Compositions and methods of treatment using CRRL 191, atrial natriuretic peptide (ANP) analogue and guanylate cyclase receptor (GC-A) activator

Abstract

The present application relates to selected engineered analogs of atrial natriuretic peptide; compositions containing these peptides and their use to treat, prevent, or alleviate symptoms of cardiovascular, heart-kidney, or metabolic diseases. The peptides are the next generation type of GC-A activators having improved binding to GC-A receptors, greater cGMP activation, enhanced resistance to neprilysin degradation, and greater efficacy in lowering blood pressure than ANP in vivo. The main analogue is CRRL 191.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTING

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 407,798, filed September 19, 2022, which is incorporated herein by reference. The file contained in the file named P35213WO00_SL.XML (in A sequence listing is provided in the format of SEQ ID NO: 1 (which measures 8,192 bytes in ), created on August 17, 2023, which is electronically submitted herewith and is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Funded Research

[0004] Portions of this disclosure were made with government support under Grant No. R01-HL136340 from the National Institutes of Health (NIH), and the government has certain rights in the invention. Technical Field

[0005] This document provides methods and materials related to selected engineered analogs of atrial natriuretic peptide (ANP). For example, this document provides one or more selected engineered analogs containing atrial natriuretic peptide (ANP) provided herein and can treat cardiovascular diseases (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction) in mammals (e.g., humans), cardiorenal diseases (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) and metabolic diseases (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) compositions. Background Art

[0006] Natriuretic polypeptides are polypeptides that can cause natriuresis - increase the excretion of sodium in the urine. Natriuretic polypeptides can be produced in the brain, heart, kidneys and / or vascular tissues. The human natriuretic polypeptide family includes the cardiac hormone atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), the black mamba (Dendroaspis) natriuretic peptide (DNP) isolated from the green mamba snake, and urodilator (URO). Natriuretic polypeptides bind to two well-characterized guanylate cyclase receptors, GC-A and GC-B, also known as natriuretic peptide receptors (NPR-A and NPR-B). GC-A is the primary receptor for ANP, BNP, DNP, and URO; and GC-B is the primary receptor for CNP). Once bound, these receptors in turn catalyze the conversion of guanosine triphosphate (GTP) to the second messenger cyclic 3'5' monophosphate guanosine (cGMP) (Kuhn, Circ. Res., 93:700-709 (2003); Tawaragi et al., Biochem. Biophys. Res. Comm., 175:645-651 (1991); and Komatsu et al., Endocrinol., 129:1104-1106 (1991)). Downstream effects of GC-A receptor activation include natriuresis, arterial vasodilation, renin and aldosterone inhibition, anti-apoptosis, anti-hypertrophy, vasodilation, angiogenesis, lipolysis, and browning of white adipocytes. Downstream effects of GC-B receptor activation include anti-fibrosis, anti-inflammation, angiogenesis, microcirculatory dilation, and vasodilation.

[0007] Previous disclosures, such as International Patent Application No. PCT / US2017 / 060808, provide alternatively spliced ​​atrial natriuretic peptide (MANP) analogs consisting of the 28 amino acids of native ANP and 12 amino acids extended at the C-terminus. Compared to ANP, MANP is more resistant to degradation, has enhanced binding to the GC-A receptor, causes increased and more sustained sodium excretion, causes more sustained suppression of aldosterone, and leads to greater and more sustained reductions in blood pressure. Summary of the invention

[0008] The present disclosure provides the design and validation of a next generation class of novel GC-A activators with improved binding to the GC-A receptor, more potent cGMP activation, enhanced resistance to enkephalinase degradation, and greater efficacy in lowering blood pressure than ANP in vivo.

[0009] The present disclosure provides and includes methods and materials related to selected engineered analogs of atrial natriuretic peptide (ANP). For example, the present disclosure provides engineered polypeptides as shown in Table 1, which comprise, consist essentially of, or consist of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0010] In one aspect, a selected engineered analog of ANP provided herein is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide comprising a sequence having at least 90% homology with any one of SEQ ID NO:3-7. In one aspect, the polypeptide sequence has at least 95% homology with any one of SEQ ID NO:3-7. In one aspect, the polypeptide sequence has at least 97% homology with any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide comprising the amino acid sequence shown in amino acids 1 to 8 of SEQ ID NO 7.

[0011] The present disclosure provides and includes substantially pure polypeptides, which are associated with selected engineered analogs of ANP. In one aspect, a selected engineered analog of ANP provided herein is a substantially pure polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a substantially pure polypeptide comprising an amino acid sequence with at least 90% homology to any one of SEQ ID NO:3-7. In one aspect, the substantially pure polypeptide has a sequence with at least 95% homology to any one of SEQ ID NO:3-7. In one aspect, the substantially pure polypeptide has a sequence with at least 97% homology to any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a substantially pure polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, wherein the polypeptide comprises one, two or three amino acid substitutions, additions or deletions. In one aspect, a selected engineered analog of ANP provided herein is a substantially pure polypeptide comprising the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO 7.

[0012] The present disclosure also provides a composition of a selected engineered analog of ANP provided herein. In one aspect, a composition provided herein, comprising a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a composition provided herein, comprising a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO:3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions. In one aspect, a composition provided herein, comprising at least two polypeptides, wherein each polypeptide in the at least two polypeptides is a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a composition provided herein, comprising an amino acid sequence shown in amino acids 1 to 8 of SEQ ID NO 7. In one aspect, the composition further comprises one or more pharmaceutically acceptable excipients. In one aspect, the composition further comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB). In one aspect, the composition further comprises furosemide.

[0013] The present disclosure also provides nucleic acids and compositions thereof of selected engineered analogs of encoding ANP provided herein. In one aspect, a nucleic acid provided herein encodes a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a composition provided herein comprises a nucleic acid encoding a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a composition provided herein comprises a nucleic acid encoding at least two polypeptides, wherein each of the at least two polypeptides is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, the composition further comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB). In one aspect, the composition further comprises furosemide. In one aspect, the nucleic acid is in the form of a non-viral vector. In one aspect, the non-viral vector is an expression plasmid. In one aspect, the nucleic acid is in the form of a viral vector.

[0014] The disclosure also provides and includes methods for treating cardiovascular diseases (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal diseases (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic diseases (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in mammals (e.g., humans) (e.g., humans) for treating mammals ...

[0015] The present disclosure provides and includes methods for lowering blood pressure, increasing natriuresis, causing arterial vasodilation, inhibiting renin and aldosterone, reducing cell apoptosis, reducing hypertrophy, increasing diastolic properties, inducing angiogenesis, increasing lipolysis, and causing browning of white adipocytes by administering a composition provided herein, the composition comprising one or more engineered analogs of ANP provided herein, comprising, consisting essentially of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0016] In one aspect, provided herein is a method for treating a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 3-7 or a nucleic acid encoding the polypeptide. In one aspect, the mammal is a human. In one aspect, the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension. In one aspect, the cardiovascular disease, cardiorenal disease or metabolic disease is refractory hypertension. In one aspect, the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB). In one aspect, the composition comprises furosemide.

[0017] In one aspect, provided herein is a method for treating a mammal at risk of developing a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH), wherein the method comprises administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 3-7 or a nucleic acid encoding the polypeptide. In one aspect, the mammal is a human. In one aspect, the cardiovascular disease, cardiorenal disease, or metabolic disease is hypertension. In one aspect, the cardiovascular disease, cardiorenal disease, or metabolic disease is refractory hypertension. In one aspect, the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB). In one aspect, the composition comprises furosemide.

[0018] In one aspect, provided herein is a method for alleviating the symptoms of a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 3-7 or a nucleic acid encoding the polypeptide. In one aspect, the mammal is a human. In one aspect, the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension. In one aspect, the cardiovascular disease, cardiorenal disease or metabolic disease is refractory hypertension. In one aspect, the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB). In one aspect, the composition comprises furosemide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various aspects of the present disclosure are described herein by way of example only with reference to the accompanying drawings. With specific reference now to the drawings in detail, it should be emphasized that the details shown are by way of example and for illustrative discussion of various aspects of the present disclosure. In this regard, the description and drawings considered individually and together make it apparent to those skilled in the art how various aspects of the present disclosure may be practiced.

[0020] Figure 1A The structure of ANP is shown.

[0021] Figure 1B The structure of CRRL 191 is shown.

[0022] Figure 2 HEK293 / GCA overexpressing cells responded to 10 -8 Figure 2 shows the cGMP production levels of ANP or CRRL 191 at different doses over 6 hours.

[0023] Figure 3 is a graph of the in vitro degradation of CRRL 191 and ANP in the neprilysin assay.

[0024] Figure 4 is a graph of cGMP production levels induced by ANP and CRRL191 in primary human cardiomyocytes.

[0025] Figure 5 is a graph of cGMP production levels induced by MANP, CRRL 191, and CRRL 101 in primary human cardiomyocytes.

[0026] Figure 6 Figure 2 is a graph showing reduction of apoptosis by increasing doses of CRRL 191 compared to control over 90 hours in primary human cardiomyocytes induced by CRRL 191 using real-time imaging.

[0027] Fig. 7A is a graph of the binding kinetics of ANPs to GC-A measured by surface plasmon resonance (SPR).

[0028] Figure 7B is a graph of the binding kinetics of MANP to GC-A measured by surface plasmon resonance (SPR).

[0029] Figure 7C is a graph of the binding kinetics of CRRL 191 to GC-A measured by surface plasmon resonance (SPR).

[0030] Fig. 8A Comparison of systolic blood pressure reduction induced by saline vehicle, 100 or 300 pmol / kg / min of CRRL 191, or 100 or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during infusion and washout periods.

[0031] Figure 8BComparison of the decrease in diastolic blood pressure induced by saline, 100 or 300 pmol / kg / min of CRRL 191, or 100 or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during the infusion and washout periods.

[0032] Figure 8C Comparison of the decrease in mean arterial pressure induced by saline, 100 or 300 pmol / kg / min of CRRL 191, or 100 or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during the infusion and washout periods.

[0033] Fig. 9A Comparison of plasma cGMP levels induced by saline, 100 or 300 pmol / kg / min of CRRL 191, or 100 or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during infusion and washout periods.

[0034] Fig. 9B Comparison of changes in plasma cGMP levels induced by saline, 100 or 300 pmol / kg / min of CRRL 191, or 100 or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during infusion and washout periods. DETAILED DESCRIPTION

[0035] This description is not intended to be a detailed catalog of all the different ways in which the present disclosure may be implemented or all the features that may be added to the present disclosure. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be deleted from the embodiment. Therefore, the present disclosure contemplates that, in some embodiments of the present disclosure, any feature or feature combination set forth herein may be excluded or omitted. In addition, according to the present disclosure, many changes and additions to the various embodiments suggested herein will be apparent to those skilled in the art, and these many changes and additions do not deviate from the present disclosure. In other cases, well-known structures, interfaces, and processes are not shown in detail to avoid unnecessarily obscuring the present disclosure. No part of this specification is intended to be interpreted as denying any part of the full scope of the present disclosure. Therefore, the following description is intended to show some specific embodiments of the present disclosure, rather than exhaustively specifying all its permutations, combinations, and variations.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Herein, the terms used in describing the present disclosure are only for the purpose of describing specific aspects or embodiments and are not intended to limit the present disclosure.

[0037] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for teachings related to the sentence and / or paragraph in which the reference is found. References to the techniques employed herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or replacements of equivalent techniques that would be apparent to those skilled in the art. In the event of a conflict, the present specification (including definitions) shall prevail.

[0038] Unless the context indicates otherwise, it is clearly intended that the various features of the present disclosure described herein can be used in any combination. In addition, the present disclosure also contemplates that in some embodiments of the present disclosure, any feature or feature combination set forth herein can be excluded or omitted.

[0039] Method disclosed herein includes and comprises one or more steps or actions for realizing described method.Method steps and / or actions can be interchanged with each other without departing from the scope of the present disclosure.In other words, unless the correct operation embodiment requires a specific order of steps or actions, otherwise the order and / or use of specific steps and / or actions can be modified without departing from the scope of the present disclosure.Although methods and materials similar or equivalent to methods and materials described herein can be used to practice the aspects of the present disclosure, suitable methods and materials are described herein.

[0040] As used in the description of the present disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0041] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative ("or").

[0042] As used herein, the terms "about" and "approximately" when referring to a measurable value such as a length, frequency, or duration, etc., are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

[0043] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

[0044] As used herein, the term "exemplary" is used to mean serving as an example, instance, or illustration. Any aspect or aspects described as "exemplary" are not necessarily to be construed as preferred or advantageous over other aspects or aspects, nor are equivalent structures and techniques known to those of ordinary skill in the art meant to be excluded. Rather, the use of the word exemplary is intended to present concepts in a concrete manner, and the disclosed subject matter is not limited to such examples.

[0045] Peptides

[0046] The present disclosure provides methods and materials related to selected engineered analogs of atrial natriuretic peptide (ANP). For example, the present disclosure provides the polypeptides shown in Table 1. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide consisting essentially of the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP provided herein is a polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NO:3-7. In one aspect, a selected engineered analog of ANP comprises the amino acid sequence shown in SEQ ID NO:3. In one aspect, a selected engineered analog of ANP consists essentially of the amino acid sequence shown in SEQ ID NO:3. In one aspect, a selected engineered analog of ANP consists of the amino acid sequence shown in SEQ ID NO:3. In one aspect, a selected engineered analog of ANP is CRRL 101. In one aspect, a selected engineered analog of ANP comprises the amino acid sequence shown in SEQ ID NO:4. In one aspect, a selected engineered analog of ANP consists essentially of the amino acid sequence shown in SEQ ID NO:4. In one aspect, a selected engineered analog of ANP consists of the amino acid sequence shown in SEQ ID NO:4. In one aspect, a selected engineered analog of ANP is CRRL 90. In one aspect, a selected engineered analog of ANP comprises the amino acid sequence shown in SEQ ID NO:5. In one aspect, a selected engineered analog of ANP consists essentially of the amino acid sequence shown in SEQ ID NO:5. In one aspect, a selected engineered analog of ANP consists of the amino acid sequence shown in SEQ ID NO:5. In one aspect, a selected engineered analog of ANP consists of CRRL 91. In one aspect, a selected engineered analog of ANP comprises the amino acid sequence shown in SEQ ID NO:6. In one aspect, a selected engineered analog of ANP consists essentially of the amino acid sequence shown in SEQ ID NO:6. In one aspect, a selected engineered analog of ANP consists of the amino acid sequence set forth in SEQ ID NO: 6. In one aspect, a selected engineered analog of ANP is CRRL 111. In one aspect, a selected engineered analog of ANP comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 7.In one aspect, a selected engineered analog of ANP comprises the amino acid sequence set forth in SEQ ID NO:7. In one aspect, a selected engineered analog of ANP consists essentially of the amino acid sequence set forth in SEQ ID NO:7. In one aspect, a selected engineered analog of ANP consists of the amino acid sequence set forth in SEQ ID NO:7. In one aspect, a selected engineered analog of ANP is CRRL 191.

[0047] Table 1. Exemplary engineered ANP polypeptides

[0048]

[0049] Without being bound by theory, selected engineered analogs of ANP with amino acid substitutions, additions and deletions may function similarly to one another. In one aspect, a selected engineered analog of ANP provided herein may comprise, or consist essentially of, or consist of an amino acid sequence shown in any one of SEQ ID NOs: 1-7, having zero, one, two or three amino acid substitutions within the hinge sequence of a sequence identifier (e.g., SEQ ID NO: 1).

[0050] In one aspect, a selected engineered analog of ANP provided herein may include one, two or three conservative substitutions. Without being bound by theory, conservative amino acid substitutions may be performed by selecting substitutions that have no significant difference in the effect on the structure of the peptide backbone in the (a) substitution region, the charge or hydrophobicity of the molecule at the target site, or the volume of the (c) side chain. For example, naturally occurring residues may be divided into several groups according to 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 affect chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine and phenylalanine). The non-limiting examples of useful conservative substitutions may include, but are not limited to, valine is substituted with alanine, lysine is substituted with arginine, glutamine is substituted with asparagine, glutamic acid is substituted with aspartic acid, serine is substituted with cysteine, asparagine is substituted with glutamine, aspartic acid is substituted with glutamic acid, proline is substituted with glycine, arginine is substituted with histidine, leucine is substituted with isoleucine, isoleucine is substituted with leucine, arginine is substituted with lysine, leucine is substituted with methionine, leucine is substituted with phenylalanine, glycine is substituted with proline, threonine is substituted with serine, serine is substituted with threonine, tyrosine is substituted with tryptophan, phenylalanine is substituted with tyrosine and / or leucine is substituted with valine. In one aspect, a selected engineered analog of ANP provided herein may include one, two or three conservative substitutions, in which one of the acidic amino acid residues is replaced by another acidic amino acid residue. In one aspect, a selected engineered analog of ANP provided herein may include one, two or three conservative substitutions, in which one of the basic amino acid residues is replaced by another basic amino acid residue.

[0051] In one aspect, a selected engineered analog of ANP provided herein can include one, two or three non-conservative substitutions. Without being bound by theory, non-conservative substitutions generally require that a member of a classification in the above-mentioned classification be exchanged for a member of another classification. Such production may be desirable for providing a large amount of or alternative embodiments of such compounds. Whether amino acid changes produce functional polypeptides can be easily determined by using, for example, the specific activity of peptide variants measured by methods disclosed herein.

[0052] In one aspect, a selected engineered analog of ANP provided herein, it includes the amino acid sequence shown in any one of SEQ ID NO:1-7, or is substantially composed of the amino acid sequence, or is composed of the amino acid sequence, is a polypeptide with zero, one, two or three amino acid additions in the hinge sequence of a sequence identifier (e.g., SEQ ID NO:1). In one aspect, a selected engineered analog of ANP provided herein, it includes the amino acid sequence shown in any one of SEQ ID NO:1-7, or is substantially composed of the amino acid sequence, or is composed of the amino acid sequence, is a polypeptide with zero, one, two or three amino acid deletions in the hinge sequence of a sequence identifier (e.g., SEQ ID NO:1). In one aspect, a selected engineered analog of ANP provided herein, it includes the amino acid sequence shown in any one of SEQ ID NO:1-7, or is substantially composed of the amino acid sequence, or is composed of the amino acid sequence, is a polypeptide with zero, one, two, three, four or five amino acid residues before the hinge sequence of a sequence identifier (e.g., SEQ ID NO:1). In one aspect, a selected engineered analog of ANP provided herein, which comprises, consists essentially of, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 1-7, is a polypeptide having zero, one, two, three, four or five amino acid residues after the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1).

[0053] In one aspect, a selected engineered analog of ANP provided herein, which comprises, consists essentially of, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 1-7, is a polypeptide having zero, one, two or three amino acid substitutions within the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1), and / or having zero, one, two or three amino acid additions within the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1), and / or having zero, one, two or three amino acid deletions within the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1), and / or having zero, one, two, three, four or five amino acid residues before the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1), and / or having zero, one, two, three, four or five amino acid residues after the hinge sequence of the sequence identifier (e.g., SEQ ID NO: 1).

[0054] Without being bound by theory, some amino acids of the selected engineered analogs of ANP provided herein are unmodified (e.g., amino acid substitutions, additions, deletions), to provide beneficial properties. For example, cysteine ​​residues are used to form disulfide bonds. In one aspect, the cysteine ​​residues of the selected engineered analogs of ANP provided herein are unsubstituted. In one aspect, the first four amino acids (e.g., amino acid TAPR) of SEQ ID NO:6 are unmodified (e.g., amino acid substitutions, additions, deletions). In one aspect, the first four amino acids (e.g., amino acid RALL) of SEQ ID NO:7 are unmodified (e.g., amino acid substitutions, additions, deletions). In one aspect, the first eight amino acids (e.g., amino acid RALLTAPR) of SEQ ID NO:7 are unmodified (e.g., amino acid substitutions, additions, deletions). Most naturally occurring amino acids are L-amino acids, and naturally occurring polypeptides are mainly composed of L-amino acids. D-amino acids are enantiomers of L-amino acids. In one aspect, a polypeptide provided herein can contain one or more D-amino acids.

[0055] In one aspect, a selected engineered analog of ANP provided herein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NO:1-7. The sequence identity percentage is calculated by dividing the number of paired positions by the total number of amino acids in the comparison by determining the number of paired positions. The paired position refers to the position where the same amino acid occurs in the same position in the amino acid sequence of the comparison. The sequence identity percentage of any nucleotide sequence can also be determined.

[0056] The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number can be determined by methods known in the art, for example, as disclosed in WO / 2018 / 089601, which is incorporated herein by reference in its entirety.

[0057] The selected engineered approaches analogs of ANP provided herein can be produced using any suitable method, including but not limited to solution phase synthesis (SPS), solid phase peptide synthesis (SPPS) and expressed protein connection (EPL). The selected engineered approaches analogs of ANP provided herein can be produced using manual techniques or automation techniques (for example, using Applied BioSystems (Foster City, California) peptide synthesizer or Biosearch Inc (San Rafael, California) automatic peptide synthesizer). The selected engineered approaches analogs of ANP provided herein can also be recombinantly produced.

[0058] Due to the disulfide bonds between the underlined cysteine ​​residues in the sequences shown in Table 1, the selected engineered analogs of ANP provided herein are generally cyclic. The disulfide bonds can be formed by any method known to those skilled in the art, including but not limited to oxidation (enzyme-mediated or by reaction of low molecular weight oxidants) or reagents (e.g., dicarbonyl and related cross-linking agents, such as glutaraldehyde). The disulfide bonds between the cysteine ​​residues can also be introduced by using KCN mild oxidation linear polypeptides, as taught in, for example, U.S. Patent No. 4,757,048.

[0059] The term "substantially pure" as used herein, relating to a polypeptide, means that the polypeptide is substantially free of other polypeptides, lipids, carbohydrates and nucleic acids that are naturally associated with it. Therefore, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and has a purity of at least 60%. A substantially pure polypeptide can be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% purity. Typically, a substantially pure polypeptide will produce a single major band on a non-reducing polyacrylamide gel. In one aspect, a substantially pure polypeptide provided herein is a polypeptide that is synthesized to have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% purity.

[0060] Any method can be used to obtain substantially pure polypeptides. 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 for obtaining substantially pure polypeptides. For example, tissues from wild-type or transgenic animals can be used as source materials. In addition, tissue culture cells engineered to overexpress specific polypeptides can be used to obtain substantially pure polypeptides. Solid phase peptide synthesis can also be used to obtain substantially pure polypeptides. In addition, polypeptides can be engineered to contain amino acid sequences that allow the polypeptide to be captured on an affinity matrix. For example, such as c-myc, hemagglutinin, polyhistidine or FLAG TM Tags such as Kodak tags can be used to aid in polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl terminus or the amino terminus or in between. Other fusions that can be used include enzymes that aid in detection of the polypeptide, such as alkaline phosphatase.

[0061] Not bound by theory, the selected engineered analogs of ANP are processed by propeptides. The selected engineered analogs of ANP provided herein can be processed in part by any propeptides. In one aspect, the selected engineered analogs of ANP can be made into propeptides, which are further processed into the selected engineered analogs of ANP provided herein naturally or non-naturally.

[0062] Without being bound by theory, the engineered analogs of ANP provided herein can play a role through one or more guanylate cyclase receptors, and natural natriuretic polypeptides also play a role through the one or more guanylate cyclase receptors. For example, polypeptides provided herein can be combined with GC-A (also referred to as NPR-A) receptors and play a role through them, and ANP, BNP and DNP also play a role through GC-A receptors, although they can also play a role through GC-B (also referred to as NPR-B) receptors, and CNP also plays a role through GC-B receptors. In one aspect, the engineered analogs of ANP provided herein can be combined with guanylate cyclase-A (GC-A) receptors. In one aspect, the engineered analogs of ANP provided herein can be combined with guanylate cyclase-A (GC-A) receptors. In one aspect, the engineered analogs of ANP provided herein can be combined with more than one guanylate cyclase receptor and play a role through them, and the more than one guanylate cyclase receptors include GC-A and GB-B. The method for assessing the function of a specific engineered analog of ANP, which receptor is involved, is known in the art. For example, glomeruli containing GC-A and GC-B can be isolated (e.g., from an experimental animal such as a dog) and incubated with an engineered analog of ANP (e.g., any one of SEQ ID NOs: 3-7), and cGMP levels can be measured. Glomeruli can be pretreated with antagonists of GC-A or GC-B to determine whether cGMP production stimulated by natriuretic polypeptides through one or the other receptor can be attenuated.

[0063] The biological activity of the selected engineered analogs of ANP provided herein can be measured using any of a variety of assays, including those described herein. For example, the activity of the engineered analogs of ANP provided can be determined in vitro by testing the impact of cGMP produced in cultured cells (e.g., cardiac fibroblasts, aortic endothelial cells, or glomerular cells cultured). Cells can be exposed to the engineered analogs of ANP provided herein (e.g., 10 -10 Up to 10 -4 M's CRRL 191), and samples can be assayed to assess the effect on cGMP production. cGMP production can be detected and measured using, for example, a competitive RIA cGMP kit (Perkin-Elmer, Boston, MA) or a cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY).

[0064] The selected engineered analogs of ANPs provided herein can be any suitable length (eg, can include any number of amino acids). For example, selected engineered analogs of ANPs provided herein can be from about 20 amino acids long to about 100 amino acids long (e.g., from about 20 to about 90 amino acids, from about 20 to about 80 amino acids, from about 20 to about 70 amino acids, from about 20 to about 60 amino acids, from about 20 to about 55 amino acids, from about 20 to about 52 amino acids, from about 20 to about 50 amino acids, from about 20 to about 100 amino acids, from about 30 to about 100 amino acids, from about 40 to about 100 amino acids, from about 50 to about 100 amino acids, from about 60 to about 100 amino acids, from about 70 to about 100 amino acids, from about 80 to about 100 amino acids, from about 90 to about 100 amino acids, from about 20 to about 80 amino acids, from about 20 to about 70 amino acids, from about 30 to about 60 amino acids, from about 35 to about 55 amino acids, from about 38 to about 52 amino acids, from about 40 to about 50 amino acids, or from about 45 to about 50 amino acids). In one aspect, the length of the selected engineered analogs of the ANP provided herein can be about 46 to about 50 amino acid sequences.For example, the length of the selected engineered analogs of the ANP provided herein and with the GC-A binding characteristic can be about 38 to about 42 amino acid sequences.For example, the length of the selected engineered analogs of the ANP provided herein and with the GC-A binding characteristic can be about 42 to about 46 amino acid sequences.For example, the length of the selected engineered analogs of the ANP provided herein and with the GC-A binding characteristic can be about 46 to about 50 amino acid sequences.

[0065] The selected engineered analogs of ANP provided herein can include one or more sequences (for example, ANP, BNP, CNP, urodilator and DNP) present in the polypeptide with natriuretic polypeptide activity. In one aspect, the selected engineered analogs of ANP can include the sequence that non-naturally occurs. In one aspect, the selected engineered analogs of ANP can include the sequence that naturally occurs and non-naturally occurs. In one aspect, the selected engineered analogs of ANP can include the sequence that is present in any species, including but not limited to mankind, horse, pig, goat, cattle, dog, cat, rat or snake.

[0066] Nucleic Acids

[0067] The present disclosure also provides nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., one or more polypeptides comprising, substantially consisting of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7). For example, the present disclosure provides vectors (e.g., plasmids and viral vectors) comprising nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., one or more polypeptides comprising, substantially consisting of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7) in a manner such that the polypeptides can be expressed in a cell.

[0068] When the vector comprising a nucleic acid encoding one or more selected engineered analogs of ANPs provided herein (e.g., one or more polypeptides comprising, substantially consisting of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7) is a non-viral vector, any suitable non-viral vector may be used. In one aspect, the non-viral vector may be an expression plasmid (e.g., a cDNA expression vector).

[0069] When the vector of the nucleic acid comprising the selected engineered analogs (e.g., one or more polypeptides comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence) encoding one or more ANPs provided herein is a viral vector, any suitable viral vector can be used. In one aspect, the viral vector can be derived from a positive-strand virus or a negative-strand virus. In one aspect, the viral vector can be derived from a virus with a single-stranded genome or a virus with a double-stranded genome. In one aspect, the viral vector can be derived from a virus with a DNA genome or an RNA genome. In one aspect, the viral vector can be a chimeric viral vector. In one aspect, the viral vector can infect dividing cells. In one aspect, the viral vector can infect non-dividing cells. Examples of viral-based vectors that can include nucleic acids encoding selected engineered analogs of ANP provided herein (e.g., a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) include, but are not limited to, viral-based vectors based on adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, measles virus, vesicular stomatitis virus, and vaccinia virus.

[0070] In addition to the nucleic acid of the selected engineered analogs (e.g., one or more polypeptides comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence) of encoding one or more ANPs provided herein, a vector (e.g., a plasmid or viral vector) can contain one or more regulatory elements operably linked to the nucleic acid of the selected engineered analogs of encoding one or more ANPs provided herein. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements for regulating nucleic acid expression (e.g., transcription or translation). The selection of regulatory elements that can be included in the vector depends on a variety of factors, including but not limited to inducibility, targeting, and required expression levels. For example, a promoter can be included in the vector to promote the transcription of the nucleic acid of the selected engineered analogs (e.g., comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence) of encoding one or more ANPs provided herein. In one aspect, the promoter can be a naturally occurring promoter or a recombinant promoter. In one aspect, the promoter can be a constitutive or inducible promoter (for example, in the presence of tetracycline), and can affect the expression of the nucleic acid of the coded polypeptide in a broad or cell / tissue-specific manner. The example of the promoter that can be used to drive the selected engineered analogs of one or more ANPs provided herein (for example, comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, being substantially composed of the amino acid sequence or being composed of the amino acid sequence) expression in the cell includes but is not limited to, CMV promoter, EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, TRE promoter and CAG promoter. As used herein, "operably connected" refers to the positioning of the regulatory element in the carrier relative to the nucleic acid of the coded polypeptide so as to allow or promote the expression of the coded polypeptide. For example, the carrier can contain the nucleic acid of the selected engineered analogs of the coding one or more ANPs provided herein. In this case, the promoter is operably linked to the nucleic acid of the selected engineered analogs of the coding one or more ANPs provided herein, so that the expression of the selected engineered analogs (class) of the ANPs in the driving cell is made.

[0071] Combinations of polypeptides and nucleic acids, compositions and preparations thereof

[0072] The disclosure also provides compositions, it comprises one or more (for example, one, two, three, four, five, six or seven) selected engineered analogs and / or nucleic acids of ANP provided herein, the nucleic acid encoding one or more (for example, one, two, three, four, five, six or seven) selected engineered analogs of ANP provided herein. In one aspect, a composition provided herein comprises one or more selected engineered analogs of ANP shown in Table 1, is substantially composed of these analogs or is composed of these analogs. In one aspect, a composition provided herein comprises one or more nucleic acids, the nucleic acid encoding comprises the amino acid sequence shown in any one of SEQ ID NO:3-7, is substantially composed of the amino acid sequence, or is composed of the amino acid sequence. In one aspect, a composition provided herein comprises one or more selected engineered analogs of ANP shown in Table 1 and one or more nucleic acids, the one or more nucleic acid encoding comprises the amino acid sequence shown in any one of SEQ ID NO:3-7, is substantially composed of the amino acid sequence, or is composed of the amino acid sequence.

[0073] In one aspect, a composition provided herein comprises at least two (e.g., two, three, four, five, six or seven) selected engineered analogs and / or nucleic acids of the ANP provided herein, which encode at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) selected engineered analogs of the ANP provided herein. In one aspect, a composition provided herein comprises at least two selected engineered analogs of the ANP shown in Table 1, is substantially composed of these analogs or is composed of these analogs. In one aspect, a composition provided herein comprises at least two nucleic acids, which encode a polypeptide consisting of, substantially composed of or composed of the amino acid sequence shown in any one of SEQ ID NO: 3-7. In one aspect, a composition provided herein comprises at least two selected engineered analogs of ANPs shown in Table 1 and at least two nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3-7.

[0074] Any appropriate method can be used to prepare compositions provided herein (for example, including the selected engineered analogs of one or more ANPs provided herein and / or the compositions of the nucleic acids encoding the selected engineered analogs of one or more ANPs provided herein). In one aspect, the selected engineered analogs of one or more ANPs provided herein can be combined with a pharmaceutically acceptable carrier and / or a pharmaceutical excipient. In one aspect, the nucleic acids encoding the polypeptides comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence can be combined with a pharmaceutically acceptable carrier and / or a pharmaceutical excipient. In one aspect, the selected engineered analogs of one or more ANPs provided herein and one or more encodings comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence can be combined with a pharmaceutically acceptable carrier and / or a pharmaceutical excipient. The term "pharmaceutically acceptable" refers to a compound that is usually nontoxic, inert, and / or physiologically compatible. The term "pharmaceutical excipient" includes materials such as carriers, pH adjusters and buffers, tonicity adjusters, wetting agents, colorants and preservatives.

[0075] In one aspect, provided herein is a composition (e.g., a composition comprising a selected engineered analog of one or more ANPs provided herein and / or a composition of a nucleic acid encoding a selected engineered analog of one or more ANPs provided herein) can be formulated with at least one other combination selected from a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB). In one aspect, the composition comprises a selected engineered analog of ANP and furosemide.

[0076] Treatment and prevention methods

[0077] Not bound by theory, the selected engineered analogs of ANP provided herein can be used for treating, preventing cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) and / or improving the symptoms of these diseases. In one aspect, the selected engineered analogs of ANP herein can be used for treating cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH). In one aspect, the selected engineered analogs of ANP provided herein can be used for preventing cardiovascular disease (for example, hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (for example, acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (for example, diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH). In one aspect, the selected engineered analogs of ANP provided herein can be used for improving cardiovascular disease (for example, hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (for example, acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (for example, diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) symptom. In one aspect, selected engineered analogs of ANP provided herein can be used to treat, prevent, and / or ameliorate symptoms of hypertension (eg, resistant hypertension) or heart failure (HF).The presence or extent of disease may be assessed using methods known in the art, including, but not limited to, general clinical examination to assess blood pressure, heart rate, rhythm, arterial oxygen, and hemoglobin levels; echocardiography to measure ejection fraction, LV and left atrial (LA) diameters, LV wall motion, LV filling pressures, and diastolic function by pulsed and tissue Doppler; measurement of cardiac output, pulmonary capillary wedge pressure, pulmonary artery pressure, right ventricular pressure, right atrial pressure, and systemic and pulmonary vascular resistance using a Swan-Ganz catheter; and assessment of renal function by determination of glomerular filtration rate, serum creatinine, and blood urea nitrogen. ;And the 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 gellanase-associated lipid transporter (NGAL), N-acetyl-β-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 one aspect, a selected engineered analog of ANP provided herein can alleviate one or more symptoms of acute or chronic HF, including edema, shortness of breath and fatigue. In order to determine the efficacy level of the selected engineered analog of ANP provided herein to improve HF symptoms, one or more of these parameters can be evaluated using methods such as known in the art (e.g., before and after treatment with ANP engineered analogs). Favorable clinical responses include cardiac unloading (i.e., reduced stress on the heart), increased glomerular filtration rate (GFR), decreased PRA, decreased angiotensin II levels, decreased proliferation of cardiac fibroblasts, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicating reduced fibrosis and hypertrophy), decreased PWCP (an indirect measure of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased aldosterone levels (indicating an antifibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end-systolic diameter.

[0078] Without being bound by theory, the selected engineered analogs of ANP provided herein can reduce blood pressure, increase natriuresis, cause arterial vasodilation, inhibit renin and aldosterone, reduce apoptosis, reduce hypertrophy, increase dilatation, induce angiogenesis, increase lipolysis and cause browning of white adipocytes. In one aspect, the selected engineered analogs of this ANP can reduce the blood pressure of mammals. In one aspect, the selected engineered analogs of ANP provided herein can increase the natriuresis of mammals. In one aspect, the selected engineered analogs of ANP provided herein can reduce the apoptosis of mammals. In one aspect, the selected engineered analogs of ANP provided herein can cause arterial vasodilation of mammals. In one aspect, the selected engineered analogs of ANP provided herein can inhibit renin and aldosterone in mammals. In one aspect, the selected engineered analogs of ANP provided herein can increase the dilatation of mammals. In one aspect, the selected engineered analogs of ANP provided herein can induce angiogenesis in mammals. In one aspect, selected engineered analogs of ANPs provided herein can increase lipolysis in mammals. In one aspect, selected engineered analogs of ANPs provided herein can cause browning of white adipocytes in mammals.

[0079] The activity of selected engineered analogs of ANP can also be assessed by, for example, testing the effects of various factors in mammals (e.g., rodents, pigs, sheep, dogs or humans), such as plasma cGMP levels, urinary cGMP excretion, net renal production 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 one aspect, such parameters can be assessed after inducing heart failure (e.g., by rapid right ventricular pacing) or hypertension.

[0080] In one aspect, selected engineered analogs of one or more ANPs provided herein (e.g., polypeptides comprising, essentially consisting of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) and / or nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., nucleic acids encoding selected engineered analogs of one or more ANPs provided herein, said engineered analogs comprising, essentially consisting of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) can be administered to a mammal (e.g., a human) to treat, prevent, and / or alleviate the symptoms of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction) in the mammal. In one aspect, selected engineered analogs of one or more ANPs provided herein (e.g., polypeptides comprising, or consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) and / or nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., nucleic acids encoding selected engineered analogs of one or more ANPs provided herein, said engineered analogs comprising, or consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) can be administered to a mammal (e.g., a human) to treat, prevent, and / or alleviate the symptoms of cardiorenal diseases (e.g., acute renal injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) in the mammal. In one aspect, selected engineered analogs of one or more ANPs provided herein (e.g., polypeptides comprising, or consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) and / or nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., nucleic acids encoding selected engineered analogs of one or more ANPs provided herein, said engineered analogs comprising, or consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7) can be administered to a mammal (e.g., a human) to treat, prevent, and / or alleviate the symptoms of metabolic diseases (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in the mammal.

[0081] As used herein, the term "treatment" is a method for obtaining a beneficial or desired clinical outcome. In one aspect, the term "treatment" means administering a selected engineered analog of one or more ANPs disclosed herein, which partially or completely alleviates, improves, alleviates, inhibits one or more symptoms, features and causes of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH), delays its onset, reduces its severity and / or reduces its incidence. The term "treatment" includes the administration of one or more selected engineered analogs of ANP disclosed herein to prevent or delay the onset of symptoms, complications or biochemical indicators of cardiovascular disease, cardiorenal disease or metabolic disease, alleviate the symptoms of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH), or prevent or inhibit the further development of these diseases. Treatment can be prophylactic (to prevent or delay the onset of, or prevent the manifestation of clinical or subclinical symptoms of, cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH)) or therapeutically suppress or alleviate symptoms after the manifestation of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH).

[0082] Any suitable mammal can be administered with a selected engineered analog of one or more ANPs provided herein (e.g., a selected engineered analog of one or more ANPs, comprising, consisting essentially of, or consisting of an amino acid sequence as shown in any of SEQ ID NOs: 3-7) and / or a nucleic acid encoding a selected engineered analog of one or more ANPs provided herein (e.g., a nucleic acid encoding a selected engineered analog of one or more ANPs, the engineered analogs comprising, consisting essentially of, or consisting of an amino acid sequence as shown in any of SEQ ID NOs: 3-7) to treat the mammal. Examples of mammals to which a selected engineered analog of one or more ANPs provided herein (and / or a nucleic acid encoding a selected engineered analog of one or more ANPs provided herein) can be administered include, but are not limited to, humans, non-human primates (e.g., monkeys or apes), horses, dogs, cats, bovines, pigs, sheep, mice, rats, hamsters, bats, foxes, goats, minks, and deer. In one aspect, it is possible to be identified as suffering from cardiovascular disease (for example, hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (for example, acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (for example, diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) or the mankind at risk of developing these diseases, use the selected engineered analogs of one or more ANPs provided herein to treat this mankind. In one aspect, it is possible to be identified as suffering from cardiovascular disease (for example, hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (for example, acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (for example, diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) or the mankind at risk of developing these diseases, use the nucleic acid of the selected engineered analogs of encoding one or more ANPs provided herein to treat this mankind. In one aspect, one or more selected engineered analogs of ANP provided herein and nucleic acids encoding one or more selected engineered analogs of ANP provided herein can be administered to a human identified as having a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) or being at risk of developing these diseases to treat the human.

[0083] In one aspect, the efficacy of treatment is measured by clinical parameters such as the alleviation of symptoms. Symptoms of cardiovascular disease, cardiorenal disease or metabolic disease include, but are not limited to, edema, shortness of breath and fatigue, as well as cardiac unloading (i.e., reduced cardiac pressure), increased glomerular filtration rate (GFR), reduced PRA, reduced angiotensin II levels, reduced cardiac fibroblast proliferation, reduced left ventricular (LV) hypertrophy, reduced LV mass (indicating reduced fibrosis and hypertrophy), reduced PWCP (indirect measurement of left atrial pressure), reduced right atrial pressure, reduced mean arterial pressure, reduced aldosterone levels (indicating an anti-fibrotic effect), reduced ventricular fibrosis, increased ejection fraction and reduced LV end-systolic diameter.

[0084] In one aspect, the materials and methods described herein can be used to delay the onset of one or more symptoms of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal at risk for developing cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In one aspect, the materials and methods described herein can be used to reduce, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the duration and / or severity of one or more symptoms of a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) present in a mammal with a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH). See, e.g., J Jamison et al., Harrison's Principles of Medicine (2018, pp.th version).

[0085] When applying the composition provided herein to a mammal (e.g., the mankind), any suitable route of administration can be used. In one aspect, a composition provided herein can be applied to a mammal (e.g., the mankind) intramuscularly (e.g., via intramuscular injection), subcutaneously (e.g., via subcutaneous injection), orally, intranasally, percutaneously or via inhalation. In one aspect, a route of administration and / or mode of a composition provided herein can be adjusted for the treated mammal. In one aspect, different doses of the composition provided herein are applied to a mammal by the same route of administration. In one aspect, different doses of the composition provided herein are applied to a mammal by different routes of administration. For example, the first dose of the selected engineered analogs of one or more ANPs provided herein (e.g., the selected engineered analogs of one or more ANPs, comprising the amino acid sequence shown in any one of SEQ ID NO:3-7, being substantially composed of the amino acid sequence or being composed of the amino acid sequence) can be administered subcutaneously, and the second dose is administered intranasally.

[0086] In one aspect, the effective amount of the selected engineered analogs of one or more ANPs provided herein (e.g., one or more selected engineered analogs of ANPs, comprising, consisting essentially of, or consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-7) can be an amount that alleviates symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) without producing significant toxicity to the mammal. In one aspect, the effective amount of the nucleic acids encoding the selected engineered analogs of one or more ANPs provided herein (e.g., nucleic acids encoding the selected engineered analogs of one or more ANPs, the engineered analogs comprising, consisting essentially of, or consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-7) can be an amount that alleviates symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) without producing significant toxicity to the mammal. In one aspect, the selected engineered analogs of one or more ANPs provided herein (e.g., selected engineered analogs of one or more ANPs, comprising, consisting essentially of, or consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-7) and nucleic acids encoding selected engineered analogs of one or more ANPs provided herein (e.g., nucleic acids encoding selected engineered analogs of one or more ANPs, the engineered analogs comprising, consisting essentially of, or consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-7) effective amount can be an amount that alleviates symptoms (e.g., shortness of breath, systolic pressure, diastolic pressure) in mammals (e.g., humans) without producing significant toxicity to the mammal. The effective amount can remain constant, or can be adjusted with a sliding ratio or variable dose according to the response of the mammal to treatment. Without being bound by theory, various factors can affect the actual effective amount for a particular application. The dosage can be adjusted by a technician or a treating physician.

[0087] In one aspect, the selected engineered analogs of one or more ANPs provided herein (e.g., the selected engineered analogs of one or more ANPs, which include the amino acid sequence shown in any of SEQ ID NO: 3-7, are substantially composed of the amino acid sequence, or are composed of the amino acid sequence) effective amount is about 1.0 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 or 100 mg. In one aspect, the selected engineered analogs of one or more ANPs provided herein (e.g., the selected engineered analogs of one or more ANPs, which include the amino acid sequence shown in any of SEQ ID NO: 3-7, are substantially composed of the amino acid sequence, or are composed of the amino acid sequence) effective amount is about 1 mg-100 mg, about 1 mg-80 mg, about 0.5 mg-80 mg, about 0.5 mg-60 mg or about 1 mg-50 mg. In one aspect, the selected engineered analogs of one or more ANPs provided herein (e.g., selected engineered analogs of one or more ANPs, comprising the amino acid sequence shown in any of SEQ ID NOs: 3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence) oral dosage is about 1 mg-100 mg, about 1 mg-80 mg, about 0.5 mg-80 mg, about 0.5 mg-60 mg, or about 1 mg-50 mg. In one aspect, the selected engineered analogs of one or more ANPs provided herein (e.g., selected engineered analogs of one or more ANPs, comprising the amino acid sequence shown in any of SEQ ID NOs: 3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence) effective amount is 1 mg-50 mg by oral administration. In one aspect, the effective amount of one or more selected engineered analogs of ANP provided herein (e.g., one or more selected engineered analogs of ANP comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 3-7) administered by subcutaneous administration is about 0.01 μg / kg-50 μg / kg, about 0.1 μg / kg-40 μg / kg, about 0.1 μg / kg-30 μg / kg, about 0.05 μg / kg-40 μg / kg, or about 0.05 μg / kg-30 μg / kg.

[0088] In one aspect, the effective frequency of administration of one or more selected engineered analogs of ANPs provided herein (e.g., one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) and / or nucleic acids encoding one or more selected engineered analogs of ANPs provided herein (e.g., nucleic acids encoding one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) can be a frequency that reduces symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) without causing significant toxicity to the mammal. In one aspect, the effective frequency of the selected engineered analogs of one or more ANPs provided herein (and / or the selected engineered analogs of encoding one or more ANPs provided herein) can be approximately once every two hours, approximately once every three hours, approximately once every four hours, approximately once every five hours, approximately once every six hours, approximately once every seven hours, approximately once every eight hours, approximately once every nine hours, approximately once every ten hours, approximately once every eleven hours, approximately once every twelve hours, approximately once every day, approximately once every two days, approximately once every three days, approximately once every four days, approximately once every five days, approximately once every six days, approximately once a week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, approximately once a month, approximately once every two months, approximately once every three months, approximately once every four months, approximately once every five months, approximately once every six months, or approximately once a year. Not bound by theory, various factors can affect the actual effective amount for specific applications. Frequency can be adjusted by technical personnel or treating physicians.

[0089] In one aspect, the effective administration duration of one or more selected engineered analogs of ANPs provided herein (e.g., one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) and / or nucleic acids encoding one or more selected engineered analogs of ANPs provided herein (e.g., nucleic acids encoding one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) can be a duration that reduces symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) without causing significant toxicity to the mammal. In one aspect, the effective duration of the selected engineered analogs of one or more ANPs provided herein (and / or the nucleic acids encoding the selected engineered analogs of one or more ANPs provided herein) can be about one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, five years, ten years or as required. Without being bound by theory, various factors can affect the actual effective amount for a particular application. The duration can be adjusted by a technician or a treating physician.

[0090] The present disclosure also provides kits comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) selected engineered analogs of ANPs provided herein (e.g., one or more substantially pure selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) and / or nucleic acids encoding one or more selected engineered analogs of ANPs provided herein (e.g., nucleic acids encoding one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7). In one aspect, a kit provided herein comprises a selected engineered analog of one or more ANPs provided herein (e.g., a selected engineered analog of one or more substantially pure ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7). In one aspect, a kit provided herein comprises a nucleic acid encoding a selected engineered analog of one or more ANPs provided herein (e.g., a nucleic acid encoding a selected engineered analog of one or more ANPs, the engineered analog comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7). In one aspect, a kit provided herein comprises a selected engineered analog of one or more ANPs provided herein (e.g., one or more substantially pure selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7) and a nucleic acid encoding one or more selected engineered analogs of ANPs provided herein (e.g., a nucleic acid encoding one or more selected engineered analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 3-7).

[0091] The examples set out herein illustrate one or more aspects of the present disclosure but should not be construed as limiting the scope of the present disclosure in any way.

[0092] Examples

[0093] Example 1: Synthesis of activator of CRRL 191:GC-A

[0094] CRRL 191 is a novel 48 amino acid (AA) designer natriuretic peptide that activates GC-A ( Figure 1B ). CRRL 191 was synthesized by Bachem Ltd (Torrance, CA) using solid-state peptide synthesis (see Table 2). After synthesis, the peptide was purified by high performance liquid chromatography (HPLC) and oxidized to form a ring between cysteine ​​amino acid residues 15 and 31. The structure is shown in Figure 1B As shown, it was confirmed by MALDI mass spectrometry and the purity was determined to be 97.9% using HPLC analysis. The average mass of cyclized CRRL 191 was 5557.27 Da.

[0095] Table 2: Analysis results of peptide synthesis

[0096]

[0097]

[0098] Example 2: Measurement of GC-A activation and cyclic GMP production in HEK293 cells

[0099] HEK293 cells stably transfected with human GC-A (cDNA clone from Origene, Rockville, Maryland) were developed using Lipofectami (Invitrogen, Grand Island, NY). Overexpression of the receptor was verified by immunofluorescence and Western blotting. HEK293 (HEK / GCA) cells transfected with GC-A were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 U / ml streptomycin, and 250 μg / ml G418.

[0100] Two in vitro cGMP assays were performed. Briefly, HEK / GCA cells were seeded into 48-well plates and cultured overnight to 80-90% confluence. The treatment buffer used in all experiments included Hank's balanced salt solution (HBSS), 0.1% bovine serum albumin (BSA), 2mM HEPES, and 0.5mM 3-isobutyl-1-methylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor) (Sigma, St. Louis, MO).

[0101] In the first experiment, different concentrations (10 -14 Up to 10 -6Cells were stimulated with ANP or CRRL191 for 10 minutes. After treatment, all cells were washed once with phosphate buffered saline (PBS) and lysed with 0.1 M HCl. Intracellular cGMP in lysates was measured using a commercial cGMP ELISA kit (Enzo Life Sciences, Farmingdale, New York) according to the manufacturer's instructions.

[0102] As shown in Table 3, at 10 min stimulation, the EC of CRRL191 was significantly higher than that of ANP. 50 Shows greater effectiveness.

[0103] Table 3: EC50 of CRRL191 or ANP in HEK293 GC-A cells after 10 min stimulation.

[0104]

[0105] In the second experiment, ANP or CRRL 191 (concentration of 10 -8 5M) was added to the treatment buffer and the cells were incubated for 6 hours. After treatment, all cells were washed once with phosphate buffered saline (PBS) and lysed with 0.1M HCl. Intracellular cGMP in the lysate was measured using a commercial cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions.

[0106] like Figure 2 As shown, both ANP and CRRL 191 activated GC-A receptors and increased cGMP production at the same rate for up to about 30 minutes. After 30 minutes, GC-A receptor activation by CRRL 191 continued to increase for at least 6 hours, as evidenced by increased cGMP production, while GC-A receptor activation by ANP entered a plateau and decreased.

[0107] Example 3: In vitro enkephalinase degradation assay

[0108] In vitro degradation of ANP and CRRL191 peptides by recombinant human neprilysin (NEP) (R&D Systems, Minneapolis, MN) was measured by cGMP production in HEK / GCA cells. Briefly, 5 μl of 2×10 -5The peptides were incubated with 5 μl (10 ng / μl) recombinant NEP in 90 μl Tris / 0.1% BSA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% BSA) at 37°C for varying lengths of incubation from 15 minutes to 24 hours. The peptides at the 0 minute time point were incubated in the absence of NEP (buffer only). After the desired incubation time (0 minutes to 24 hours), 100 μl 0.5 N perchloric acid was added to the reaction solution to inactivate NEP and stop degradation, and 20 μl 2.5 N NaOH was added to neutralize the solution. An aliquot of the neutralized reaction was added to HEK / GCA cells and the undegraded peptide (diluted to a final concentration of 5.5X 10 -8 M) Residual capacity to produce cGMP. The degradation of ANP and CRRL 191 was calculated by the loss of cGMP production capacity in HEK / GCA cells compared to the basal cGMP production capacity at 0 min.

[0109] like Figure 3 As shown, CRRL 191 is highly resistant to degradation by enkephalinase compared to ANP as measured by this in vitro assay. After 30 minutes of enkephalinase degradation, CRRL 191 retained approximately 90% of its original bioactivity, while ANP retained less than 5% of its original bioactivity. After 6 hours of enkephalinase degradation, CRRL 191 retained 84% of its original bioactivity, demonstrating significant resistance and stability compared to ANP.

[0110] Example 4: Human cardiomyocyte (HCM) cGMP analysis

[0111] Human cardiomyocytes (HCM) were purchased from ScienCell (Catalog #6200) and maintained in cardiomyocyte culture medium (Catalog #6101, ScienCell) containing 10% FBS. In vitro cGMP assays were performed. Briefly, HCM cells were seeded in 6-well plates and cultured overnight to 80-90% confluence. The treatment buffer used in all experiments included Hank's balanced salt solution (HBSS), 0.1% BSA, 2mM HEPES, and 0.5mM 3-isobutyl-1-methylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor) (Sigma, St. Louis, MO). For test samples, HCM were incubated in the presence of ANP or CRRL 191 (at a concentration of 10 -12 M, 10 -11 M, 10 -10 M, 10-9 M, 10 -8 M, 10 -7 M and 10 -6 The cells were incubated in 5% paraformaldehyde (2% HCl) and 4% paraformaldehyde (2% HCl) for 10 minutes. For the negative control, HCM was incubated only in the treatment buffer (vehicle). After 10 minutes of treatment, all cells were washed once with phosphate buffered saline (PBS), lysed with 0.1 M HCl, and sonicated for 10 minutes. Intracellular cGMP in the lysate was measured using a commercial cGMP ELISA kit (Enzo Life Sciences, Farmingdale, New York) according to the manufacturer's instructions.

[0112] like Figure 4 As shown, CRRL 191 induces higher levels of cGMP production in human primary cells. -12 M to 10 -11 M, CRRL 191 and ANP induced similar levels of cGMP production. -10 M to 10 -6 M, CRRL 191 induced more cGMP production than ANP.

[0113] like Figure 5 As shown, CRRL 191 induced higher levels of cGMP production in human primary cells compared with two other GC-A activators, MANP and CRRL 101. -9 M to 10 -6 M, CRRL191 induced higher levels of cGMP production than MANP or CRRL101.

[0114] Example 5: Real-time apoptosis analysis of human cardiomyocytes (HCM)

[0115] HCM cells were seeded in 96-well plates at approximately 80% confluence. The cells were treated with different concentrations of staurosporine (SP) to induce apoptosis. Based on the dose-response curve, 0.05 μM of SP was selected to induce apoptosis of HCM cells.

[0116] On the day of the experiment, HCM was incubated with cell growth medium containing 0.05 μM SP, different concentrations of ANP or CRRL 191 and 5 μmol / L IncuCyte Caspase-3 / 7Green Reagent (Essen Bioscience, Ann Arbor, Michigan). The plate was then transferred to the time-lapse real-time imaging system IncuCyte S3 (Essen BioScience, Ann Arbor, Michigan), and apoptosis was monitored every 2 hours for a total of 90 hours. All treatment groups were performed in quintuplicate and repeated twice. Images were collected using the IncuCyte S3 live cell analysis system software (Essen Bioscience, Ann Arbor, Michigan), and the data of the green object area of ​​the apoptotic body in each well were analyzed.

[0117] like Figure 6 As shown, 10 -10 and 10 -8 M dose of CRRL 191 reduced SP-induced cardiomyocyte apoptosis, while 10 -6 The M dose of CRRL 191 had the greatest effect on reducing SP-induced cardiomyocyte apoptosis.

[0118] Example 6: GC-A binding studies

[0119] Surface plasmon resonance (SPR) measurements were performed on a BI-4500SPR instrument (Biosensing Instrument Inc., Tempe, Arizona) at 25°C. According to the instrument manual, 400 μM nickel sulfate in deironized water was used as a linker to fix the extracellular domain of GC-A recombinant protein (MyBioSource, Inc., San Diego, California) with a C-terminal His tag to a Ni-NTA sensor chip (Biosensing Instrument Inc., Tempe, Arizona). Then, 40 μg / ml recombinant GC-A was fixed on a Ni-NTA sensor chip. After washing the chip with buffer (150 mM NaCl, 50 μM EDTA, pH 7.4, 0.1% DMSO), 100 μL of serial dilutions of CRRL 191 or ANP (0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM) were injected at a rate of 60 μL / min and allowed to dissociate for 60 seconds. Data were collected as sensorgrams. Binding kinetics were calculated from the sensorgrams using the BI data analysis program (Biosensing Instrument, Tempe, AZ).

[0120] As shown in the binding curve, CRRL 191( Figure 7C ) showed a higher Fig. 7A ) and MANP( Figure 7B ) Stronger binding to GC-A receptor. K of CRRL 191 D The value was 128 pM, while the K D The values ​​were 280 pM and 233 pM, respectively. These data indicate that CRRL 191 binds to human GC-A better than ANP or MANP.

[0121] Example 7: In vivo experiments in rats

[0122] All rat experiments were performed in accordance with the Animal Welfare Act, and the protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). A total of 19 spontaneously hypertensive rats (SHR, male, 250-300 g) were randomly assigned to receive one of the following infusions: (i) vehicle (0.9% saline, n = 3), (ii) low-dose ANP ((100 pmol / kg / min, n = 4), (iii) high-dose ANP (300 pmol / kg / min, n = 4), (iv) low-dose CRRL 191 (100 pmol / kg / min, n = 4), and (v) high-dose CRRL 191 (300 pmol / kg / min, n = 4). Rats were included only if they were deemed healthy by the institutional veterinary department. The investigator performing the in vivo studies was aware of the treatments, but biochemical analyses were performed by another investigator who was blinded to the treatments.

[0123] On the day of the experiment, SHR rats were anesthetized with sodium thiobutyrate (inactin, 120 mg / kg, intraperitoneal injection, Sigma, St. Louis, MO). The rats were then placed on a heating pad at 38°C to maintain normal body temperature throughout the study. Vascular and bladder catheterization was performed. Briefly, a polyethylene 50 (PE-50) catheter was placed in one jugular vein for peptide infusion, and another PE-50 tube was placed in the carotid artery for blood pressure (BP) monitoring (Sonometrics, London, Ontario, Canada) and blood sampling. After the instrument was installed, a 15-minute equilibration period followed, and a 30-minute pre-infusion period was performed to collect baseline urine samples. The pre-infusion period was followed by a 45-minute continuous infusion of CRRL 191, ANP, or vehicle (15 minutes for drug infusion and 30 minutes for clearance during drug infusion).

[0124] Immediately after cessation of peptide infusion, another 30-minute washout period was initiated before termination and sacrifice. Four blood samples were collected to determine circulating cGMP levels: at baseline, before the end of drug infusion, during the washout period, and before the end of the washout period. Any blood loss in the rats was replaced with an equal volume of saline. After the start of peptide infusion, BP was monitored and recorded every 15 minutes. All blood collected was stored at -80°C until assayed.

[0125] For biochemical analysis, plasma cGMP levels were measured using a cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY).

[0126] Compared with ANP and vehicle, CRRL 191 is a more effective antihypertensive drug. As shown in the figure, 100 pmol / kg and 300 pmol / kg doses of CRRL 191 reduced systolic blood pressure in spontaneously hypertensive rats ( Fig. 8A ), diastolic blood pressure( Figure 8B ) and mean arterial pressure ( Figure 8C ) was more effective than 100 pmol / kg or 300 pmol / kg of ANP and had a more sustained effect over time after peptide administration.

[0127] Finally, plasma cGMP levels increased in spontaneously hypertensive rats when ANP or CRRL 191 was administered ( Fig. 9A However, compared with 100 pmol / kg or 300 pmol / kg ANP, 100 pmol / kg and 300 pmol / kg doses of CRRL 191 were more effective in activating cGMP in plasma and had a more sustained activation effect after peptide administration ( Fig. 9B ).

[0128] Other embodiments

[0129] It should be understood that although the present disclosure has been described in conjunction with the specific description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. A list of exemplary embodiments is provided:

[0130] Example 1. A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

[0131] Embodiment 2. A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions.

[0132] Embodiment 3. A polypeptide comprising, consisting essentially of, or consisting of a sequence having at least 90% homology to any one of SEQ ID NOs: 3-7.

[0133] Embodiment 4. The polypeptide according to embodiment 3, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3-7.

[0134] Embodiment 5. The polypeptide according to embodiment 3, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3-7.

[0135] Embodiment 6. A polypeptide comprising, essentially consisting of, or consisting of the amino acid sequence shown as amino acids 1 to 8 of SEQ ID NO 7.

[0136] Embodiment 7. A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

[0137] Embodiment 8. A substantially pure polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% homology to any one of SEQ ID NOs: 3-7.

[0138] Embodiment 9. The substantially pure polypeptide according to embodiment 8, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3-7.

[0139] Embodiment 10. The substantially pure polypeptide according to embodiment 8, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3-7.

[0140] Embodiment 11. A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide comprises one, two or three amino acid substitutions, additions or deletions.

[0141] Example 12. A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence shown as amino acids 1 to 8 of SEQ ID NO 7.

[0142] Embodiment 13. A composition comprising a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0143] Embodiment 14. A composition comprising, essentially consisting of, or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions.

[0144] Embodiment 15. A composition comprising at least two polypeptides, wherein each of the at least two polypeptides is a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3-7.

[0145] Embodiment 16. A composition comprising, consisting essentially of, or consisting of the amino acid sequence shown as amino acids 1 to 8 of SEQ ID NO 7.

[0146] Embodiment 17. The composition according to any one of embodiments 13 to 16, further comprising one or more pharmaceutically acceptable excipients.

[0147] Embodiment 18. The composition according to any one of embodiments 13 to 17, further comprising an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

[0148] Embodiment 19. The composition according to any one of embodiments 13 to 18, further comprising furosemide.

[0149] Embodiment 20. A nucleic acid encoding a polypeptide having, consisting essentially of, or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

[0150] Embodiment 21. A composition comprising a nucleic acid encoding a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence.

[0151] Embodiment 22. A composition comprising a nucleic acid encoding at least two polypeptides, wherein each of the at least two polypeptides is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 3-7, consisting essentially of the amino acid sequence, or consisting of the amino acid sequence.

[0152] Embodiment 23. The composition according to any one of embodiments 21 to 22, further comprising an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

[0153] Embodiment 24. The composition according to any one of embodiments 21 to 23, further comprising furosemide.

[0154] Embodiment 25. The composition according to any one of embodiments 21 to 24, wherein the nucleic acid is in the form of a non-viral vector.

[0155] Embodiment 26. The composition according to embodiment 25, wherein the non-viral vector is an expression plasmid.

[0156] Embodiment 27. The composition according to any one of embodiments 21 to 24, wherein the nucleic acid is in the form of a viral vector.

[0157] Embodiment 28. A method for treating a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0158] Embodiment 29. The method of embodiment 28, wherein the mammal is a human.

[0159] Embodiment 30. The method according to any one of embodiments 28 to 29, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

[0160] Embodiment 31. The method according to any one of embodiments 28 to 30, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

[0161] Embodiment 32. The method according to any one of embodiments 28 to 31, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

[0162] Embodiment 33. The method according to any one of embodiments 28 to 32, wherein the composition comprises furosemide.

[0163] Embodiment 34. A method for treating a mammal at risk for developing a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH), wherein the method comprises administering to the mammal a composition comprising a polypeptide or a nucleic acid encoding the polypeptide, the polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0164] Embodiment 35. The method of embodiment 34, wherein the mammal is a human.

[0165] Embodiment 36. The method according to any one of embodiments 34 to 35, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

[0166] Embodiment 37. The method according to any one of embodiments 34 to 36, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

[0167] Embodiment 38. The method according to any one of embodiments 34 to 37, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

[0168] Embodiment 39. The method according to any one of embodiments 34 to 38, wherein the composition comprises furosemide.

[0169] Embodiment 40. A method for alleviating the symptoms of a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 3-7.

[0170] Embodiment 41. The method of embodiment 40, wherein the mammal is a human.

[0171] Embodiment 42. The method according to any one of embodiments 40 to 41, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

[0172] Embodiment 43. The method according to any one of embodiments 40 to 42, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

[0173] Embodiment 44. The method according to any one of embodiments 40 to 43, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

[0174] Embodiment 45. The method according to any one of embodiments 40 to 44, wherein the composition comprises furosemide.

Claims

1. A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

2. A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions.

3. A polypeptide comprising a sequence having at least 90% homology to any one of SEQ ID NOs: 3-7.

4. The polypeptide of claim 3, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3-7.

5. The polypeptide of claim 3, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3-7.

6. A polypeptide comprising the amino acid sequence shown in amino acids 1 to 8 of SEQ ID NO 7.

7. A substantially pure polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

8. A substantially pure polypeptide comprising an amino acid sequence having at least 90% homology to any one of SEQ ID NOs: 3-7.

9. The substantially pure polypeptide of claim 8, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3-7.

10. The substantially pure polypeptide of claim 8, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3-7.

11. A substantially pure polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide comprises one, two or three amino acid substitutions, additions or deletions.

12. A substantially pure polypeptide comprising the amino acid sequence shown in amino acids 1 to 8 of SEQ ID NO 7.

13. A composition comprising a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

14. A composition comprising a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7, wherein the polypeptide further comprises one, two or three amino acid substitutions, additions or deletions.

15. A composition comprising at least two polypeptides, wherein each of the at least two polypeptides is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

16. A composition comprising the amino acid sequence shown in amino acids 1 to 8 of SEQ ID NO 7.

17. The composition according to any one of claims 13 to 16, further comprising one or more pharmaceutically acceptable excipients.

18. The composition according to any one of claims 13 to 17, further comprising an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

19. The composition according to any one of claims 13 to 18, further comprising furosemide.

20. A nucleic acid encoding a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

21. A composition comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

22. A composition comprising a nucleic acid encoding at least two polypeptides, wherein each of the at least two polypeptides is a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7.

23. The composition according to any one of claims 21 to 22, further comprising an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB) and a calcium channel blocker (CCB).

24. The composition according to any one of claims 21 to 23, further comprising furosemide.

25. The composition of any one of claims 21 to 24, wherein the nucleic acid is in the form of a non-viral vector.

26. The composition of claim 25, wherein the non-viral vector is an expression plasmid.

27. A composition according to any one of claims 21 to 24, wherein the nucleic acid is in the form of a viral vector.

28. A method for treating a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 3-7 or a nucleic acid encoding the polypeptide.

29. The method of claim 28, wherein the mammal is a human.

30. The method of any one of claims 28 to 29, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

31. The method of any one of claims 28 to 30, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

32. The method of any one of claims 28 to 31, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

33. The method of any one of claims 28 to 32, wherein the composition comprises furosemide.

34. A method for treating a mammal at risk of developing a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH), wherein the method comprises administering to the mammal a composition comprising a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7 or a nucleic acid encoding the polypeptide.

35. The method of claim 34, wherein the mammal is a human.

36. The method of any one of claims 34 to 35, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

37. The method of any one of claims 34 to 36, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

38. The method of any one of claims 34 to 37, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

39. The method of any one of claims 34 to 38, wherein the composition comprises furosemide.

40. A method for alleviating the symptoms of a cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction), a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction), or a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in a mammal, wherein the method comprises administering to the mammal a composition comprising a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-7 or a nucleic acid encoding the polypeptide.

41. The method of claim 40, wherein the mammal is a human.

42. The method of any one of claims 40 to 41, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is hypertension.

43. The method of any one of claims 40 to 42, wherein the cardiovascular disease, cardiorenal disease or metabolic disease is resistant hypertension.

44. The method of any one of claims 40 to 43, wherein the composition comprises an agent selected from the group consisting of a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

45. The method of any one of claims 40 to 44, wherein the composition comprises furosemide.

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