WO2023039453A2

WO2023039453A2 - Dual-function polypeptides and methods for use thereof

Info

Publication number: WO2023039453A2
Application number: PCT/US2022/076083
Authority: WO
Inventors: Xiao MA; John C. Burnett, Jr.
Original assignee: Mayo Foundation For Medical Education And Research
Priority date: 2021-09-09
Filing date: 2022-09-08
Publication date: 2023-03-16
Also published as: WO2023039453A3

Abstract

Chimeric polypeptides that exhibit pGC-A gain of function and inhibit PKC are provided herein. For example, this document provides chimeric polypeptides containing an alternatively spliced form of atrial natriuretic peptide (MANP) and a PKC inhibitor. Methods for using the chimeric polypeptides to treat cardiovascular disease also are provided.

Description

DUAL-FUNCTION POLYPEPTIDES AND METHODS FOR USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional Application Serial No. 63/242,329, filed September 9, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2076W01_SL_ST26.XML.” The XML file, created on September 8, 2022, is 29,850 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to methods and materials for treating mammals having cardiovascular disease, and more particularly to methods and materials for treating mammals having cardiovascular disease using a dual function, chimeric peptide that can activate the particulate guanylyl cyclase A (pGC-A) receptor and inhibit the protein kinase C (PKC) signaling pathway.

BACKGROUND

Hypertension (HTN) is a leading cause of adverse cardiovascular outcomes and global mortality. Despite existing therapeutic options (e.g., angiotensin converting enzyme inhibitors, angiotensin II type I receptor (AT1R) blockers, and aldosterone antagonists), a substantial portion of the hypertensive population has uncontrolled blood pressure (BP) and is at high risk for developing cardiovascular disease. SUMMARY

This document provides methods and materials for treating cardiovascular disease. For example, this document provides chimeric polypeptides that can activate the pGC-A receptor (also referred to as NPR A) and inhibit the PKC pathway. The methods and materials provided herein can maximize the therapeutic benefits of pGC-A/cyclic guanosine monophosphate (cGMP) signaling by focusing on the interaction between the angiotensin II (ANGII)/ANGII receptor type 1(AT1R) and pGC-A/cGMP pathways. The disclosed methods and materials are likely to have a broad impact on clinical implementation of all pGC-A/cGMP ligands and neprilysin (NEP) inhibitors.

As demonstrated herein, in healthy humans, plasma cGMP was positively associated with ANP or BNP only in the presence of low (< 4.5 pg/mL) ANGII levels, but not in the presence of high (> 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, but ANP-mediated cGMP generation was reduced in the presence of ANGII. The suppressive effect of ANGII on pGC-A was recapitulated in human embryonic kidney (HEK) 293 cells overexpressing both pGC-A and AT1R, but not in HEK293 cells over expressing pGC-A alone. In addition, PKC, a well-established downstream target of AT1R, was a key mediator for this observed crosstalk, as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor.

Further, MANP-PKCi, a dual functional polypeptide containing the amino acid sequences of PKC 19-36 and MANP (SEQ ID NO:3; FIG. 3), can activate pGC-A and inhibit PKC, preventing desensitization of pGC-A that otherwise would be induced by ANGII, and leading to enhanced activation of the pGC-A/cGMP pathway under hypertensive conditions. MANP-PKCi had superior potency with regard to cGMP generation, as compared to MANP, currently the best-in-class ANP-analog, in vitro.

In general, this document features isolated polypeptides having the ability to activate pGC-A and the ability to inhibit PKC.

In a first aspect, this document features a polypeptide containing, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON2, SEQ ID NO: 13, SEQ ID NO:14, or SEQ ID NO: 15. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features a polypeptide containing, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 5 or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 5. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:5. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO: 5. The polypeptide can have the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features a polypeptide containing, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NON or the amino acid sequence set forth in SEQ ID NON with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NON. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NON. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NON. The polypeptide can have the amino acid sequence set forth in SEQ ID NON. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features an isolated nucleic acid encoding a polypeptide described herein. The isolated nucleic acid can contain, consist essentially of, or consist of the nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30. In another aspect, this document features a vector containing a nucleic acid encoding a polypeptide described herein. The vector can contain a nucleic acid having a nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30.

In another aspect, this document features a host cell containing a nucleic acid encoding a polypeptide described herein. The nucleic acid can contain, consist essentially of, or consist of a nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30. The host cell can be a eukaryotic host cell.

This document also features a pharmaceutical composition containing a pharmaceutically acceptable carrier and a polypeptide described herein.

In yet another aspect, this document features a method for treating a cardiovascular disease in a mammal in need thereof. The method can include, or consist essentially of, administering to the mammal an effective amount of a composition containing a polypeptide that includes, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO: 3. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The cardiovascular disease can be hypertension, and wherein composition can be administered in an amount effective to reduce the blood pressure of the mammal. The cardiovascular disease can be heart failure. The mammal can be a human. The composition can be administered at a dose of 0.01 ng/kg to 50 pg/kg. The method can include administering the composition intravenously. The method can further include identifying the mammal as being in need of the treatment.

In another aspect, this document features a method for treating a cardiovascular disease in a mammal in need thereof, where the method includes, or consists essentially of, administering to the mammal an effective amount of a composition containing a polypeptide that includes, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO:5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 5. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO: 5. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO: 5. The polypeptide can have the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. The cardiovascular disease can be hypertension, and wherein composition can be administered in an amount effective to reduce the blood pressure of the mammal. The cardiovascular disease can be heart failure. The mammal can be a human. The composition can be administered at a dose of 0.01 ng/kg to 50 pg/kg. The method can include administering the composition intravenously. The method can further include identifying the mammal as being in need of the treatment.

In another aspect, this document features a method for treating a cardiovascular disease in a mammal in need thereof, where the method includes administering to the mammal an effective amount of a composition containing a polypeptide that includes, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NON, or the amino acid sequence set forth in SEQ ID NON with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NON. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NON. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NON. The polypeptide can have the amino acid sequence set forth in SEQ ID NON. The cardiovascular disease can be hypertension, and wherein composition can be administered in an amount effective to reduce the blood pressure of the mammal. The cardiovascular disease can be heart failure. The mammal can be a human. The composition can be administered at a dose of 0.01 ng/kg to 50 pg/kg. The method can include administering the composition intravenously. The method can further include identifying the mammal as being in need of the treatment.

In another aspect, this document features the use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder. The polypeptide can contain, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 3. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The cardiovascular disorder can be hypertension or heart failure. The medicament can be formulated for intravenous administration.

In another aspect, this document features the use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder, wherein the polypeptide contains, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO:5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:5. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:5. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO: 5. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NOT E The cardiovascular disorder can be hypertension or heart failure. The medicament can be formulated for intravenous administration.

In another aspect, this document features the use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder, wherein the polypeptide contains, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:4, or the amino acid sequence set forth in SEQ ID NO:4 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:4. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:4. The cardiovascular disorder can be hypertension or heart failure. The medicament can be formulated for intravenous administration. In still another aspect, this document features the use of a chimeric polypeptide for treating a cardiovascular disorder, wherein the polypeptide contains, consists essentially of, or consists the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO:3. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The cardiovascular disorder can be hypertension or heart failure.

In another aspect, this document features the use of a chimeric polypeptide for treating a cardiovascular disorder, wherein the polypeptide contains, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 5. The polypeptide can have one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:5. The polypeptide can have one to three conservative amino acid substitutions as compared to SEQ ID NO:5. The polypeptide can have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. The cardiovascular disorder can be hypertension or heart failure.

In another aspect, this document features the use of a chimeric polypeptide for treating a cardiovascular disorder, wherein the polypeptide contains, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NON, or the amino acid sequence set forth in SEQ ID NON with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NON. The polypeptide can have the amino acid sequence set forth in SEQ ID NON. The cardiovascular disorder can be hypertension or heart failure.

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.

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

DESCRIPTION OF DRAWINGS

FIG. 1 shows the amino acid sequence of MANP (SEQ ID NO: 1)

FIG. 2 is a diagram illustrating the effects of angiotensin II (ANGII) on PKC via AT1R, with subsequent inhibition of MANP-mediated pGC-A/cGMP signaling.

FIG. 3 shows the amino acid sequence of the MANP-PKCi-1 chimeric polypeptide (SEQ ID NO:3).

FIGS. 4A and 4B are graphs plotting the correlation between circulating pGC-A ligands (ANP, FIG. 4A; and BNP, FIG. 4B) and cGMP, grouped by the median level of ANGII in healthy subjects.

FIGS. 5A-5C are graphs plotting BP (FIG. 5A), plasma cGMP (FIG. 5B), and urinary cGMP (FIG. 5C) in acute studies of normotensive rats treated with ANP (filled circles), ANGII (open triangles), ANP+ ANGII (open circles), or saline (filled squares). *, P < 0.05 compared to baseline; #, P < 0.05 compared to ANP group. ANP: 300 pmol/kg/min, ANGII: 50 pmol/kg/min.

FIG. 6A is a graph plotting the binding affinity between ANGII and pGC-A as determined by surface plasmon resonance (SPR). FIG. 6B is a series of graphs plotting relative cGMP production in vitro. Cells were pretreated with vehicle (open bars) or ANGII (filled bars) for 30 minutes before being treated with the indicated concentrations of ANP for 10 minutes, ns, P > 0.05. FIG. 7A is a graph plotting cGMP levels in HEK293/pGC-A⁺/ATlR⁺ cells treated as indicated. Valsartan and Go6983 rescued ANP-induced cGMP production from the effects of ANGII in vitro, ns, P > 0.05. FIG. 7B is an image of a western blot showing expression of PKCs in the cytosol and membrane fractions of HEK293/pGC-A⁺ and HEK293/pGC-A⁺/ATlR⁺ cells, with or without ANGII treatment.

FIG. 8 shows the amino acid sequences of MANP-PKCi polypeptides (MANP- PKCi-1, SEQ ID NO:3; MANP-PKCi-2, SEQ ID NO:4; MANP-PKCi-3, SEQ ID NO:5; MANP-PKCi-4, SEQ ID NO:6; MANP-PKCi-5, SEQ ID NO:7; MANP-PKCi-6, SEQ ID NO:8; MANP-PKCi-7, SEQ ID NO:9; MANP-PKCi-8, SEQ ID NO: 10; MANP-PKCi-9, SEQ ID NO: 11; MANP-PKCi- 10, SEQ ID NO: 12; MANP-PKCi-11, SEQ ID NO: 13; MANP-PKCi-12, SEQ ID NO:14; MANP-PKCi-13, SEQ ID NO: 15.

FIG. 9 is a graph plotting cGMP levels in HEK293 cells stimulated by two different MANP-PKCi peptides.

FIGS. 10A-10C are graphs plotting the binding affinity between pGC-A and MANP (FIG. 10A), pGC-A and MANP-PKCi-1 (FIG. 10B), and pGC-A and MANP- PKCi-2 (FIG. 10C), determined using a surface plasmon resonance (SPR) assay.

FIG. 11 is a graph plotting the in vitro effects of three MANP-PKCi peptides on HEK-GCA cells.

FIG. 12 is a graph plotting the in vitro effects of five MANP-PKCi peptides on human renal proximal tubular cells (HRPTC).

FIG. 13 is a schematic depicting the protocol for in vivo studies in a hypertensive rat model (SHR).

FIGS. 14A-14F are graphs plotting in vivo data for MANP-PKCi-1, including systolic BP (FIG. 14A), diastolic BP (FIG. 14B), mean arterial pressure (FIG. 14C), urine excretion (FIG. 14D), plasma cGMP (FIG. 14E), and urine cGMP (FIG. 14F).

FIGS. 15A-15F are graphs plotting in vivo data for MANP-PKCi-2, including systolic BP (FIG. 15A), diastolic BP (FIG. 15B), mean arterial pressure (FIG. 15C), urine excretion (FIG. 15D), plasma cGMP (FIG. 15E), and urine cGMP (FIG. 15F). FIGS. 16A-16F are graphs plotting in vivo data for MANP-PKCi-4, including systolic BP (FIG. 16A), diastolic BP (FIG. 16B), mean arterial pressure (FIG. 16C), urine excretion (FIG. 16D), plasma cGMP (FIG. 16E), and urine cGMP (FIG. 16F).

FIGS. 17A-17F are graphs plotting in vivo data for MANP-PKCi-5, including systolic BP (FIG. 17A), diastolic BP (FIG. 17B), mean arterial pressure (FIG. 17C), urine excretion (FIG. 17D), plasma cGMP (FIG. 17E), and urine cGMP (FIG. 17F).

FIGS. 18A-18F are graphs plotting in vivo data for MANP-PKCi-6, including systolic BP (FIG. 18A), diastolic BP (FIG. 18B), mean arterial pressure (FIG. 18C), urine excretion (FIG. 18D), plasma cGMP (FIG. 18E), and urine cGMP (FIG. 18F).

DETAILED DESCRIPTION

This disclosure provides methods and materials related to chimeric polypeptides containing an MANP sequence and a PKC inhibitor sequence, where the chimeric polypeptides have natriuretic activity and can inhibit PKC activity. For example, this document provides substantially pure polypeptides having a natriuretic polypeptide activity - particularly the ability to activate the pGC-A receptor - and having the ability to inhibit the PKC pathway. This document also provides compositions containing such polypeptides, nucleic acid molecules encoding the polypeptides, and host cells containing isolated nucleic acid molecules that encode the polypeptides provided herein. In addition, this document provides methods and materials for treating cardiovascular disorders in mammals (e.g., humans, non-human primates, rodents, pigs, sheep, dogs, or cats).

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are naturally occurring ligands for the pGC-A/cGMP pathway. In animal models, deletion of the ANP gene or the BNP gene can result in hypertensive phenotypes (Holditch et al., Sci Rep, 6:25623, 2016; and John et al. Am J Physiol, 271 :R109-114, 1996). In humans, gain- of-function genetic variants in either gene have been associated with lower BP and reduced risk of HTN and obesity (Newton-Cheh et al., Nat Genet, 41 :348-353, 2009). BP lowering effects can be achieved by directly promoting pGC-A/cGMP signaling for its vasodilatory and natriuretic properties. Moreover, activation of pGC-A/cGMP signaling can antagonize actions of the renin angiotensin system (RAS), leading to cardiorenal protection and BP reduction (Lee and Burnett, Heart Fail Rev, 12: 131-142, 2007).

MANP is an ANP-analogue resulting from a frameshift mutation in the human ANP gene (McKie et al., J Am Coll Cardiol, 54: 1024-1032, 2009; Hodgson-Zingman et al., NEJM, 359(2): 158-165, 2008). MANP is 40 amino acids in length, and includes the 28 amino acid core structure of ANP and a 12 amino acid extended C-terminus (SEQ ID NO: 1; FIG. 1). In vitro, MANP activates pGC-A/cGMP signaling with a potency equal to that of ANP, and is highly resistant to degradation by neprilysin (NEP), the major enzyme that degrades endogenous ANP and BNP. In vivo, MANP has longer-lasting BP lowering, natriuretic, and aldosterone suppressing effects than ANP. When administered subcutaneously, MANP activates plasma cGMP to an extent that is 10-fold greater than ANP, with a 4-fold longer half-life. Moreover, MANP potently reduces BP and enhances renal function in multiple experimental models of HTN (McKie et al., Hypertension, 56: 1152-1159, 2010; and Chen et al., Am J Physiol Regul Integr Comp Physiol, 318:R669-R676, 2020).

PKC is a family of protein kinases that regulates cellular signaling transduction (Mochly-Rosen et al., Nat Rev Drug Discov, 11 :937-957, 2012), and can desensitize the pGC-A receptor via allosteric modulation (Potter and Garbers, J Biol Chem, 269: 14636- 14642, 1994) (FIG. 2). PKC also is a downstream target of ANGII/AT1R signaling. An 18 amino acid polypeptide inhibitor for PKC (PKC19-36; (House and Kemp, Science, 238: 1726-1728, 1987)) can act as a pseudo-substrate for PKC and is a potent inhibitor of PKC action. PKC 19-36 also can enhance ANP-induced cGMP production in vitro (Yasunari et al., Hypertension, 28: 159-168, 1996).

In some cases, the polypeptides provided herein can be effective to increase plasma cGMP levels, increase urinary cGMP excretion, increase net renal cGMP generation, increase urine flow, increase urinary sodium excretion, increase urinary potassium excretion, increase hematocrit, increase plasma BNP immunoreactivity, increase renal blood flow, increase plasma ANP immunoreactivity, decrease renal vascular resistance, decrease proximal and distal fractional reabsorption of sodium, decrease mean arterial pressure, decrease pulmonary wedge capillary pressure, decrease right atrial pressure, decrease pulmonary arterial pressure, decrease plasma renin activity, decrease plasma ANGII levels, decrease plasma aldosterone levels, decrease renal perfusion pressure, and/or decrease systemic vascular resistance, and also can be effective to inhibit the PKC pathway.

The 40 amino acid sequence for MANP is SLRRSSCFGGRMDRIGAQSGLGCN SFRYRITAREDKQGWA (SEQ ID NO: 1; FIG. 1). Like other mature natriuretic polypeptides, MANP includes a 17-amino acid ring structure with a cysteine bond between the cysteine residues at positions 1 and 17 (underlined in the above sequence) of the ring. The amino acid sequence of PKC 19-36 is RFARKGALRQKNVHEVKN (SEQ ID NO:2), and the amino acid sequence of MANP-PKCi (also referred to herein as MANP-PKCi-1) is RFARKGALRQKNVHEVKNSLRRSSCFGGRMDRIGAQSGLGC NSFRYRITAREDKQGWA (SEQ ID NO:3; FIG. 3).

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.

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.

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.

In some cases, the chimeric polypeptides provided herein can include variants of the MANP and/or PKC19-36 sequences set forth in SEQ ID NO: 1 and SEQ ID NO:2, respectively. For example, the polypeptides provided herein can contain the entire amino acid sequence set forth in SEQ ID NO:3, except that the amino acid sequence can contain from one to five (e.g., five, four, three, two, one, one to five, one to four, one to three, or one to two) amino acid additions, subtractions, and substitutions, or modifications. For example, a polypeptide can contain the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five single amino acid residue additions, subtractions, or substitutions. In some cases, a polypeptide can contain the amino acid sequence set forth in SEQ ID NO:4 with one, two, three, four, or five single amino acid residue additions, subtractions, or substitutions. In some cases, a polypeptide can contain the amino acid sequence set forth in SEQ ID NO: 5 with one, two, three, four, or five single amino acid residue additions, subtractions, or substitutions. Any amino acid residue set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 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:3, SEQ ID NO:4, or SEQ ID NO:5. 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 as provided herein can contain one or more D-amino acids. In some embodiments, a polypeptide can contain chemical structures such as 8- amino hexanoic acid; hydroxylated amino acids such as 3-hydroxyproline, 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.

Chimeric polypeptides having one or more amino acid additions, subtractions, or substitutions relative to the MANP and PKC19-36 amino acid sequences set forth in SEQ ID NO: 1 and/or SEQ ID NO:2 (also referred to herein as “variant” chimeric polypeptides) 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 (norleucine, 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. Non-limiting examples of useful conservative substitutions can include, without limitation, substitution of valine for alanine, lysine 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 for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenyalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine for valine.

Further examples of conservative substitutions that can be made at any position within the polypeptides provided herein are set forth in TABLE 1. TABLE 1

Examples of conservative amino acid substitutions

In some cases, a chimeric polypeptide can include one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Such production can be desirable to provide large quantities or alternative embodiments of such compounds. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the peptide variant using, for example, methods disclosed herein. In some cases, a polypeptide as provided herein can have a length of, for example,

53 to 63 amino acid residues (e.g., 53 to 55, 54 to 56, 55 to 57, 56 to 58, 57 to 59, 58 to 60, 59 to 61, 60 to 62, or 61 to 63 amino acid residues). In some cases, a polypeptide as provided herein can have a length of, for example, 42 to 50 amino acid residues (e.g., 42 to 44, 43 to 45, 44 to 46, 45 to 47, 46 to 48, 47 to 49, or 48 to 50 amino acid residues).

In some cases, a chimeric polypeptide provided herein can include an amino acid sequence with at least 90% (e.g., at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or 100%) sequence identity to a region of a reference chimeric polypeptide sequence (e g., SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 12). 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.

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

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 by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:3 or SEQ ID NO:5), followed by multiplying the resulting value by 100. For example, an amino acid sequence that has 55 matches when aligned with the sequence set forth in SEQ ID NO:3 is 94.8 percent identical to the sequence set forth in SEQ ID NO:3 (i.e., 55 58 x 100 = 94.8). It is noted that the percent sequence identity value is rounded to the nearest tenth. F or example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. It also is noted that the length value will always be an integer.

In some cases, the PKC19-36 amino acid sequence (or a variant thereof) can make up the N-terminal portion of a chimeric polypeptide provided herein, and the MANP amino acid sequence (or a variant thereof) can make up the C-terminal portion of the chimeric polypeptide (e.g., as set forth in SEQ ID NO:3). In some cases, the MANP amino acid sequence (or a variant thereof) or the ANP sequence (or a variant thereof) can make up the N-terminal portion of a chimeric polypeptide provided herein, and the PKC19-36 amino acid sequence (or a variant thereof) can make up the C-terminal portion of the chimeric polypeptide (e.g., as set forth in SEQ ID NOS:5, 9, 10, and 11, which do not include the MANP tail; FIG. 8).

Isolated polypeptides can be produced using any suitable method, such as 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. Chimeric polypeptides also can be produced recombinantly, or obtained commercially.

The chimeric polypeptides provided herein typically are cyclic due to disulfide bonds between the cysteine residues underlined in the sequences shown above. In some embodiments, a sulfhydryl group on a cysteine residue can be replaced with an alternative group (e.g., -CH2CH2-). To replace a sulfhydryl group with a -CH2- group, for example, a cysteine residue can be replaced by alpha-aminobutyric acid. Such cyclic analog polypeptides can be generated, for example, in accordance with the methodology of Lebl and Hruby ((1984) Tetrahedron Lett. 25:2067-2068), or by employing the procedure disclosed in U.S. Patent No. 4,161,521.

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 include, without limitation those described in Schiller et al. (1985) Biochem. Biophys. Res. Comm. 127:558, and Schiller et al. (1985) hit. J. Peptide Protein Res. 25: 171 (both incorporated herein by reference in their entirety). 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 in, for example, Spatola et al. (1986) Life Sci. 38: 1243; Spatola (1983) Vega Data 1(3); Morley (1980) Trends Pharm. Sci. 463-468; Hudson et al. (1979) Ini. J. Pept. Prot. Res. 14:177; Spatola, in Chemistry and Biochemistry of Amino Acid Peptides and Proteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Hann (1982) J. Chem. Soc. Perkin Trans. 1 :307; Almquist et al. (1980) J. Med. Chem. 23: 1392; Jennings- White et al. (1982) Tetrahedron Lett. 23:2533; European Patent Application EP 45665; Holladay et al. (1983) Tetrahedron Lett. 24:4401; and Hruby (1982) Life Sci. 31 : 189.

N-acyl derivatives of an amino group of a polypeptide can be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives can be prepared for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.

In some cases, a chimeric 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 chimeric polypeptide. Methods for modifying a polypeptide by linkage to PEG (also referred to as “PEGylation”) or other polymers include, without limitation, 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 include, without limitation, those set forth in U.S. Patent Publication No. 20040086976, and Wang et al. (2004) Pharm. Res. 21 :2105-2111, both of which are incorporated herein by reference in their entirety. In some cases, a polypeptide 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.

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).

The chimeric polypeptides provided herein typically function (in part) through activation of one or more of the guanylyl cyclase receptors through which native natriuretic polypeptides function. For example, the polypeptides provided herein can bind to and function through the pGC- A receptor through which ANP and BNP function, although they also may function through the pGC-B receptor (also referred to as NPR-B) through which CNP functions. Further, in some cases, a chimeric polypeptide provided herein can bind to and function through more than one guanylyl cyclase receptor, including NPR-A and NPR-B, for example. Any appropriate method can be used to evaluate which receptor is involved in the function of a particular chimeric polypeptide. For example, glomeruli, which contain both NPR-A and NPR-B, can be isolated (e.g., from a laboratory animal such as a dog) and incubated with a chimeric polypeptide, and cGMP levels can be measured. Glomeruli can be pretreated with antagonists of NPR-A or NPR-B to determine whether cGMP production stimulated by a chimeric polypeptide through one or the other receptor can be attenuated.

In some cases, a chimeric polypeptide provided herein can be used to treat cardiovascular disease. For example, the polypeptides provided herein can be used to treat hypertension or heart failure. The presence or extent of disease can be evaluated using methods such as, without limitation, general clinical examination to evaluate 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; assessment of kidney function by determination of glomerular filtration rate, serum creatinine, and blood urea nitrogen; and measurement of biomarkers such as BNP, amino-terminal 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), kidney injury molecule- 1 (KIM-1), angiotensin-II, renin, aldosterone, and inflammatory cytokines (e.g., interleukin (IL)-6, IL- 18, etc.). In some cases, a chimeric polypeptide provided herein can reduce one or more symptoms of acute HF, including clinical parameters such as edema, shortness of breath, and fatigue, as well as cardiac unloading (i.e., reduced pressure in the heart), increased glomerular filtration rate (GFR), decreased PRA, decreased levels of angiotensin II, decreased proliferation of cardiac fibroblasts, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicative of reduced fibrosis and hypertrophy), decreased PWCP (an indirect measure of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased levels of aldosterone (indicative of an anti-fibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end systolic diameter. To determine whether a chimeric polypeptide is capable of inhibiting or reducing a symptom of acute HF, one or more of these parameters can be evaluated (e.g., before and after treatment with the chimeric polypeptide).

Chimeric polypeptides, including variant chimeric polypeptides having conservative and/or non-conservative substitutions with respect to SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 12 (e g., polypeptides containing any of SEQ ID NOS:6, 8, 10, 11, 13, 14, or 15), as well as fragments of variants of SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, or SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 12 (e.g., fragments of any of SEQ ID NOS: 6, 8, 10, 11, 13, 14, or 15), can be screened for biological activity using any of a number of assays, including those described herein. For example, the activity of a chimeric polypeptide provided herein can be evaluated in vitro by testing its effect on cGMP production in cultured cells (e.g., cultured cardiac fibroblasts, aortic endothelial cells, or glomerular cells). Cells can be exposed to a chimeric polypeptide (e.g., IO'¹⁰ to 10'⁴ M chimeric polypeptide), and samples can be assayed to evaluate the chimeric polypeptide’s effects on cGMP generation. cGMP generation can be detected and measured using, for example, a competitive RIA cGMP kit (Perkin-Elmer, Boston, MA).

The activity of a chimeric polypeptide also can be evaluated in vivo by, for example, testing its effects on factors such as plasma cGMP levels, urinary cGMP excretion, net renal generation of cGMP, glomerular filtration rate, blood pressure, heart rate, hemodynamic function such as cardiac output, pulmonary wedge pressure, systemic vascular resistance, and renal function such as renal blood flow, urine volume, and sodium excretion rate in a mammal (e.g., a rodent, pig, sheep, dog, or human). In some cases, such parameters can be evaluated after inducing heart failure (e.g., by rapid right ventricular pacing) or hypertension.

The effect of a chimeric polypeptide on PKC activity can be evaluated in vitro and/or in vivo using any appropriate assay, including those described herein. For example, western blotting can be used to detect PKC isoforms as an indication of protein expression and activity.

This document also provides nucleic acid molecules encoding the polypeptides provided herein. 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 singlestranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

For example, this document provides nucleic acid molecules encoding a chimeric polypeptide having the sequence set forth in SEQ ID NO:3, SEQ ID NO: 5, or SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 12 as well as nucleic acid molecules encoding chimeric polypeptides that are variants of the polypeptide having the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 12 (e.g., any of SEQ ID NOS: 4, 6, 8, 10, 11, 13, 14, and 15, or variants of any of SEQ ID NOS:4, 6, 8, 10, 11, 13, 14, and 15). Thus, a nucleic acid molecule as provided herein can encode a polypeptide that contains the amino acid sequence set forth in SEQ ID NO:3, except that the amino acid sequence contains one to five (e.g., five, four, three, two, one, one to four, one to three, one to two, two to four, two to three, three to four, three to five, or four to five) amino acid additions, subtractions, and substitutions as described herein.

Representative examples of nucleic acids sequences encoding polypeptides disclosed herein are set forth below.

A representative nucleotide sequence encoding the MANP amino acid sequence of SEQ ID NO: 1 is set forth in SEQ ID NO: 16: AGCCTGCGGAGATCCAGCTGCTTC GGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGC TTCCGGTACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGGGCC.

A representative nucleotide sequence encoding the PKC 19-36 amino acid sequence of SEQ ID NO: 2 is set forth in SEQ ID NO: 17: CGCTTCGCCCGCAAAGG GGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAAC. A representative nucleotide sequence encoding the MANP-PKCi amino acid sequence of SEQ ID NO: 3 is set forth in SEQ ID NO: 18: CGCTTCGCCCGCAAAGGG GCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACAGCCTGCGGAGATCC AGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGC TGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGG GCC.

A representative nucleotide sequence encoding the MANP-PKCi-2 amino acid sequence of SEQ ID NO: 4 is set forth in SEQ ID NO: 19: CAAGAAGTGGAGCACGTG CAAGAAGACGGAGTCGCGGGGAAACGCCCGCTTCGCAGCCTGCGGAGATCC AGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGC TGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGG GCC.

A representative nucleotide sequence encoding the MANP-PKCi-3 amino acid sequence of SEQ ID NO:5 is set forth in SEQ ID NO:20: AGCCTGCGGAGATCCAG CTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGT AACAGCTTCCGGTACCGCTTCGCCCGCAAAGGGGCGCTGAGGCAGAAGAAC GTGCACGAGGTGAAGAAC.

A representative nucleotide sequence encoding the MANP-PKCi-4 amino acid sequence of SEQ ID NO:6 is set forth in SEQ ID NO:21 : CGCTTCGCCCGCAAAGGG TCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACAGCCTGCGGAGATCC AGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGC TGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGG GCC.

A representative nucleotide sequence encoding the MANP-PKCi-5 amino acid sequence of SEQ ID NO:7 is set forth in SEQ ID NO:22: CGCTTCGCCCGCAAAGG GGCGCTGAGGCAGAAGAACGTGAGCCTGCGGAGATCCAGCTGCTTCGGGGG CAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGG TACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGGGCC.

A representative nucleotide sequence encoding the MANP-PKCi-6 amino acid sequence of SEQ ID NO: 8 is set forth i

GGTCGCTGAGGCAGAAGAACGTGAGCCTGCGGAGATCCAGCTGCTTCGGGGG CAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGG TACCGGATAACAGCCAGGGAGGACAAGCAGGGCTGGGCC.

A representative nucleotide sequence encoding the MANP-PKCi-7 amino acid sequence of SEQ ID NO:9 is set forth in SEQ ID NO:24: AGCCTGCGGAGATCCAGC TGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTA ACAGCTTCCGGTACCAAGAAGTGGAGCACGTGCAAGAAGACGGAGTCGCGG GGAAACGCCCGCTTCGC.

A representative nucleotide sequence encoding the MANP-PKCi-8 amino acid sequence of SEQ ID NO: 10 is set forth in SEQ ID NO:25: AGCCTGCGGAGATCCAG CTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGT AACAGCTTCCGGTACCGCTTCGCCCGCAAAGGGTCGCTGAGGCAGAAGAACG TGCACGAGGTGAAGAAC.

A representative nucleotide sequence encoding the MANP-PKCi-9 amino acid sequence of SEQ ID NO:11 is set forth in SEQ ID NO:26: AGCCTGCGGAGATCCA GCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCT GTAACAGCTTCCGGTACCAAGAAGTGGAGCACGTGCAAGAAGACGGAGTCG CTGGGAAACGCCCGCTTCGC.

A representative nucleotide sequence encoding the MANP-PKCi-10 amino acid sequence of SEQ ID NO: 12 is set forth in SEQ ID NO:27: CGCTTCGCCCGCAAAGG GGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACTCGTACAGCCTGCG GAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGG ACTGGGCTGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCA GGGCTGGGCC.

A representative nucleotide sequence encoding the MANP-PKCi- 11 amino acid sequence of SEQ ID NO: 13 is set forth in SEQ ID NO:28: CGCTTCGCCCGCAAAGG GGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACAGGTCGAGCCTGCG GAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGG ACTGGGCTGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCA GGGCTGGGCC. A representative nucleotide sequence encoding the MANP-PKCi-12 amino acid sequence of SEQ ID NO: 14 is set forth in SEQ ID NO:29: CGCTTCGCCCGCAAAG GGGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACTACTCGAGCCTGC GGAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCG GACTGGGCTGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGC AGGGCTGGGCC.

A representative nucleotide sequence encoding the MANP-PKCi-13 amino acid sequence of SEQ ID NO: 15 is set forth in SEQ ID NO:30: CGCTTCGCCCGCAAAGG GGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGAACTCGAGGAGCCTGCG GAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGG ACTGGGCTGTAACAGCTTCCGGTACCGGATAACAGCCAGGGAGGACAAGCA GGGCTGGGCC.

The term “isolated” as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5’ end and one on the 3’ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

In some cases, a nucleic acid provided herein can include a nucleotide sequence having at least 90% (e.g., at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or 100%) sequence identity to a region of a reference nucleotide sequence (e.g, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:27), such as a nucleotide sequence set forth in any of SEQ ID NOS:21, 23, 25, 26, 28, 29, or 30. Percent sequence identity is calculated as described herein.

In some cases, an isolated nucleic acid molecule encoding a chimeric polypeptide provided herein can be about 159 to about 189 bases in length (e.g, about 159 to about 165, about 165 to about 171, about 171 to about 177, about 177 to about 183, or about 183 to about 189 bases in length) and hybridize, under moderately or highly stringent hybridization conditions, to the sense or antisense strand of a nucleic acid having a sequence that encodes a chimeric polypeptide with the sequence set forth in SEQ ID NO:3, or a variant thereof.

For the purpose of this document, moderately stringent hybridization conditions mean the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO4 (pH 7.4), 5X SSC, 5X Denhart’s solution, 50 pg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5xl0⁷ cpm/pg), while the washes are performed at about 50°C with a wash solution containing 2X SSC and 0.1% sodium dodecyl sulfate.

Highly stringent hybridization conditions mean the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO4 (pH 7.4), 5X SSC, 5X Denhart’s solution, 50 pg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5xl0⁷ cpm/pg), while the washes are performed at about 65°C with a wash solution containing 0.2X SSC and 0.1% sodium dodecyl sulfate.

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 a chimeric polypeptide 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 complementary DNA (cDNA) strands. Ligase chain reaction, strand displacement amplification, self- sustained sequence replication, or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254: 1292.

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.

Isolated nucleic acids (e.g., nucleic acids encoding variant chimeric polypeptides provided herein) also can be obtained by mutagenesis. For example, a reference sequence can be mutated using standard techniques including oligonucleotide-directed mutagenesis and site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992. Non-limiting examples of variant chimeric polypeptides are provided herein.

Vectors containing the nucleic acids 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.

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

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).

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.

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 a chimeric polypeptide having the amino acid sequence set forth in SEQ ID NO:3, or a variant of the amino acid sequence set forth in SEQ ID NO:3). As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by any appropriate technique. Suitable methods for transforming and transfecting host cells can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2^nd edition), 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.

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.

Any appropriate method can be used to identify cells containing an isolated nucleic acid molecule or vector 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.

The chimeric polypeptides described herein, or nucleic acids encoding the chimeric polypeptides described herein, can be incorporated into compositions for administration to a subject (e.g., a subject identified as having, or being at risk for having, a cardiovascular disease such as HTN). Any appropriate method can be used to formulate and subsequently administer a therapeutic composition provided herein. Dosages typically are dependent on the responsiveness of the subject to the compound, with the course of treatment lasting from several days to several months, or until a suitable response is achieved. Optimum dosages can vary depending on the relative potency of an antibody, and generally can be estimated based on the EC50 found to be effective in in vitro and/or in vivo animal models. Compositions containing the chimeric polypeptides (e.g., MANP-PKCi polypeptides) and nucleic acids provided herein may be given once or more daily, weekly, monthly, or even less often, or can be administered continuously for a period of time (e.g., hours, days, or weeks). For example, a chimeric polypeptide or a composition containing a chimeric polypeptide provided herein can be administered to a patient at a dose of at least about 0.01 ng chimeric polypeptide/kg to about 100 mg chimeric polypeptide/kg of body mass, or can be administered continuously as an infusion for one to seven days (e.g., at a dose of about 0.01 ng chimeric polypeptide/kg/minute to about 0.5 pg chimeric polypeptide/kg/minute).

The chimeric polypeptides and nucleic acids can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, receptor or cell targeted molecules, or oral, topical or other formulations for assisting in uptake, distribution and/or absorption.

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

Compositions and formulations for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions (e.g., sterile physiological saline), which also can contain buffers, diluents and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers).

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.

Formulations for topical administration include, for example, sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents and other suitable additives. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be useful. In some embodiments, transdermal delivery of chimeric polypeptides as provided herein can be particularly useful. Methods and compositions for transdermal delivery include those described in, for example, Wermeling et al. (2008) Proc. Natl. Acad. Sci. USA 105:2058- 2063; Goebel and Neubert (2008) Skin Pharmacol. Physiol. 21 :3-9; Banga (2007) Pharm. Res. 24: 1357-1359; Malik et al. (2007) Curr. Drug Deliv. 4: 141-151; and Prausnitz (2006) Nat. Biotechnol. 24:416-417 (all of which are incorporated herein by reference in their entirety).

Nasal preparations can be presented in a liquid form or as a dry product. Nebulized aqueous suspensions or solutions can include carriers or excipients to adjust pH and/or tonicity.

Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, for example, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Emulsion formulations are particularly useful for oral delivery of therapeutic compositions due to their ease of formulation and efficacy of solubilization, absorption, and bioavailability. Liposomes can be particularly useful due to their specificity and the duration of action they offer from the standpoint of drug delivery.

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., chimeric polypeptide). Accordingly, for example, this document provides pharmaceutically acceptable salts of the chimeric polypeptides provided herein, as well as 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 chimeric polypeptides useful in methods provided herein (i.e., salts that retain the desired biological activity of the parent chimeric polypeptides 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.

In some cases, a chimeric polypeptide provided herein can be formulated as a sustained release dosage form. For example, a chimeric polypeptide 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 a polymeric substances, liposomes, microemulsions, microparticles, nanoparticles, or waxes.

Pharmaceutical formulations as disclosed herein, which can be presented conveniently in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients (i.e., the antibodies) 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. In some embodiments, a chimeric polypeptide 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 polypeptide can be mixed with one or more excipients that are pharmaceutically acceptable and are compatible with the polypeptide in amounts suitable for use in the methods described herein. For example, a polypeptide can be combined 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.

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

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).

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

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.

In some embodiments, a chimeric polypeptide can be incorporated into a hydrogel delivery system. For example, a polypeptide can be formulated for subcutaneous delivery to a patient via a xerogel-hydrogel system that can release the polypeptide in a continuous sustained manner over an extended period of time. See, for example, U.S. Patent No. 5,226,325, and PCT Publication No. WO 2004/071736.

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.

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

In some embodiments, a pharmaceutical composition for controlled release delivery of a chimeric polypeptide in a subject can include (a) a complex of the polypeptide (where the polypeptide has at least one basic functional group) and a polyanion derived from hexahydroxycyclohexane (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 delivery of a pharmaceutical agent in vivo over a period of time (e.g., several days to weeks or months) following administration. Sustained controlled release delivery of an MANP polypeptide can be demonstrated by, for example, continued therapeutic effects of the polypeptide over time (e.g., continued reductions in symptoms over time). Sustained delivery of the polypeptide also can be demonstrated by detecting the presence of the polypeptide in vivo over time. The compositions can provide a low initial burst delivery, followed by stable, controlled release of the polypeptide in vivo for prolonged periods of time (e.g., from days to months).

In such embodiments, a physically and chemically stable complex can form upon appropriate combining of a chimeric polypeptide 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.

A stable complex between a chimeric polypeptide and a polyanion can be incorporated into a pharmaceutically acceptable carrier containing a biodegradable waterinsoluble 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. 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.

Biodegradable water-insoluble polymers also can include end capped, end uncapped, or mixtures of end capped and end uncapped polymers. An end capped polymer generally is defined as having capped carboxyl end groups, while an uncapped polymer has free carboxyl end groups.

Factors to consider when determining suitable molecular weights for the polymer can include desired polymer degradation rate, mechanical strength, and rate of dissolution of polymer in solvent. Useful molecular weights for polymers can be from about 2,000 Daltons to about 150,000 Daltons, for example, with a polydispersity of from 1.1 to 2.8, depending upon which polymer is selected for use.

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.

Any appropriate method can be used to form various pharmaceutically acceptable polymer carriers. See, for example, 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.

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).

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

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.

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.

In some embodiments, a chimeric polypeptide can be formulated in a depot, which can provide constantly high exposure levels and may reach high exposure levels rapidly (with a short or no lag phase). See, e.g., U.S. Publication No. 2010/0266704. Depot formulations can include an MANP polypeptide 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.

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

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

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

The polypeptide content of the depot formulation (the loading) can be in a range of 1% to 30% (e.g., 10% to 25%, more preferred 15% to 20%. The loading is defined as the weight ratio of polypeptide to the total mass of the PLGA formulation. 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 the polypeptide. Such excipients can be present in the composition in an amount of about 0.1% to about 50%. Suitable excipients include, without limitation, polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose sodium, dextrin, PEG, surfactants such as poloxamers (also known as poly(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.

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 two PLGAs are present as a polymer blend can be particularly useful.

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.

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).

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.

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

(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;

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

(ii) preparing an external aqueous phase containing one or more stabilizers (e.g., poly(vinylalcohol), hydroxy ethyl cellulose, hydroxypropyl cellulose, poly( vinyl pyrrolidone), or gelatin) and optionally a buffer salt;

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

(iv) hardening the microparticles by solvent evaporation or solvent extraction, washing the microparticles (e.g., with water), collecting and drying the microparticles (e.g., by freeze-drying or drying under vacuum), and sieving the microparticles (e.g., through 140 pm).

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.

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).

Also provided herein are articles of manufacture containing one or more chimeric polypeptides or pharmaceutical compositions as described herein (e.g., a depot formulation containing a MANP-PKCi polypeptide) in a 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.

This document also provides methods for treating a cardiovascular disorder (e.g., hypertension, resistant hypertension, or myocardial infarction) in a mammal by administration (e.g., parenteral or subcutaneous administration) of a chimeric polypeptide provided 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.

Any appropriate mammal can be treated as described herein. For example, humans, non-human primates, dogs, cats, horses, cows, pigs, sheep, mice, rabbits, and rats can be treated using the methods described herein.

In some cases, before administering a chimeric 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 a cardiovascular disease. After identifying a mammal as having a need for such treatment, the mammal can be treated with a composition provided herein. For example, a composition containing a chimeric polypeptide can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce one or more symptoms of a cardiovascular disease, or to prevent or delay worsening of one or more such symptoms). In some cases, a chimeric polypeptide or a composition containing a chimeric polypeptide can be administered at a dose of at least about 0.01 ng chimeric polypeptide/kg to about 100 mg chimeric polypeptide/kg of body mass (e.g., about 10 ng chimeric polypeptide/kg to about 50 mg chimeric polypeptide/kg, about 20 ng chimeric polypeptide/kg to about 10 mg chimeric polypeptide/kg, about 0.1 ng chimeric polypeptide/kg to about 20 ng chimeric polypeptide/kg, about 3 ng chimeric polypeptide/kg to about 10 ng chimeric polypeptide/kg, or about 50 ng chimeric polypeptide/kg to about 100 pg/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 chimeric polypeptide/kg/minute to about 500 ng chimeric polypeptide/kg/minute (e.g., about 0.5 ng chimeric polypeptide/kg/minute, about 1 ng chimeric polypeptide/kg/minute, about 2 ng chimeric polypeptide/kg/minute, about 3 ng chimeric polypeptide/kg/minute, about 5 ng chimeric polypeptide/kg/minute, about 7.5 ng chimeric polypeptide/kg/minute, about 10 ng chimeric polypeptide/kg/minute, about 12.5 ng chimeric polypeptide/kg/minute, about 15 ng chimeric polypeptide/kg/minute, about 20 ng chimeric polypeptide/kg/minute, about 25 ng chimeric polypeptide/kg/minute, about 30 ng chimeric polypeptide/kg/minute, about 50 ng chimeric polypeptide/kg/minute, about 100 ng chimeric polypeptide/kg/minute, or about 300 ng chimeric polypeptide/kg/minute).

A chimeric polypeptide or a composition containing a chimeric polypeptide can be administered once (e.g., by implantation or injection of a depot composition), or more than once (e.g., by repeated injections, or by use of a series of transdermal drug patches). When administered more than once, the frequency of administration can range from about four times a day to about once every other month (e.g., twice a day, once a day, three to five times a week, about once a week, about twice a month, about once a month, or about once every other month). In addition, the frequency of administration can remain constant or can be variable during the duration of treatment. Various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, route of administration, and severity of condition may require an increase or decrease in administration frequency. In some embodiments, a chimeric polypeptide or a composition containing a chimeric polypeptide 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., topically or subcutaneously) for a second period of time. For example, a composition containing a chimeric polypeptide can be intravenously administered to a mammal (e.g., a human) at a dose of about 0.1 ng chimeric polypeptide/kg/minute to about 300 ng chimeric polypeptide/kg/minute (e.g., about 1 ng chimeric polypeptide/kg/minute to about 15 ng chimeric polypeptide/kg/minute, about 3 ng chimeric polypeptide/kg/minute to about 10 ng chimeric polypeptide/kg/minute, or about 10 ng chimeric polypeptide/kg/minute to about 30 ng chimeric polypeptide/kg/minute) for one to seven days (e.g., one, two, three, four, five, six, or seven days), and subsequently can be subcutaneously administered to the mammal at a dose of about 10 ng chimeric polypeptide/kg/day to about 100 ng chimeric polypeptide/kg/day (e.g., about 10 ng chimeric polypeptide/kg/day, about 20 ng chimeric polypeptide/kg/day, about 25 ng chimeric polypeptide/kg/day, about 30 ng chimeric polypeptide/kg/day, about 50 ng chimeric polypeptide/kg/day, or about 100 ng chimeric polypeptide/kg/day) for five to 30 days (e.g., seven, 10, 14, 18, 21, 24, or 27 days).

The methods provided herein can include administering to a mammal an effective amount of a chimeric polypeptide (e.g., MANP-PKCi or a variant thereof) or a nucleic acid encoding such a chimeric polypeptide, or an effective amount of a composition containing such a chimeric polypeptide. 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 a chimeric polypeptide can be an amount of the chimeric polypeptide that is sufficient 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 immunoreactivity, renal vascular resistance, proximal and distal fractional reabsorption of sodium, mean arterial pressure, pulmonary wedge capillary pressure, 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 a chimeric polypeptide can be an amount that increases cGMP levels in a treated mammal by at least 10% as compared to the level of cGMP in the mammal prior to administration of the chimeric polypeptide, or as compared to the level of sodium excretion in a control, untreated mammal.

In some embodiments, an “effective amount” of a chimeric polypeptide can be an amount of the chimeric polypeptide that is sufficient to reduce the occurrence of a symptom of cardiovascular disease by at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%). In some cases, for example, an “effective amount” of a chimeric polypeptide as provided herein can be an amount that reduces a symptom of cardiovascular disease in a treated mammal by at least 10% as compared to the level of the symptom in the mammal prior to administration of the chimeric polypeptide or without administration of the chimeric polypeptide, or as compared to the level of the symptom in a control, untreated mammal. The presence or extent of such symptoms can be evaluated using any appropriate method. In some cases, an “effective amount” of a chimeric polypeptide provided herein can be an amount that reduces blood pressure in a mammal identified as having hypertension by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%) as compared to the blood pressure in the mammal prior to administration of the chimeric polypeptide or without administration of the chimeric polypeptide, or as compared to the level of the symptom in a control, untreated mammal.

In some cases, the amount and frequency of administration for a chimeric polypeptide administered to a mammal can be titrated in order to, for example, identify a dosage that is most effective to treat hypertension or another cardiovascular disease while having the least amount of adverse effects. For example, an effective amount of a composition can be any amount that reduces fibrillation within a mammal without having significant toxicity in the mammal. If a particular mammal fails to respond to a particular amount, then the amount can be increased by, for example, two-fold, three-fold, 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.

The frequency of administration can be any frequency that reduces a symptom of cardiovascular disease within a mammal without producing significant toxicity in the mammal. For example, the frequency of administration can be from about four times a day to about once every other month, or from about once a day to about once a month, or from about once every other day to about once a week. In addition, the frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, route of administration, and severity of renal condition may require an increase or decrease in administration frequency.

An effective duration of administration can be any duration that reduces hypertension or a symptom of cardiovascular disease within a mammal without producing significant toxicity in the mammal. The effective duration can vary 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 treatment or prevention regimen. For example, an effective duration can vary with the frequency of administration, amount administered, route of administration, and severity of a renal condition.

After administering a polypeptide or composition provided herein to a mammal, the mammal can be monitored to determine whether or not the cardiovascular disease has improved. For example, a mammal can be assessed after treatment to determine whether or not one or more symptoms of the disease have decreased. Any suitable method can be used to assess improvements in function. If a mammal fails to respond to a particular dose, 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 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.

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

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

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

EXAMPLES

Example 1 - Evidence for crosstalk between ANGII and pGC-A/cGMP in humans Circulating levels of ANP, BNP, cGMP and ANGII [median(IQR)] in 128 healthy subjects were 15 pg/mL (6-34 pg/mL), 26 pg/mL (15-39 pg/mL), 1.6 pmol/mL (1.2-2.4 pmol/mL), and 4.5 pg/mL (3.4-6. 1 pg/mL), respectively. Interaction tests suggested that ANGII levels affected the correlations between cGMP and ANP (^interaction = 0.14) or BNP (interaction < 0.001), after adjusting for age and sex. Further stratification analysis revealed that cGMP was positively associated with ANP or BNP only in the subgroup with ANGII levels < 4.5pg/ml, but not in the subgroup with ANGII levels > 4.5pg/ml (FIGS. 4A and 4B)

Example 2 - Inhibitory effect of ANGII on ANP-induced cGMP production in vivo Acute intravenous infusion of ANP resulted in elevated plasma and urinary cGMP, reduced BP, and enhanced diuresis and natriuresis in normotensive rats. Coinfusion of ANGII with ANP attenuated ANP’s induction of cGMP in both plasma and urine, antagonized ANP’s BP lowering effects, and had no influences on ANP’s diuretic and natriuretic effects (FIGS. 5A and 5B). Infusion of saline or ANGII alone had no effects on plasma and urinary cGMP (FIG. 5C). In FIGS. 5A and 5B, two-way ANOVA was conducted for intergroup and intragroup comparison. For FIG. 5C, unpaired t test was conducted for comparison.

Example 3 - Molecular characterization of ANGII inhibition of ANP-induced cGMP production in vitro

Three potential mechanisms at the cellular level were investigated in vitro to determine how ANGII suppresses pGC-A actions: (1) direct allosteric inhibition of pGC- A; (2) indirect inhibition via AT1R signaling; and (3) indirect inhibition via its type 2 receptor (AT2R) signaling. A SPR assay was performed at 25°C. The extracellular domain of pGC-A recombinant protein was immobilized onto a Ni-NTA sensorchip, and sequentially diluted polypeptides were injected onto the chip at constant rate followed by dissociation. Data were collected as sensor grams and binding kinetics indices derived. For each result, K_a reports the association affinity, Ka reports the dissociation affinity, and binding affinity (KD) was calculated by Ka/K_a. Smaller KD values indicated stronger binding affinity between the peptide and the pGC-A receptor.

Using this SPR assay, the KD between ANGII and pGC-A was determined to be 258 nM (FIG. 6A), which was substantially weaker than the binding affinity between ANP and pGC-A (KD = 61 nM). These data further indicated that AT1R was required for ANGII to exert its inhibitory effects on ANP-induced cGMP, as such inhibition was only recapitulated in HEK293 cells overexpressing both pGC-A and AT1R (HEK293/pGC- A⁺/AT1R⁺), and not in HEK293 cells overexpressing pGC-A alone (HEK293/pGC-A⁺) or overexpressing both pGC-A and AT2R (FIG. 6B). Unpaired t test was conducted for comparison between groups.

Example 4 - Involvement of PKC underlying the crosstalk between ANGII and pGC-A/cGMP pathway

In HEK293/pGC-A⁺/ATlR⁺ cells, the inhibitory effect of ANGII on ANP-induced cGMP production was largely ablated by treatment with Valsartan (AT1R blocker) or Go6983 (pan PKC inhibitor), further confirming that the process is mediated by AT1R and suggesting that the process may also involve the AT1R downstream target, PKC (FIG. 7A). One-way ANOVA was conducted, followed by multiple comparisons. Compared to HEK293/pGC-A⁺ cells, the overexpression of AT1R in HEK293/pGC- A⁺/AT1R⁺ cells enhanced the membrane localization of at least two PKC isoforms (FIG. 7B). Interestingly, treatment of ANGII did not further alter the expression of PKC isoforms in either cell type, indicating that the effect of ANGII on PKC activity occurs at post-translational levels.

Example 5 - Synthesis of PKCi-MANP

An 18 amino acid sequence from PKC was added to the N-terminus of MANP to generate MANP-PKCi (SEQ ID NO:3; FIG. 3), a polypeptide with dual functions of PKC inhibition and pGC-A activation. The MANP-PKCi polypeptide was synthesized following procedures similar to those described elsewhere (Whelton et al., Circulation, 138:e426-e483, 2018; and Lee and Burnett, J Investig Med, 57(1): 18-21, 2009). Briefly, the polypeptide was synthesized by Fmoc solid phase methods to acquire a crude linear form. After purification with preparative reversed-phase-high-performance liquid chromatography, the polypeptide was oxidized with clear-oxresin to form the cyclized ring between cysteine residues 25 and 41. The cyclized MANP-PKCi was purified again, and the quality was further verified by electrospray ionization-time-of-flight mass spectrometry.

Example 6 - Alternative MANP-PKCi polypeptides

The structure of the MANP-PKCi polypeptide (SEQ ID NO:3; also referred to as MANP-PKCi- 1) was modified for additional studies. While the PKC 19-36 amino acids were initially positioned at the N-terminus of MANP (FIG. 3), several modifications were generated, including: (1) flipping the PKC 19-36 sequences to generate MANP- PKCi-2 (SEQ ID NO:4; FIG. 8); (2) replacing the key functional alanine at position 7 to serine for more potency on PKC inhibition (SEQ ID NO: 6; FIG. 8); and/or (3) deleting five redundant amino acids from the C-terminal end of the PKCi portion of the polypeptide, to make the polypeptide shorter and more potent for PKC inhibition (SEQ ID NOS:7-8; FIG. 8). To assess cGMP activation by MANP-PKCi polypeptides, HEK293/pGC- A⁺/AT1R⁺ cells were cultured to 80% confluence and pretreated with vehicle or ANGII for 30 minutes, followed by treatment with 10'⁷M MANP, MANP-PKCi- 1, or MANP- PKCi-2 for 10 minutes. Cells were then lysed and subjected to cGMP measurement by ELISA. As shown in FIG. 9, among MANP, MANP-PKCi- 1, and MANP-PKCi-2, MANP-PKCi-1 stimulated the greatest cGMP response. One-way ANOVA was conducted, followed by multiple comparisons.

SPR assays were performed at 25°C to determine the binding affinity of MANP (FIG. 10A), MANP-PKCi-1 (FIG. 10B), and MANP-PKCi-2 (FIG. 10C) for the extracellular domain of pGC-A, as described in Example 3. These studies demonstrated that MANP, MANP-PKCi-1, and MANP-PKCi-2 had similar binding affinity for pGC-A, and that MANP-PKCi-2 had the strongest KD of 168 pM (FIG. 10C).

Example 1 -In vitro effect of MANP-PKCi and MANP on NEP degradation

To assess NEP degradation, equimolar concentrations (e.g., IO'¹⁰, 10'⁸, 10'⁷, and/or 10'⁶M) of MANP-PKCi (referred to collectively as “MANP-PKCi”) and MANP are incubated with 50 ng of recombinant NEP at 37°C for 30, 60, 120, and 240 minutes. At each time point, an equal volume of perchloric acid is added to the reaction to inactive NEP and stop degradation. Residual polypeptide is added to HEK293/pGC-A^+/ATlR⁺ cells to determine the ability for cGMP stimulation. As control experiments, an NEP inhibitor, phosphoramidon, is incubated with NEP and polypeptides to block any degradation specifically incurred by NEP digestion.

Example - In vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies

The PK and PD characteristics of various MANP-PKCi polypeptides (referred to collectively as “MANP-PKCi”) and MANP are evaluated in vivo under normal and hypertensive conditions. In particular, an acute study is conducted on both normotensive and spontaneously hypertensive (SHR) rats following cannulation using a procedure described elsewhere (Chen et al., supra, and Sangaralingham et al., Hypertension, 57:201-207, 2011). A single bolus of MANP-PKCi or MANP (3.88 mg/kg) is injected subcutaneously at the beginning of the study (0 min). Rats are subjected to continuous BP monitoring and urine collection, and are sacrificed at 15, 60, 120, or 240 minutes for blood sampling. PK characteristics are determined by measuring circulating levels of ANP-like peptide with an antibody (Burnett et al., Science, 231 : 1145-1147, 1986) to detect the ANP ring structure. PD characteristics are evaluated by measuring BP, plasma and urinary levels of cGMP, and levels of RAS components including renin, ANGII, and aldosterone. Both PK and PD are assessed over a 240 minute period.

All analyses are performed with JMP or SAS, and in collaboration with a Master level statistician. Student’s t test, 1-way and 2-way ANOVA followed by multiple comparisons are performed when appropriate. Specifically, N=16 per group achieves adequate statistical power based on plasma cGMP changes in rat studies described elsewhere (Chen et al., supra). The detectable effect size between groups with 80% power is about 1 for both designs. The in vivo studies are conducted on 120% of the proposed numbers of rats in practice to compensate for any unexpected dropping of animals.

Example 9 - Additional alternative MANP-PKCi polypeptides

The structure of the MANP-PKCi polypeptide (SEQ ID NO:3; also referred to as MANP-PKCi- 1) is further modified for additional studies. Modifications that are generated include, for example: (1) moving the PKC19-36 amino acids to the C-terminus of MANP to replace the MANP tail (SEQ ID NOS:5 and 9-11; FIG. 8); and (2) adding digestion sites (S-Y or R-S) for Corin, a protease that processes the ANP precursor, at the link between PKC19-39 and MANP (SEQ ID NOS: 12-15; FIG. 8) such that the entire peptide can derive into two functional peptides in vivo.

The additional variant MANP-PKCi polypeptides are evaluated in the in vitro and in vivo studies described herein (e.g., in culture with HEK 293 cells to determine their effect on cGMP production, in SPR assays, and in in vivo studies). Example 10 - fa vitro effect of MANP-PKCi and MANP on cGMP generation

To evaluate the potency of MANP-PKCi polypeptides in terms of GC-A receptor engagement in vitro, cGMP was measured by ELISA after the treatment of MANP-PKCi polypeptides in two different cultured cells (FIGS. 11 and 12). Specifically, equimolar concentrations (e.g., IO'¹⁰, 10'⁸, and 10'⁶ M) of MANP-PKCi (referred to collectively as “MANP-PKCi”) and MANP were incubated on human embryonic cell lines overexpressing the GC-A protein (HEK-GCA) (FIG. 11) or on primary cultures of human renal proximal tubular cells (FIG. 12) for 10 minutes before the cells were lysed for cGMP quantification. In these in vitro settings, the MANP-PKCi polypeptides showed dose-dependent activation of cGMP, providing evidence of their ability to stimulate GC-A activation. The generation of cGMP stimulated by MANP-PKCi appeared to be equal to (or higher for some peptide candidates) cGMP generated stimulated by MANP, suggesting that addition of the PKC inhibitor peptide to the N- terminus of MANP had minimal or no influence on the biological activity of MANP in vitro.

Example 11 - fa vivo PK and PD studies

The PK and PD characteristics of various MANP-PKCi polypeptides (referred to collectively as “MANP-PKCi”) were evaluated in vivo in spontaneously hypertensive rats (SHR) - highly clinically relevant animal model of human essential hypertension. The plasma and urinary responses of MANP-PKCi were used as primary indices for their in vivo effects.

A total of 8 SHR rats (male, 250-300 g) were randomly assigned for infusion with one of the following: vehicle (0.9% saline, n=2), MANP-PKCi-1 (600 pmol/kg/min, n=l), MANP-PKCi-2 (600 pmol/kg/min, n=l), MANP-PKCi-4 (600 pmol/kg/min, n=2), MANP-PKCi-5 (600 pmol/kg/min, n=l), or MANP-PKCi-6 (600 pmol/kg/min, n=l). Rats were included only if deemed healthy by the institutional veterinary department. Investigators who conducted the in vivo study data were not blinded to treatments, but biochemical analysis was performed by a different investigator who was blinded to treatment. A detailed protocol used to assess the in vivo drug effects of MANP-PKCi polypeptides is shown in FIG. 13. On the day of the experiment, SHR rats were anesthetized with inactin (120 mg/kg, intraperitoneal injection; Sigma, St. Louis, MO). Rats were then kept on a heating pad at 38°C to maintain a normal body temperature for the entire study. Vessel and bladder cannulation procedures were conducted as described elsewhere (Chen et al., supra, and Sangaralingham et al., supra). Briefly, a polyethylene (PE)-50 tube catheter was placed into one jugular vein for peptide infusion and another PE-50 tube was placed into the carotid artery for blood pressure (BP) monitoring (Sonometrics; London, Ontario, Canada) and blood sampling. Urine samples were passively collected from the bladder through a PE-90 tube catheter. Instrumentation was followed by a 15 minute equilibrium period, and then a 30 minute preinfusion period was performed to collect baseline urine samples. The preinfiision period was followed by a 45 minute continuous infusion of MANP-PKCi or vehicle (15 minute lead-in drug infusion, 30 minute clearance during drug infusion). Immediately after discontinuation of peptide infusion, another 30 minute washout clearance period was allowed to elapse before termination and sacrifice. Two additional urine samples were collected during the 30 minute clearance and 30 minute washout clearance periods, respectively. Four blood samples were collected at baseline, by the end of drug infusion, at the middle of the washout clearance period, and by the end of the washout clearance period to determine the dynamics of circulating cGMP levels; the blood collected from each rat was replaced with an equal volume of saline. BP was monitored and recorded every 15 minutes after the initiation of peptide infusion. All collected blood and urine samples were stored at -80°C until they were assayed. For the biochemical analyses, plasma and urinary cGMP were measured with a cGMP ELISA kit (Enzo Life Sciences; Farmingdale, NY).

Compared to vehicle, infusion of MANP-PKCi-1 at 600 pmol/kg/min dramatically reduced systolic blood pressure (FIG. 14A), diastolic blood pressure (FIG. 14B), and mean arterial pressure (FIG. 14C). During infusion and washout periods, the volume of urine excretion was dramatically higher in the MANP-PKCi-1 group compared to the vehicle group (FIG. 14D). Further, infusion of MANP-PKCi-1 stimulated cGMP generation in vivo in this experimental model of hypertension, as cGMP was detected to be higher in both plasma samples (FIG. 14E) and urine samples (FIG. 14F) compared to vehicle.

Compared to vehicle, infusion of MANP-PKCi-2 at 600 pmol/kg/min dramatically reduced systolic blood pressure (FIG. 15A) and mean arterial pressure (FIG. 15C), and mildly reduced diastolic blood pressure (FIG. 15B). During the infusion and washout periods, the volume of urine excretion was dramatically higher in the MANP-PKCi-2 group compared to the vehicle group (FIG. 15D). Infusion of MANP- PKCi-2 stimulated cGMP generation in vivo in this experimental model of hypertension, as cGMP was detected to be higher in both plasma samples (FIG. 15E) and urine samples (FIG. 15F) compared to vehicle.

Compared to vehicle, infusion of MANP-PKCi-4 at 600 pmol/kg/min dramatically reduced systolic blood pressure (FIG. 16A), diastolic blood pressure (FIG. 16B), and mean arterial pressure (FIG. 16C). During the infusion and washout periods, the volume of urine excretion was dramatically higher in the MANP-PKCi-4 group compared to the vehicle group (FIG. 16D). Infusion of MANP-PKCi-4 also stimulated cGMP generation in vivo in this experimental model of hypertension, as cGMP was detected to be higher in both plasma samples (FIG. 16E) and urine samples (FIG. 16F) compared to vehicle.

Compared to vehicle, infusion of MANP-PKCi-5 at 600 pmol/kg/min dramatically reduced systolic blood pressure (FIG. 17A) and mean arterial pressure (FIG. 17C), and mildly reduced diastolic blood pressure (FIG. 17B). During the infusion and washout periods, the volume of urine excretion was dramatically higher in the MANP-PKCi-5 group compared to the vehicle group (FIG. 17D). Infusion of MANP- PKCi-5 stimulated cGMP generation in vivo in this experimental model of hypertension, as cGMP was detected to be higher in both plasma samples (FIG. 17E) and urine samples (FIG. 17F) compared to vehicle.

Compared to vehicle, infusion of MANP-PKCi-6 at 600 pmol/kg/min dramatically reduced systolic blood pressure (FIG. 18A), diastolic blood pressure (FIG. 18B), and mean arterial pressure (FIG. 18C). During the infusion and washout periods, the volume of urine excretion was dramatically higher in the MANP-PKCi-6 group compared to the vehicle group (FIG. 18D). Infusion of MANP-PKCi-6 stimulated cGMP generation in vivo in this experimental model of hypertension, as cGMP was detected to be higher in both plasma samples (FIG. 18E) and urine samples (FIG. 18F) compared to vehicle.

OTHER EMBODIMENTS

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. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3.

2. The polypeptide of claim 1, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3.

3. The polypeptide of claim 1, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:3.

4. The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

5. The polypeptide of any one of claims 1 to 4, wherein said polypeptide is a substantially pure polypeptide.

6. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5 or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:5.

7. The polypeptide of claim 6, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO: 5.

8. The polypeptide of claim 6, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO: 5.

9. The polypeptide of claim 6, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NOTO, or SEQ ID NO: 11.

10. The polypeptide of any one of claims 6 to 9, wherein said polypeptide is a substantially pure polypeptide.

11. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 or the amino acid sequence set forth in SEQ ID NO:4 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:4.

12. The polypeptide of claim 11, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:4.

13. The polypeptide of claim 11, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:4.

14. The polypeptide of claim 11, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

15. The polypeptide of any one of claims 11 to 14, wherein said polypeptide is a substantially pure polypeptide.

16. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 15.

17. The isolated nucleic acid of claim 16, wherein the isolated nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30.

18. A vector comprising a nucleic acid encoding the polypeptide of any one of claims 1 to 15.

19. The vector of claim 18, wherein the vector comprises a nucleic acid having a nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30.

20. A host cell comprising a nucleic acid encoding the polypeptide of any one of claims 1 to 15.

21. The host cell of claim 20, wherein the nucleic acid comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 16 to 30.

22. The host cell of claim 20 or claim 21, wherein said host cell is a eukaryotic host cell.

23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polypeptide of any one of claims 1 to 15.

24. A method for treating a cardiovascular disease in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition comprising a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 3.

25. The method of claim 24, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3.

26. The method of claim 24, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:3.

27. The method of claim 24, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

28. A method for treating a cardiovascular disease in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition comprising a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO:5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO: 5.

29. The method of claim 28, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO: 5.

30. The method of claim 28, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO: 5.

31. The method of claim 28, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

32. A method for treating a cardiovascular disease in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition comprising a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:4, or the amino acid sequence set forth in SEQ ID NO:4 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:4.

33. The method of claim 32, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:4.

34. The method of claim 32, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:4.

35. The method of claim 32, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

36. The method of any one of claims 24 to 35, wherein said cardiovascular disease is hypertension, and wherein said composition is administered in an amount effective to reduce the blood pressure of said mammal.

37. The method of any one of claims 24 to 35, wherein said cardiovascular disease is heart failure.

38. The method of any one of claims 24 to 37, wherein said mammal is a human.

39. The method of any one of claims 24 to 38, wherein said composition is administered at a dose of 0.01 ng/kg to 50 pg/kg.

40. The method of any one of claims 24 to 39, comprising administering said composition intravenously.

41. The method of any one of claims 24 to 40, further comprising identifying said mammal as being in need of said treatment.

42. Use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3.

43. The use of claim 42, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3.

44. The use of claim 42, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:3.

45. The use of claim 42, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

46. Use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO:5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:5.

47. The use of claim 46, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:5.

48. The use of claim 46, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO: 5.

49. The use of claim 46, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

50. Use of a chimeric polypeptide in the manufacture of a medicament for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4, or the amino acid sequence set forth in SEQ ID NO:4 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NON.

51. The use of claim 50, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

52. The use of any one of claims 42 to 51, wherein said cardiovascular disorder comprises hypertension or heart failure.

53. The use of any one of claims 42 to 52, wherein said medicament is formulated for intravenous administration.

54. Use of a chimeric polypeptide for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:3.

55. The use of claim 54, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:3.

56. The use of claim 54, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO:3.

57. The use of claim 54, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

58. Use of a chimeric polypeptide for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NO:5.

59. The use of claim 58, wherein said polypeptide comprises one, two, three, four, or five conservative amino acid substitutions as compared to SEQ ID NO:5.

60. The use of claim 58, wherein said polypeptide comprises one to three conservative amino acid substitutions as compared to SEQ ID NO: 5.

61. The use of claim 58, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

62. Use of a chimeric polypeptide for treating a cardiovascular disorder, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NON, or the amino acid sequence set forth in SEQ ID NON with one, two, three, four, or five amino acid additions, subtractions, or substitutions as compared to SEQ ID NON.

63. The use of claim 62, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NON.

64. The use of any one of claims 54 to 63, wherein said cardiovascular disorder comprises hypertension or heart failure.