual effective frequency of administration.

[0141] In some cases, an effective duration of administration of an ISP-NP provided herein or a pharmaceutical composition containing an ISP-NP can be a duration that reduces one or more symptoms associated with a disorder (e.g., DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury7, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity ) in a mammal, without producing significant toxicity to the mammal. In some cases, an effective duration of administration of an ISP-NP provided herein or a pharmaceutical composition containing an ISP-NP can be a duration that reduces one or more symptoms associated with a disorder (e.g., DM, cardiovascular disease, metabolic syndrome, fibrosis, MASLD, MASH, or obesity) in a mammal as compared to a control mammal having a comparable disorder and not treated with the ISP-NP or the composition. For example, an effective duration of administration of an ISP-NP provided herein or a pharmaceutical composition containing an ISP-NP 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 cousage with other therapeutic or prophylactic treatments, and the judgment of the treating clinician may require an increase or decrease in the actual effective duration of administration of an ISP-NP described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the ISP-NP or the pharmaceutically acceptable salt as described herein.

[0142] After administering an ISP-NP or composition provided herein to a mammal having a disorder (e.g., DM, cardiovascular disease, metabolic syndrome, fibrosis (e.g., hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, IPF, cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, and / or Peyronie’s disease), MASLD, MASH, or obesity), the mammal can be monitored to determine whether or not the 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. For example, fibrosis in a mammal can be assessed using a method such as, without limitation, biopsy, fibroscan, ultrasound elastography, magnetic resonance imaging elastography, vibration-controlled transient elastography. computerized tomography (CT) scan, x-ray. echocardiogram, bronchoscopy, nuclear magnetic resonance (NMR), and / or ultrasound. If a mammal fails to respond to a particular dose, then the amount can be increased by, for example, twofold, 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.

[0143] The methods provided herein can further include monitoring the concentration of an ISP-NP 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 the ISP-NP administered after measuring its concentration.

[0144] Any suitable method can be used to measure serum levels of an ISP-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.

[0145] Antibodies can be generated and purified using any suitable method. For example, monoclonal antibodies can be prepared using hybridoma, recombinant, or phage display technology7, 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).

[0146] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0147] Example 1 - ISP-NPs

[0148] Candidate polypeptides having a putative pGC-activating domain (e.g., based on a natriuretic peptide sequence) and a GLP-l-like domain having insulin-secreting properties were generated by direct synthesis and then tested in vitro and in vivo. The sequences of several of the resulting polypeptides. CRRL-101 (SEQ ID NO: 1). CRRL- 094 (SEQ ID NO:2), and CRRL-090 (SEQ ID NO:3) are shown in FIG. 1.

[0149] As described herein, CRRL-101 was demonstrated to activate cGMP via the natriuretic peptide A receptor (NPRA), induce insulin secretion in pancreatic beta cells, activate cAMP in pancreatic beta cells at a level similar to GLP-1, reduce alpha smooth muscle actin (a-SMA) production in human cardiac fibroblast (HCF) cells, effectively counter fibrosis (a property not observed with GLP-1), inhibit insulin-induced apoptosis in cardiomyocytes (a property not observed with GLP-1, and increase insulin secretion and decrease plasma glucose in a rat model of diabetes when administered by acute infusion.

[0150] Also as described herein, CRRL-094 was demonstrated to activate cGMP via the natriuretic peptide B receptor (NPRB), induce insulin secretion in pancreatic beta cells, activate cAMP in pancreatic beta cells at a level similar to GLP-1, and increase insulin secretion and decrease plasma glucose in a rat model of diabetes when administered by acute infusion.

[0151] Example 2 - CRRL-101 induces insulin secretion and activates the natriuretic peptide A receptor

[0152] In vitro studies were conducted to explore the biological properties of CRRL-101 in various cell types and to elucidate its antiapoptotic properties, insulin-secreting properties, and cardioprotective effects. In particular, the properties of CRRL-101 were investigated in INS-1 rat beta cells and in AC16 human cardiomyocytes. Insulin production and cAMP levels were determined under high glucose (20 mM) conditions in INS-1 cells treated with CRRL-101 or GLP-1. In addition, apoptosis was evaluated in the AC 16 cell line using the IncuCyte assay after exposure to insulin (10 nmol / L) or a combination of insulin and CRRL-101 or insulin and GLP-1. In addition, a binding assay was conducted to determine the affinity of CRRL-101 for the GCA / GCB receptor. In the INS-1 cells, CRRL-101 significantly increased insulin production and cAMP levels, similar to GLP-1 (TABLE 1 and FIGS. 2A-2B). In AC16 cardiomyocytes, insulin-induced apoptosis was dose-dependently inhibited by CRRL-101, while GLP-1 had no effect on insulin-induced apoptosis (FIG. 3). The receptor binding assay highlighted the predominant affinity of CRRL-101 for the NP-A receptor. Thus, these in vitro studies demonstrated that CRRL-101 exhibited insulin-secreting actions that were similar to those of GLP-1 in beta cells, but unlike GLP-1, CRRL-101 suppressed insulin- induced cardiomyocyte apoptosis and exhibited a high affinity for the NP-A receptor.

[0153] TABLE 1: Effects of CRRL-101 vs. GLP-1 in INS-1 cells

[0154] Example 3 - CRRL-101 as a therapy for diabetic cardiomyopathy

[0155] In vivo studies were conducted in male Wistar rats injected with streptozotocin to induce diabetes after two weeks of a high-fat diet. Animals were treated with different doses of CRRL-101 (1 pmol / kg / min or 10 pmol / kg / min), and compared to a placebo group. Blood samples were collected at multiple times during the infusion, and plasma insulin and cGMP were measured.

[0156] In these acute experiments, plasma insulin was significantly increased in the treated groups compared to the controls, accompanied by a statistically significant reduction in blood glucose (FIG. 4). Moreover, there was a significant increase in cGMP levels in the treated animals, which was not observed with the placebo control. These studies demonstrated that CRRL-101 has bispecific biological actions of inducing insulin secretion and activating cGMP (the second messenger of NPs).

[0157] Example 4 - Glucose-lowering and cardiorenal effects of CRRL-094 vs. exogenous insulin in experimental diabetes

[0158] Studies were conducted to compare the cardiorenal actions of CRRL-094 to those of exogenous insulin. Wistar rats (male, aged 6-8 weeks) were split into four groups: nondiabetic controls (n=17) and three groups that were made diabetic via intraperitoneal Streptozotocin (65 mg / kg). After 8 weeks, groups received placebo (untreated diabetic (UDM), n=17, and controls). CRRL-094 (CDM. n=l l, 10 pmol / kg / min), or insulin (IDM, n=9, 10 nmol / mL). After four weeks of treatment (12 total weeks), blood and tissues were collected.

[0159] These studies demonstrated that treatment of rats with CRRL-094 restored insulin levels to those similar to nondiabetic controls (FIG. 5A) while attenuating DM-associated hyperglycemia (FIG. 5B) (p <0.0001). CRRL-094 also increased plasma cGMP in rats versus control and untreated diabetic animals, while insulin reduced plasma cGMP (FIG. 6) (p<0.0001). While diabetes was associated with increased left ventricular (LV) fibrosis in the animals, CRRL-094 reversed such effects while insulin exacerbated them (FIGS. 7A and 7B). In addition, CRRL-094 protected against the DM-associated decline in GFR (FIG. 8A) and trended toward protecting against proteinuria (FIG. 8B). CRRL-094 also protected against glomerular hyperfiltration associated with insulin therapy.

[0160] Thus, CRRL-094 successfully promoted endogenous insulin secretion and partially attenuated DM-associated hyperglycemia. CRRL-094 also reduced DM- associated left ventricular fibrosis, protected against the DM-associated decline in GFR, and mitigates DM-associated proteinuria. These studies demonstrated that CRRL-094 is a bispecific polypeptide that potently promotes endogenous insulin secretion and simultaneously activates the NP system, and is further associated with cardiorenal protective effects.

[0161] Example 5 - Metabolic effects of CRRL-094 and CRRL-101

[0162] Lipolysis has been shown to be beneficial in cardiometabolic diseases. To evaluate the effects of CRRL-094 and CRRL-101 on lipolysis, studies were conducted to measure release of glycerol and NEFA from adipocytes treated with several concentrations of these ISP-NPs for 6 hours. As shown in FIGS. 9A-9D, both CRRL-101 and CRRL-094 had increased lipolysis actions as compared to GLP-1, and CRRL-101 was more lipolytic than CRRL-094.

[0163] Browning of adipose tissues also can be beneficial in cardiometabolic diseases. The effects of CRRL-101 and CRRL-094 on browning were evaluated in adipocytes based on expression of uncoupling protein 1 (UCP1), which is a mitochondrial carrier protein found in brown adipose tissue. As shown in FIGS. 10A-10C, UCP1 expression was increased in response to both CRRL-101 and CRRL-094 as compared to GLP-1, indicating that both of these ISP-NPs had increased browning actions.

[0164] Example 6 - Effects of ISP-NPs in MASH An in vitro model of MASH was used to evaluate the effects of CRRL-094 and CRRL-101 on this disorder. This model is generated by exposing HepG2 (human hepatocyte) cells to oleic acid (OA, a lipid), which causes the OA to be taken up into the cells. Studies were conducted using this model by treating HepG2 cells with OA for 18 hours, at which point the cells were also treated with NPs or ISP-NPs. These studies demonstrated that CRRL-101 and CRRL-094 both resulted in decreased lipid uptake into the HepG2 cells, which was similar to the effect of GLP-1 on lipid uptake and better than the effect of the natriuretic peptides ANP and CNP (FIGS. 11A and 11B).

[0165] Example 7 - CRRL-094 in MASLD

[0166] A preliminary’ cohort analysis was carried out using male Sprague-Dawley rats aged 4-6 weeks that were divided into three groups: controls, MASLD controls, and MASLD treated with CRRL-094. MASLD was induced in tyvo groups (MASLD controls and MASLD + CRRL-094) by feeding the rats a high-fat, high-glucose / fructose diet for 8 weeks. After this initial period, all groups underwent pump implantation. Saline pumps yvere implanted in chow-fed controls (designated as controls) and untreated MASLD rats (designated as UM), while MASLD rats receiving CRRL-094 were implanted with CRRL-094 pumps (designated as MASLD + CRRL-094). Treatment continued for 4 additional weeks. Echocardiography was performed at baseline. 8 weeks, and 12 weeks. At approximately 12 weeks, rats were anesthetized for an acute procedure, during which arterial blood was collected from the carotid artery for hemodynamic and renal function analyses. Cardiac, renal, pancreatic, hepatic, and adipose tissues yvere harvested for subsequent fibrosis and apoptosis studies. Due to the limited number of rats in the control and untreated MASLD groups, formal statistical analyses were not performed at this stage.

[0167] At the end of the treatment period, the folloyving observations were made:

[0168] The MASLD+CRRL-094 group appeared to have experienced more w eight loss than to the tw o other groups (FIG. 12).

[0169] Ratios of heart weight to body weight (FIG. 13A) and left ventricle weight to body weight (FIG. 13B) w ere lower in the treatment cohort than in the other groups. Histological analysis suggested that CRRL-094 may have imparted antifibrotic (FIG. 14A) and anti-apoptotic (FIG. 14B) effects in the left ventricle in the setting of MASLD. These results suggested favorable remodeling of the heart with CRRL-094.

[0170] No significant difference in LVEF was observed between the groups (FIG. 15). The kidney weight to body weight ratio was lower in the treatment cohort (FIG. 16). Histological analysis indicated that CRRL-094 may have imparted antifibrotic effects in the renal cortex (FIG. 17). This suggested favorable remodeling of the kidney with CRRL-094 treatment.

[0171] With CRRL-094, plasma creatinine appeared to be lower (FIG. 18), suggesting an improvement in renal function.

[0172] The liver-to-body weight ratio was lower in the treatment group compared to the untreated MASLD cohort (FIG. 19). This suggested favorable remodeling of the liver with CRRL-094 treatment.

[0173] Plasma insulin levels were higher in the treatment cohort (FIG. 20), which was consistent with the expected insulin-secreting properties of CRRL-094.

[0174] Total cholesterol appeared to be lower in the treatment group compared to the untreated MASLD group (FIG. 21A). There did not appear to be a significant difference in plasma HDL levels (FIG. 21B), while plasma LDL (FIG. 21C) and triglyceride (FIG. 21D) levels did not show a clear association with CRRL-094 treatment.

[0175] OTHER EMBODIMENTS

[0176] 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 a polypeptide in the manufacture of a medicament for treating fibrosis, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH), cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus, wherein said polypeptide comprises:(a) the amino acid sequence set forth in SEQ ID NO:2,(b) the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2,(c) the amino acid sequence set forth in SEQ ID NO: 1, or(d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1.

2. The use of claim 1. wherein said medicament is for treating fibrosis.

3. The use of claim 2, wherein said fibrosis comprises one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthro fibrosis, and Peyronie’s disease.

4. The use of claim 1, wherein said medicament is for treating MASLD.

5. The use of claim 1, wherein said medicament is for treating cardiovascular disease.

6. The use of claim 5. wherein said cardiovascular disease comprises hypertension.

7. The use of claim 1, wherein said medicament is for treating diabetes mellitus, metabolic syndrome, nonalcoholic fatty7liver disease, and / or obesity7.

8. The use of any one of claims 1 to 7, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2.

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

10. The use of any one of claims 1 to 7, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

11. The use of any one of claims 1 to 7, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

12. The use of any one of claims 1 to 11, wherein said medicament is formulated for subcutaneous, intravenous, or oral administration.

13. A method for treating a mammal having fibrosis, Metabolic Dysfunction- Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH), cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus, wherein said method comprises administering, to said mammal, an effective amount of a composition comprising a polypeptide comprising:(a) the amino acid sequence set forth in SEQ ID NO: 2,(b) the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2,(c) the amino acid sequence set forth in SEQ ID NO: 1, or(d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1.

14. The method of claim 13, wherein said mammal has fibrosis, and wherein said composition is administered to said mammal in an amount effective to reduce said fibrosis in said mammal.

15. The method of claim 14, wherein said fibrosis comprises one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthro fibrosis, and Peyronie’s disease.

16. The method of claim 13, wherein said mammal has MASH, and wherein said composition is administered in an amount effective to reduce a symptom of said MASH in said mammal.

17. The method of claim 13, wherein said mammal has diabetes mellitus, and wherein said composition is administered to said mammal in an amount effective to reduce blood glucose levels and / or increase blood insulin levels in said mammal.

18. The method of claim 13 or claim 17, wherein said composition is administered in an amount effective to increase blood cGMP levels in said mammal.

19. The method of claim 13, wherein said mammal has cardiovascular disease, metabolic syndrome, and / or obesity7.

20. The method of any one of claims 13 to 19. wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2.

21. The method of any one of claims 13 to 19, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NOT.

22. The method of any one of claims 13 to 19, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

23. The method of any one of claims 13 to 19, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NOT.

24. The method of any one of claims 13 to 23. comprising administering said composition subcutaneously, intravenously, or orally.

25. The method of any one of claims 13 to 24, wherein said mammal is a human.

26. The method of any one of claims 13 to 25, further comprising identifying said mammal as being in need of said treating.

27. A method for increasing natriuretic activity within a mammal having fibrosis. Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH), cardiovascular disease, metabolic syndrome, obesity, and / or diabetes mellitus, wherein said method comprises administering, to said mammal, an effective amount of a composition comprising a polypeptide comprising:(a) the amino acid sequence set forth in SEQ ID NO:2,(b) the amino acid sequence set forth in SEQ ID NO: 2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2,(c) the amino acid sequence set forth in SEQ ID NO: 1, or(d) the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1.

28. The method of claim 27, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or the amino acid sequence set forth in SEQ ID NO:2 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:2.

29. The method of claim 27, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or the amino acid sequence set forth in SEQ ID NO: 1 but with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 1.

30. The method of claim 27, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

31. The method of claim 27, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

32. The method of any one of claims 27 to 31. comprising administering said composition subcutaneously, intravenously, or orally.

33. The method of any one of claims 27 to 32, wherein said mammal is a human.

34. The method of any one of claims 27 to 33, further comprising identifying said mammal as being in need of increased natriuretic activity.

35. The method of any one of claims 27 to 34, wherein said mammal has fibrosis.

36. The method of claim 35, wherein said fibrosis comprises one or more of hepatic fibrosis, renal fibrosis, cardiac fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pneumoconiosis, radiation-induced lung injury, gliosis, mediastinal fibrosis, retroperitoneal fibrosis, arthro fibrosis, and Peyronie’s disease.

37. The method of any one of claims 27 to 36, wherein said mammal has MASLD.

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