Therapeutic compositions and methods using CRRL 191 (an atrial natriuretic peptide (ANP) analog and guanylyl cyclase receptor (GC-A) activator)

Abstract

This application relates to artificial analogs of selected atrial natriuretic peptides, compositions containing these peptides, and their use in the treatment, prevention, or symptomatic relief of cardiovascular, cardiorenal, or metabolic diseases. These peptides are next-generation GC-A activators with improved GC-A receptor binding, stronger cGMP activation, increased resistance to degradation by neprilysin, and a stronger antihypertensive effect in vivo than atrial natriuretic peptide (ANP). The main analog is CRRL 191.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS This application claims priority to U.S. Provisional Application No. 63 / 407,798, filed September 19, 2022, which is incorporated herein by reference in its entirety. The Sequence Listing contained in the file named P35213WO00_SL.XML, created August 17, 2023, and 8,192 bytes in size (measured in MS-Windows), is submitted electronically herewith and is incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This disclosure was made in part with government support under Grant No. R01-HL136340 from the National Institutes of Health (NIH), and the government has certain rights in this invention.

[0003] The present specification provides methods and materials related to artificial analogs of selected atrial natriuretic peptides (ANPs). For example, the present specification provides compositions containing one or more artificial analogs of selected atrial natriuretic peptides (ANPs) provided herein, and capable of 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), and metabolic diseases (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, nonalcoholic steatohepatitis (NASH)) in mammals (e.g., humans). [Background technology]

[0004] Natriuretic polypeptides are polypeptides that can cause natriuresis (increased sodium excretion in urine). They can be produced in the brain, heart, kidney, and / or vascular tissue. In humans, the natriuretic polypeptide family includes the cardiac hormones atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP) isolated from the green mamba snake, and urodilatin (URO). Natriuretic polypeptides bind to two well-characterized guanylyl cyclase receptors, GC-A and GC-B (also called natriuretic peptide receptors (NPR-A and NPR-B)). GC-A is the primary receptor for ANP, BNP, DNP, and URO; GC-B is the primary receptor for CNP. Once bound, these receptors sequentially catalyze the conversion of guanosine triphosphate (GTP) to the second messenger cyclic 3'5' guanosine monophosphate (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 dilation, renin and aldosterone suppression, anti-apoptosis, anti-hypertrophy, systolic relaxation, revascularization, lipolysis, and browning of white adipocytes. Downstream effects of GC-B receptor activation include anti-fibrosis, anti-inflammation, revascularization, microcirculatory dilation, and systolic relaxation.

[0005] Previous disclosures, such as International Patent Application No. PCT / US2017 / 060808, provided an alternatively spliced ​​analog of atrial natriuretic peptide (MANP) consisting of the 28 amino acids of native ANP plus a 12 amino acid C-terminal extension. Compared to ANP, MANP is more resistant to degradation, has enhanced binding to GC-A receptors, causes increased and more sustained sodium excretion, causes more sustained suppression of aldosterone, and results in greater and more sustained blood pressure reduction. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure provides the design and validation of next-generation novel GC-A activators that have improved binding affinity to the GC-A receptor, more potent cGMP activation, increased resistance to degradation by neprilysin, and a stronger blood pressure lowering effect in vivo than ANP. [Means for solving the problem]

[0007] The present disclosure provides and includes methods and materials related to artificial analogs of selected atrial natriuretic peptides (ANPs). For example, the present disclosure provides the artificial polypeptides set forth in Table 1, which comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7.

[0008] In one embodiment, an artificial analog of a selected ANP provided herein is a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-7. In one embodiment, an artificial analog of a selected ANP provided herein is a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-7, wherein the polypeptide further comprises one, two, or three amino acid substitutions, additions, or deletions. In one embodiment, an artificial analog of a selected ANP provided herein is a polypeptide comprising a sequence having at least 90% homology to any one of SEQ ID NOs: 3-7. In one embodiment, the polypeptide sequence has at least 95% homology to any one of SEQ ID NOs: 3-7. In one embodiment, the polypeptide sequence comprises at least 97% homology to any one of SEQ ID NOs: 3-7. In one embodiment, an artificial analog of a selected ANP provided herein is a polypeptide comprising the amino acid sequence set forth in amino acids 1-8 of SEQ ID NO: 7.

[0009] The present disclosure provides and includes substantially pure polypeptides related to artificial analogs of selected ANPs. In one embodiment, the artificial analogs of selected ANPs provided herein are substantially pure polypeptides comprising the amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the artificial analogs of selected ANPs provided herein are substantially pure polypeptides comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOS: 3-7. In one embodiment, the substantially pure polypeptide comprises a sequence at least 95% identical to any one of SEQ ID NOS: 3-7. In one embodiment, the substantially pure polypeptide comprises a sequence at least 97% identical to any one of SEQ ID NOS: 3-7. In one embodiment, the artificial analogs of selected ANPs provided herein are substantially pure polypeptides comprising the amino acid sequence set forth in any one of SEQ ID NOS: 3-7, wherein the polypeptide comprises one, two, or three amino acid substitutions, additions, or deletions. In one embodiment, the artificial analogs of selected ANPs provided herein are substantially pure polypeptides comprising the amino acid sequence set forth in amino acids 1-8 of SEQ ID NOS: 7.

[0010] The present disclosure also provides compositions of artificial analogs of selected ANPs provided herein. In one embodiment, the compositions provided herein include a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the compositions provided herein include a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3-7, wherein the polypeptide further comprises one, two, or three amino acid substitutions, additions, or deletions. In one embodiment, the compositions provided herein include at least two polypeptides, each of which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the compositions provided herein comprise an amino acid sequence set forth in amino acids 1-8 of SEQ ID NOS: 7. In one embodiment, the compositions further comprise one or more pharmaceutically acceptable excipients. In one embodiment, the compositions further comprise an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB). In one embodiment, the composition further comprises furosemide.

[0011] The present disclosure also provides nucleic acids encoding selected artificial analogs of ANPs provided herein and compositions thereof. In one embodiment, the nucleic acid provided herein encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the composition provided herein comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the composition provided herein comprises a nucleic acid encoding at least two polypeptides, each of the at least two polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the composition further comprises a drug selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB). In one embodiment, the composition further comprises furosemide. In one embodiment, the nucleic acid is in the form of a non-viral vector. In one embodiment, the non-viral vector is an expression plasmid. In one embodiment, the nucleic acid is in the form of a viral vector.

[0012] The present disclosure also provides and includes methods 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, nonalcoholic steatohepatitis (NASH)) in a mammal (e.g., human). For example, administering a composition comprising one or more selected artificial analogs of ANP provided herein to a mammal (e.g., human) can lower blood pressure. For example, administering a composition comprising one or more selected artificial analogs of ANP provided herein to a mammal (e.g., human) can increase cGMP activation.

[0013] The present disclosure provides and includes methods of lowering blood pressure, increasing natriuresis, causing arterial dilation, suppressing renin and aldosterone, decreasing apoptosis, decreasing hypertrophy, increasing systolic relaxation, inducing revascularization, increasing lipolysis, and causing browning of white adipocytes by administering a composition comprising one or more artificial analogs of ANP provided herein, the composition comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7.

[0014] In one aspect, provided herein is a method 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, nonalcoholic steatohepatitis (NASH)) in a mammal, the method comprising administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3 to 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 calcium channel blocker (CCB). In one embodiment, the composition comprises furosemide.

[0015] 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, nonalcoholic steatohepatitis (NASH)), the method comprising administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3 to 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, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB). In one embodiment, the composition comprises furosemide.

[0016] In one aspect, provided herein is a method for alleviating 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, nonalcoholic steatohepatitis (NASH)) in a mammal, the method comprising administering to the mammal a composition comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 3 to 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 calcium channel blocker (CCB). In one embodiment, the composition comprises furosemide.

[0017] Aspects of the present disclosure are described herein, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, it is emphasized that the particulars shown are by way of example only and are for purposes of illustrative explanation of aspects of the present disclosure. In this regard, the description and drawings considered separately and together will make apparent to one skilled in the art how aspects of the present disclosure may be practiced. [Brief explanation of the drawings]

[0018] [Figure 1A] The structure of ANP is shown. [Figure 1B] The structure of CRRL 191 is shown. [Figure 2] 1 is a graph of cGMP production levels in HEK293 / GCA overexpressing cells in response to a 10 −8 M dose of ANP or CRRL 191 over a 6 hour period. [Figure 3] 1 is a graph of in vitro degradation of CRRL 191 and ANP in a neprilysin assay. [Figure 4] 1 is a graph of cGMP production levels in primary human cardiomyocytes induced by ANP and CRRL 191. [Figure 5] 1 is a graph of cGMP production levels in primary human cardiomyocytes induced by MANP, CRRL 191, and CRRL 101. [Figure 6] 1 is a graph of the reduction in apoptosis with increasing doses of CRRL 191 compared to control over a 90 hour period in primary human cardiomyocytes induced by CRRL 191 using real-time imaging. [Figure 7A] 1 is a graph of the binding kinetics of ANP to GC-A as measured by surface plasmon resonance (SPR). [Figure 7B] 1 is a graph of the binding kinetics of MANP to GC-A measured by surface plasmon resonance (SPR). [Figure 7C]1 is a graph of the binding kinetics of CRRL 191 to GC-A as measured by surface plasmon resonance (SPR). [Figure 8A] 1 is a graph comparing the reduction in systolic blood pressure induced by saline vehicle, 100 pmol / kg / min or 300 pmol / kg / min CRRL 191, or 100 pmol / kg / min or 300 pmol / kg / min ANP during and after cessation of treatment in spontaneously hypertensive rats. [Figure 8B] 1 is a graph comparing the decrease in diastolic blood pressure induced by saline, 100 pmol / kg / min or 300 pmol / kg / min of CRRL 191, or 100 pmol / kg / min or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during and after cessation of treatment. [Figure 8C] 1 is a graph comparing the reduction in mean arterial pressure induced by saline, 100 pmol / kg / min or 300 pmol / kg / min of CRRL 191, or 100 pmol / kg / min or 300 pmol / kg / min of ANP during and after cessation of treatment in spontaneously hypertensive rats. [Figure 9A] FIG. 1 is a graph comparing plasma cGMP levels induced by saline, 100 pmol / kg / min or 300 pmol / kg / min of CRRL 191, or 100 pmol / kg / min or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during and after treatment. [Figure 9B] 1 is a graph comparing the changes in plasma cGMP levels induced by saline, 100 pmol / kg / min or 300 pmol / kg / min of CRRL 191, or 100 pmol / kg / min or 300 pmol / kg / min of ANP in spontaneously hypertensive rats during and after cessation of administration. DETAILED DESCRIPTION OF THE INVENTION

[0019] 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 omitted from that embodiment. Thus, the present disclosure contemplates that some embodiments of the present disclosure may exclude or omit any feature or combination of features described herein. Furthermore, numerous modifications and additions to the various embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure, and do not depart from the present disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the present disclosure. It is intended that no portion of this specification should be interpreted in a way that disclaims any portion of the full scope of the present disclosure. Therefore, the following description is intended to illustrate some specific embodiments of the present disclosure, but is not intended to exhaustively identify all permutations, combinations, and modifications thereof.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used to describe the present disclosure herein are used only for the purpose of describing particular aspects or embodiments and are not intended to limit the present disclosure.

[0021] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is provided. References to technology used herein are intended to refer to technology as generally understood in the art, and include modifications of those technologies or equivalent technology substitutions that would be apparent to those skilled in the art. In the event of a conflict, the present specification, including definitions, will control.

[0022] Unless the context indicates otherwise, it is specifically contemplated that the various features of the present disclosure described herein can be used in any combination. The present disclosure also contemplates that some embodiments of the present disclosure can exclude or omit any feature or combination of features described herein.

[0023] The methods disclosed herein comprise and consist of one or more steps or actions for achieving the described method. Method steps and / or actions may be interchanged with one another without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of an embodiment, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the present disclosure. Although methods and materials similar or equivalent to those described herein can also be used to practice aspects of the present disclosure, suitable methods and materials are described herein.

[0024] As used in the description of this 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.

[0025] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and also includes the absence of a combination in the alternative ("or").

[0026] As used herein, the terms "about" and "approximately," when referring to a measurable value such as length, frequency, or duration, are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0027] 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."

[0028] The term "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or feature described as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or features, nor is it intended to exclude equivalent structures and techniques known to those skilled in the art. Rather, use of the term "exemplary" is intended to present concepts in a concrete form, and the disclosed subject matter is not limited by such examples.

[0029] Polypeptides The present disclosure provides methods and materials related to artificial analogs of selected atrial natriuretic peptides (ANPs). For example, the present disclosure provides polypeptides listed in Table 1. In one embodiment, the artificial analogs of selected ANPs provided herein are polypeptides comprising the amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the artificial analogs of selected ANPs provided herein are polypeptides consisting essentially of the amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the artificial analogs of selected ANPs provided herein are polypeptides consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 3-7. In one embodiment, the artificial analogs of selected ANPs comprise the amino acid sequence set forth in SEQ ID NOS: 3. In one embodiment, the artificial analogs of selected ANPs consist essentially of the amino acid sequence set forth in SEQ ID NOS: 3. In one embodiment, the artificial analogs of selected ANPs consist of the amino acid sequence set forth in SEQ ID NOS: 3. In one embodiment, the artificial analogs of selected ANPs are CRRL 101. In one embodiment, the artificial analogs of selected ANPs comprise the amino acid sequence set forth in SEQ ID NOS: 4. In one embodiment, the artificial analogs of selected ANPs consist essentially of the amino acid sequence set forth in SEQ ID NOS: 4. In one embodiment, the artificial analog of the selected ANP consists of the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the artificial analog of the selected ANP is CRRL 90. In one embodiment, the artificial analog of the selected ANP comprises the amino acid sequence set forth in SEQ ID NO:5. In one embodiment, the artificial analog of the selected ANP consists essentially of the amino acid sequence set forth in SEQ ID NO:5. In one embodiment, the artificial analog of the selected ANP is CRRL 91. In one embodiment, the artificial analog of the selected ANP comprises the amino acid sequence set forth in SEQ ID NO:6. In one embodiment, the artificial analog of the selected ANP consists essentially of the amino acid sequence set forth in SEQ ID NO:6. In one embodiment, the artificial analog of the selected ANP is CRRL 111.In one embodiment, the artificial analog of the selected ANP comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the artificial analog of the selected ANP comprises the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the artificial analog of the selected ANP consists essentially of the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the artificial analog of the selected ANP is CRRL 191. [Table 1]

[0030] Without being bound by theory, artificial analogs of selected ANPs containing amino acid substitutions, additions, and deletions can function similarly to one another. In one aspect, artificial analogs of selected ANPs provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1-7, containing zero, one, two, or three amino acid substitutions within the specified sequence of the sequence identifier (e.g., SEQ ID NO: 1).

[0031] In one embodiment, the artificial analog of the selected ANP provided herein can contain one, two, or three conservative substitutions. Without being bound by theory, conservative amino acid substitutions can be made by selecting substitutions that do not significantly affect (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. For example, naturally occurring residues can be divided into groups based on the properties of their side chains: (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). Non-limiting examples of useful conservative substitutions include, but are not limited to, valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenylalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and / or leucine for valine. In one embodiment, the artificial analogs of selected ANPs provided herein can contain one, two, or three conservative substitutions, in which an acidic amino acid residue is substituted for another acidic amino acid residue. In one embodiment, an artificial analog of a selected ANP provided herein can contain one, two, or three conservative substitutions, in which a basic amino acid residue is replaced with another basic amino acid residue.

[0032] In one aspect, the artificial analog of the selected ANP provided herein can contain one, two, or three non-conservative substitutions. Without being bound by theory, non-conservative substitutions usually involve replacing a member of one of the above-mentioned classes with a member of another class. Such production may be desirable for mass production of such compounds or for providing alternative embodiments. Whether amino acid changes result in functional polypeptides can be easily determined, for example, by measuring the specific activity of peptide variants using the methods disclosed herein.

[0033] In one embodiment, an artificial analog of a selected ANP provided herein that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOS: 1-7 is a polypeptide that comprises zero, one, two, or three amino acid additions within the specified sequence of a sequence identifier (e.g., SEQ ID NO: 1). In one embodiment, an artificial analog of a selected ANP provided herein that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOS: 1-7 is a polypeptide that comprises zero, one, two, or three amino acid deletions within the specified sequence of a sequence identifier (e.g., SEQ ID NO: 1). In one embodiment, an artificial analog of a selected ANP provided herein that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOS: 1-7 is a polypeptide that comprises zero, one, two, three, four, or five amino acid residues before the specified sequence of a sequence identifier (e.g., SEQ ID NO: 1). In one aspect, the artificial analog of a selected ANP provided herein that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-7 is a polypeptide that includes 0, 1, 2, 3, 4, or 5 amino acid residues following the specified sequence of the sequence identifier (e.g., SEQ ID NO: 1).

[0034] In one aspect, an artificial analog of a selected ANP provided herein comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 1-7 is a polypeptide comprising 0, 1, 2, or 3 amino acid substitutions within the sequence set forth in the sequence identifier (e.g., SEQ ID NO: 1), and / or 0, 1, 2, or 3 amino acid additions within the sequence identifier (e.g., SEQ ID NO: 1), and / or 0, 1, 2, or 3 amino acid deletions within the sequence identifier (e.g., SEQ ID NO: 1), and / or 0, 1, 2, 3, 4, or 5 amino acid residues before the sequence identifier (e.g., SEQ ID NO: 1) and / or 0, 1, 2, 3, 4, or 5 amino acid residues after the sequence identifier (e.g., SEQ ID NO: 1).

[0035] Without being bound by theory, certain amino acids of the artificial analogs of selected ANPs provided herein are not modified (e.g., amino acid substitution, addition, deletion) to provide beneficial properties. For example, cysteine ​​residues are used to form disulfide bonds. In one embodiment, cysteine ​​residues of the artificial analogs of selected ANPs provided herein are not substituted. In one embodiment, the first four amino acids of SEQ ID NO: 6 (e.g., amino acid TAPR) are not modified (e.g., amino acid substitution, addition, deletion). In one embodiment, the first four amino acids of SEQ ID NO: 7 (e.g., amino acid RALL) are not modified (e.g., amino acid substitution, addition, deletion). In one embodiment, the first eight amino acids of SEQ ID NO: 7 (e.g., amino acid RALLTAPR) are not modified (e.g., amino acid substitution, addition, deletion). The majority of naturally occurring amino acids are L-amino acids, and naturally occurring polypeptides are primarily composed of L-amino acids. D-amino acids are enantiomers of L-amino acids. In one embodiment, the polypeptides provided herein can contain one or more D-amino acids.

[0036] In one embodiment, the artificial analog of a selected 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 set forth in any one of SEQ ID NOs: 1-7. The percentage of sequence identity is calculated by determining the number of matching positions in the aligned amino acid sequences, dividing the number of matching positions by the total number of aligned amino acids, and multiplying by 100. A matching position refers to a position where an identical amino acid exists at the same position in the aligned amino acid sequences. The percentage of sequence identity can also be determined for any nucleic acid sequence.

[0037] 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, such as the methods disclosed in WO / 2018 / 089601, which is incorporated herein by reference in its entirety.

[0038] Artificial analogs of selected ANPs 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 ligation (EPL). Artificial analogs of selected ANPs provided herein can be produced using manual or automated techniques (e.g., using a peptide synthesizer from Applied BioSystems (Foster City, CA) or an automated peptide synthesizer from Biosearch Inc. (San Rafael, CA). Artificial analogs of selected ANPs provided herein can also be produced recombinantly.

[0039] The selected artificial analogs of ANP provided herein are typically cyclic due to disulfide bonds between the cysteine ​​residues underlined in the sequences shown in Table 1. Disulfide bonds can be formed by any method known to those skilled in the art, including, but not limited to, oxidation (whether enzyme-mediated or by reaction with low molecular weight oxidizing agents) or methods using reagents (e.g., dicarbonyl and related cross-linking agents such as glutaraldehyde). Disulfide bonds between cysteine ​​residues can also be introduced by mild oxidation of linear polypeptides with KCN, as described, for example, in U.S. Pat. No. 4,757,048.

[0040] The term "substantially pure" as used herein in reference to a polypeptide means that the polypeptide is substantially free from other polypeptides, lipids, carbohydrates, and nucleic acids with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that has been removed from its natural environment and is at least 60% pure. A substantially pure polypeptide can be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In one aspect, a substantially pure polypeptide provided herein is a polypeptide that has been synthesized to contain at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% purity.

[0041] Any method can be used to obtain a substantially pure polypeptide. For example, polypeptide purification techniques such as affinity chromatography and high-performance liquid chromatography (HPLC) can be used, as well as polypeptide synthesis techniques. Any material can be used as a source material to obtain a substantially pure polypeptide. For example, tissues from wild-type or transgenic animals can be used as source material. Tissue culture cells engineered to overexpress a particular polypeptide can also be used to obtain a substantially pure polypeptide. Solid-phase peptide synthesis can also be used to obtain a substantially pure polypeptide. Furthermore, polypeptides can be engineered to contain amino acid sequences that allow them to be captured on an affinity matrix. For example, c-myc, hemagglutinin, polyhistidine, or FLAG can be used. (商標) Tags, such as 'tag' (Kodak), can be used to aid in the purification of the polypeptide. Such tags can be inserted anywhere within the polypeptide, at either the carboxyl or amino terminus, or anywhere in between. Other fusions that can be used include enzymes, such as alkaline phosphatase, that aid in the detection of the polypeptide.

[0042] Without being bound by theory, the artificial analogs of the selected ANPs are processed from propeptides. The artificial analogs of the selected ANPs provided herein can be processed, in part, from any propeptide. In one embodiment, the artificial analogs of the selected ANPs are produced as propeptides, which are further processed into the artificial analogs of the selected ANPs provided herein by natural or non-natural methods.

[0043] Without being bound by theory, the artificial analogs of ANP provided herein can function through one or more guanylyl cyclase receptors through which natural natriuretic polypeptides function. For example, the polypeptides provided herein can bind to and function through the GC-A (also referred to as NPR-A) receptor through which ANP, BNP, and DNP function, but can also function through the GC-B (also referred to as NPR-B) receptor through which CNP functions. In one embodiment, the artificial analogs of ANP provided herein can bind to the guanylyl cyclase-A (GC-A) receptor. In one embodiment, the artificial analogs of ANP provided herein can bind to the guanylyl cyclase-A (GC-A) receptor. In one embodiment, the artificial analogs of ANP provided herein can bind to and function through one or more guanylyl cyclase receptors, including GC-A and GB-B. Methods for evaluating the receptors involved in the function of a particular artificial analog of ANP are known in the art. For example, glomeruli containing both GC-A and GC-B can be isolated (e.g., from experimental animals such as dogs), incubated with an artificial analog of ANP (e.g., any of SEQ ID NOS: 3-7), and cGMP levels can be measured. Glomeruli can be pretreated with an antagonist of GC-A or GC-B to determine whether the cGMP production stimulated by natriuretic polypeptides via one or the other receptor is attenuated.

[0044] The biological activity of the selected artificial analog of ANP provided herein can be determined using any number of assays, including those described herein.For example, the activity of the artificial analog of ANP provided herein can be determined in vitro by testing its effect on cGMP production in cultured cells (e.g., cultured cardiac fibroblasts, aortic endothelial cells, or glomerular cells).The cells are cultured with the artificial analog of ANP provided herein (e.g., 10 -10 From 10 -4Samples can be exposed to M. cruzi (CRRL 191) and assayed to assess the effect on cGMP production. cGMP production can be detected and measured, for example, using a competitive RIAcGMP kit (Perkin-Elmer, Boston, MA) or a cGMP ELISA kit (EnzoLifeSciences, Farmingdale, NY).

[0045] Artificial analogs of selected ANPs provided herein can be of any suitable length (e.g., can include any number of amino acids). For example, artificial analogs of selected ANPs provided herein can be from about 20 amino acids in length to about 100 amino acids in length (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 4 ... The length of the artificial analog of the selected ANP provided herein can be about 100 amino acids, about 50 to about 100 amino acids, about 60 to about 100 amino acids, about 70 to about 100 amino acids, about 80 to about 100 amino acids, about 90 to about 100 amino acids, about 20 to about 80 amino acids, about 20 to about 70 amino acids, about 30 to about 60 amino acids, about 35 to about 55 amino acids, about 38 to about 52 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids. In one embodiment, the artificial analog of the selected ANP provided herein can be about 46 to about 50 amino acids in length. For example, the artificial analog of the selected ANP provided herein with GC-A binding properties can be about 38 to about 42 amino acids in length. For example, the artificial analog of the selected ANP provided herein with GC-A binding properties can be about 42 to about 46 amino acids in length. For example, artificial analogs of selected ANPs containing the GC-A binding properties provided herein can be from about 46 to about 50 amino acids in length.

[0046] Artificial analogs of selected ANPs provided herein can comprise one or more sequences present in polypeptides comprising natriuretic polypeptide activity (e.g., ANP, BNP, CNP, urodilatin, and DNP). In one embodiment, artificial analogs of selected ANPs can comprise non-naturally occurring sequences. In one embodiment, artificial analogs of selected ANPs can comprise naturally occurring and non-naturally occurring sequences. In one embodiment, artificial analogs of selected ANPs can comprise sequences present in any species, including, but not limited to, humans, horses, pigs, goats, cows, dogs, cats, mice, or snakes.

[0047] nucleic acid The present disclosure also provides nucleic acids encoding artificial analogs of one or more selected ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7). For example, the present disclosure provides vectors (e.g., plasmids and viral vectors) containing nucleic acids encoding artificial analogs of one or more ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7), in an manner that allows the polypeptides to be expressed intracellularly.

[0048] When the vector containing a nucleic acid encoding one or more artificial analogs of the selected ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is a non-viral vector, any suitable non-viral vector can be used. In one embodiment, the non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

[0049] When the vector containing a nucleic acid encoding one or more selected artificial analogs of ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is a viral vector, any appropriate viral vector can be used. In one embodiment, the viral vector can be derived from a positive-stranded virus or a negative-stranded virus. In one embodiment, the viral vector can be derived from a virus having a single-stranded genome or a virus having a double-stranded genome. In one embodiment, the viral vector can be derived from a virus containing a DNA genome or a virus containing an RNA genome. In one embodiment, the viral vector can be a chimeric viral vector. In one embodiment, the viral vector is capable of infecting dividing cells. In one embodiment, the viral vector is capable of infecting non-dividing cells. Examples of viral-based vectors that can contain nucleic acids encoding artificial analogs of selected ANPs provided herein (e.g., polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence 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.

[0050] In addition to a nucleic acid encoding one or more selected artificial analogs of ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 3-7), a vector (e.g., a plasmid or viral vector) can contain one or more regulatory elements operably linked to the nucleic acid encoding one or more selected artificial analogs of 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 that regulate the expression (e.g., transcription or translation) of the nucleic acid. The selection of regulatory elements to be included in a vector depends on several factors, including, but not limited to, inducibility, targeting, and the desired expression level. For example, a promoter can be included in a vector to promote transcription of a nucleic acid encoding one or more selected artificial analogs of ANPs provided herein (e.g., one or more polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 3-7). In one embodiment, the promoter can be a naturally occurring promoter or a recombinant promoter. In one embodiment, the promoter can be constitutive or inducible (e.g., in the presence of tetracycline) and can affect expression of the nucleic acid encoding the polypeptide in a general or cell / tissue-specific manner. Examples of promoters that can be used to drive expression of one or more selected artificial analogs of ANP provided herein (e.g., polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7) in a cell include, but are not limited to, a CMV promoter, an EF1a promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a TRE promoter, and a CAG promoter.As used herein, "operably linked" refers to the positioning of regulatory elements within a vector relative to a nucleic acid encoding a polypeptide in a manner that allows or promotes expression of the encoded polypeptide. For example, a vector can include a promoter and a nucleic acid encoding one or more selected artificial analogs of ANPs provided herein. In this case, the promoter is operably linked to the nucleic acid encoding one or more selected artificial analogs of ANPs provided herein, thereby driving the expression of the selected artificial analog(s) of ANPs in a cell.

[0051] Polypeptide and nucleic acid combinations, compositions and formulations thereof The present disclosure also provides compositions comprising artificial analogs of one or more (e.g., one, two, three, four, five, six, or seven) selected ANPs provided herein and / or nucleic acids encoding one or more (e.g., one, two, three, four, five, six, or seven) selected ANPs provided herein. In one embodiment, a composition provided herein comprises, consists essentially of, or consists of an artificial analog of one or more selected ANPs listed in Table 1. In one embodiment, a composition provided herein comprises one or more nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS:3-7. In one embodiment, a composition provided herein comprises one or more nucleic acids encoding an artificial analog of one or more selected ANPs listed in Table 1 and a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS:3-7.

[0052] In one embodiment, a composition provided herein comprises artificial analogs of at least two (e.g., two, three, four, five, six, or seven) selected ANPs provided herein and / or nucleic acids encoding at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) selected ANPs provided herein. In one embodiment, a composition provided herein comprises, consists essentially of, or consists of artificial analogs of at least two selected ANPs listed in Table 1. In one embodiment, a composition provided herein comprises at least two nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs:3-7. In one aspect, the compositions provided herein comprise at least two selected artificial analogs of ANPs listed in Table 1 and at least two nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs:3-7.

[0053] Any suitable method can be used to prepare the compositions provided herein (e.g., compositions comprising one or more artificial analogs of selected ANPs provided herein and / or nucleic acids encoding one or more artificial analogs of selected ANPs provided herein). In one embodiment, one or more artificial analogs of selected ANPs provided herein can be combined with a pharmaceutically acceptable carrier and / or pharmaceutical excipient. In one embodiment, one or more nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7 can be combined with a pharmaceutically acceptable carrier and / or pharmaceutical excipient. In one embodiment, one or more artificial analogs of selected ANPs provided herein and one or more nucleic acids encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7 can be combined with a pharmaceutically acceptable carrier and / or pharmaceutical excipient. The term "pharmaceutically acceptable" generally refers to a non-toxic, inert, and / or physiologically compatible compound. The term "pharmaceutical excipient" includes substances such as carriers, pH adjusting and buffering agents, osmotic pressure adjusting agents, wetting agents, coloring agents, and preservatives.

[0054] In one embodiment, a composition provided herein (e.g., a composition comprising one or more selected artificial analogs of ANPs provided herein and / or a nucleic acid encoding one or more selected artificial analogs of ANPs provided herein) can be formulated with at least one other concomitant medication selected from a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB). In one embodiment, the composition comprises a selected artificial analog of ANP and furosemide.

[0055] Treatment and prevention methods Without being bound by theory, the artificial analogs of selected ANPs provided herein can be used to treat, prevent, and / or alleviate symptoms of 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, non-alcoholic steatohepatitis (NASH)). In one aspect, the artificial analogs of selected ANPs described herein can be used to treat 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 one embodiment, the artificial analogs of selected ANPs provided herein can be used to prevent 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 one embodiment, the artificial analogs of selected ANPs provided herein can be used to alleviate symptoms of 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 one aspect, the artificial analogs of selected ANPs provided herein can be used to treat, prevent, and / or alleviate symptoms of hypertension (e.g., resistant hypertension) or heart failure (HF).The presence or extent of disease can be assessed using methods known in the art, including, but not limited to, general clinical examinations to assess blood pressure, heart rate, heart rhythm, arterial oxygen, and hemoglobin levels; echocardiography to measure ejection fraction, left ventricular (LV) and left atrial (LA) diameters, LV wall motion, LV diastolic filling pressure, and diastolic potentiation by pulse wave and tissue Doppler; use of a Swan-Ganz catheter to measure cardiac output, pulmonary wedge capillary pressure, pulmonary artery pressure, right ventricular pressure, right atrial pressure, and systemic and pulmonary vascular resistance; glomerular filtration rate, serum creatinine, and and assessment of renal function by measuring blood urea nitrogen and blood urea nitrogen; and measurement of biomarkers such as BNP, amino-terminal pro-BNP (NT-proBNP), troponin-T, troponin-I, C-reactive protein (CRP), and creatine kinase, serum cystatin-C, albuminuria, neutrophil gelatinization-associated lopocalin (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, the artificial analogs of selected ANPs provided herein can reduce one or more symptoms of acute or chronic heart failure (HF), including edema, shortness of breath, and fatigue. To determine the effectiveness of the selected artificial analogues of ANP provided herein in improving heart failure symptoms, one or more of these parameters can be evaluated (for example, before and after treatment with the artificial analogues of ANP) using methods known in the art.Good clinical responses include: cardiac unloading (i.e., reduced intracardiac pressure), increased glomerular filtration rate (GFR), decreased plasma renin activity (PRA), decreased angiotensin II levels, reduced cardiac fibroblast proliferation, reduced left ventricular (LV) hypertrophy, reduced LV mass (indicating reduced fibrosis and hypertrophy), reduced pulmonary wedge capillary pressure (PWCP) (an indirect indicator 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.

[0056] Without being bound by theory, the artificial analogs of selected ANPs provided herein can lower blood pressure, increase natriuresis, cause arterial dilation, suppress renin and aldosterone, reduce apoptosis, reduce hypertrophy, improve dilation enhancement, induce revascularization, promote lipolysis, and cause browning of white adipocytes. In one embodiment, the artificial analogs of selected ANPs provided herein can lower blood pressure in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can increase natriuresis in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can reduce apoptosis in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can cause arterial dilation in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can suppress renin and aldosterone in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can improve dilation enhancement in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can induce revascularization in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can promote lipolysis in a mammal. In one embodiment, the artificial analogs of selected ANPs provided herein can induce browning of white adipocytes in a mammal.

[0057] The activity of a selected artificial analog of ANP can also be assessed in vivo by examining its effects on factors such as plasma cGMP levels, urinary cGMP excretion, net renal cGMP production, glomerular filtration rate, hemodynamic functions such as blood pressure, heart rate, cardiac output, pulmonary wedge pressure, and systemic vascular resistance, and renal functions such as renal blood flow, urine volume, and sodium excretion rate in mammals (e.g., rodents, pigs, sheep, dogs, or humans). In one embodiment, such parameters can be assessed after inducing heart failure (e.g., by rapid right ventricular pacing) or hypertension.

[0058] In one aspect, by administering to a mammal (e.g., a human) an artificial analog of one or more selected ANPs provided herein (e.g., a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7) and / or a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein (e.g., a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7), symptoms of cardiovascular disease (e.g., hypertension, heart failure, cardiomyopathy, valvular disease, myocardial infarction) in the mammal can be treated, prevented, and / or alleviated. In one aspect, by administering to a mammal (e.g., a human) an artificial analog of one or more selected ANPs provided herein (e.g., a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7) and / or a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein (e.g., a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7), symptoms of a cardiorenal disease (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, chemotherapy-induced cardiorenal dysfunction) in the mammal can be treated, prevented, and / or alleviated.In one aspect, administering to a mammal (e.g., a human) an artificial analog of one or more selected ANPs provided herein (e.g., a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) and / or a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein (e.g., a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) can treat, prevent, and / or alleviate symptoms of a metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH) in the mammal.

[0059] As used herein, the term "treating" or "treatment" refers to an approach to obtain beneficial or desired clinical results. In one embodiment, the term "treating" or "treatment" refers to administering one or more selected artificial analogs of ANP disclosed herein, thereby partially or completely alleviating, ameliorating, alleviating, inhibiting, delaying the onset, reducing the severity, and / or reducing the incidence of one or more symptoms, characteristics, 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 disease, chemotherapy-induced cardiorenal dysfunction), or metabolic disease (e.g., diabetes, obesity, metabolic syndrome, hyperlipidemia, insulin resistance, primary aldosteronism, NASH). The term "treating" or "treatment" includes administering one or more selected artificial analogs of ANP disclosed herein to prevent or delay the onset of symptoms, complications, or biochemical indicators of cardiovascular, cardiorenal, or metabolic disease, alleviating the symptoms of, or arresting or inhibiting the progression 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).Treatment may be prophylactic (to prevent or delay the onset of, or to prevent the onset 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 may be therapeutic suppression or alleviation of symptoms after the onset of 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).

[0060] Any suitable mammal can be treated by administering to the mammal an artificial analog of one or more selected ANPs provided herein (e.g., an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7) and / or a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein (e.g., a nucleic acid encoding an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7). Examples of mammals to which the artificial analog of one or more selected ANPs provided herein (and / or a nucleic acid encoding the artificial analog of one or more selected ANPs provided herein) can be administered include, but are not limited to, humans, non-human primates (e.g., monkeys or apes), horses, dogs, cats, bovine species, pigs, sheep, mice, rats, hamsters, bats, foxes, goats, minks, and deer. In one embodiment, a human identified as having or 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) can be treated by administering to the human one or more selected artificial analogs of ANP provided herein. In one embodiment, a human identified as having or 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) can be treated by administering to the human a nucleic acid encoding one or more selected artificial analogs of ANP provided herein.In one aspect, a human identified as having or 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) can be treated by administering to the human one or more selected artificial analogs of ANP provided herein and nucleic acids encoding the one or more selected artificial analogs of ANP provided herein.

[0061] In one aspect, the effectiveness of treatment is measured by clinical parameters such as the alleviation of symptoms.Symptoms of cardiovascular, cardiorenal, or metabolic diseases include, but are not limited to, edema, shortness of breath, and fatigue, as well as cardiac unloading (i.e., reduced intracardiac pressure), increased glomerular filtration rate (GFR), decreased plasma renin activity (PRA), decreased angiotensin II level, decreased cardiac fibroblast proliferation, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicating reduced fibrosis and hypertrophy), decreased pulmonary wedge capillary pressure (PWCP) (an indirect indicator of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased aldosterone level (indicating anti-fibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end-systolic diameter.

[0062] 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) by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in a mammal at risk of developing a cardiovascular, cardiorenal, or metabolic disease. In one aspect, the methods and substances described herein can be used to reduce the duration and / or severity of one or more symptoms of a cardiovascular disease, cardiorenal disease, or metabolic disease present in a mammal, including 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), by, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. See, e.g., J. Jamison et al., Harrison's Principles of Medicine (20th ed., 2018).

[0063] When administering the compositions provided herein to a mammal (e.g., a human), any suitable route of administration can be used. In one embodiment, the compositions provided herein can be administered to a mammal (e.g., a human) intramuscularly (e.g., by intramuscular injection), subcutaneously (e.g., by subcutaneous injection), orally, nasally, transdermally, or by inhalation. In one embodiment, the route and / or method of administration of the compositions provided herein can be tailored to the mammal being treated. In one embodiment, different doses of the compositions provided herein are administered to a mammal via the same route of administration. In one embodiment, different doses of the compositions provided herein are administered to a mammal via different routes of administration. For example, a first dose of one or more selected artificial analogs of ANPs provided herein (e.g., one or more selected artificial analogs of ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) can be administered subcutaneously, and a second dose can be administered intranasally.

[0064] In one embodiment, an effective amount of an artificial analog of one or more selected ANPs provided herein (e.g., an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one 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) and does not cause significant toxicity to the mammal. In one embodiment, an effective amount of a nucleic acid encoding an artificial analog of one or more selected ANPs provided herein (e.g., a nucleic acid encoding an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one 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) and does not cause significant toxicity to the mammal. In one embodiment, an effective amount of the artificial analogs of one or more selected ANPs provided herein (e.g., artificial analogs of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 3-7) and nucleic acids encoding the artificial analogs of one or more selected ANPs provided herein (e.g., nucleic acids encoding artificial analogs of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one 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 causing significant toxicity to the mammal. The effective amount may remain constant or may be adjusted as a smooth incremental or variable dose depending on the mammal's response to treatment. Without being bound by theory, various factors can affect the actual effective amount used for a particular application. The dose can be adjusted by one of ordinary skill in the art or a treating physician.

[0065] In one embodiment, an effective amount of an artificial analog of one or more selected ANPs provided herein (e.g., an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) 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 mg, or 100 mg. In one embodiment, an effective amount of an artificial analog of one or more selected ANPs provided herein (e.g., an artificial analog of one or more selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is about 1 mg to 100 mg, about 1 mg to 80 mg, about 0.5 mg to 80 mg, about 0.5 mg to 60 mg, or about 1 mg to 50 mg. In one embodiment, the oral dosage of one or more artificial analogs of selected ANPs provided herein (e.g., one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is about 1 mg to 100 mg, about 1 mg to 80 mg, about 0.5 mg to 80 mg, about 0.5 mg to 60 mg, or about 1 mg to 50 mg. In one embodiment, the effective oral dosage of one or more artificial analogs of selected ANPs provided herein (e.g., one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is 1 mg to 50 mg. In one embodiment, an effective amount by subcutaneous administration of one or more artificial analogs of selected ANPs provided herein (e.g., one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) is about 0.01 μg / kg to 50 μg / kg, about 0.1 μg / kg to 40 μg / kg, about 0.1 μg / kg to 30 μg / kg, about 0.05 μg / kg to 40 μg / kg, or about 0.05 μg / kg to 30 μg / kg.

[0066] In one aspect, an effective administration frequency of one or more artificial analogs of selected ANPs provided herein (e.g., one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) and / or a nucleic acid encoding one or more artificial analogs of selected ANPs provided herein (e.g., a nucleic acid encoding one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) can be a frequency that alleviates symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) and does not cause significant toxicity to the mammal. In one embodiment, the effective administration frequency of one or more selected artificial analogs of ANP provided herein (and / or nucleic acids encoding one or more selected artificial analogs of ANP provided herein) can be about once every 2 hours, about once every 3 hours, about once every 4 hours, about once every 5 hours, about once every 6 hours, about once every 7 hours, about once every 8 hours, about once every 9 hours, about once every 10 hours, about once every 11 hours, about once every 12 hours, about once every day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, or about once every year. Without being bound by theory, various factors can affect the actual effective administration frequency used in a particular application. The frequency of administration can be adjusted by one skilled in the art or treating physician.

[0067] In one aspect, an effective administration period for one or more artificial analogs of selected ANPs provided herein (e.g., one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) and / or a nucleic acid encoding one or more artificial analogs of selected ANPs provided herein (e.g., a nucleic acid encoding one or more artificial analogs of selected ANPs comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3-7) can be a period that relieves symptoms (e.g., shortness of breath, systolic blood pressure, diastolic blood pressure) in a mammal (e.g., a human) and does not cause significant toxicity to the mammal. In one embodiment, the effective administration frequency of one or more selected artificial analogs of ANP provided herein (and / or nucleic acids encoding one or more selected artificial analogs of ANP provided herein) can be about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or as long as necessary. Without being bound by theory, various factors can affect the actual effective administration period used in a particular application. The administration period can be adjusted by a skilled artisan or treating physician.

[0068] The present disclosure also provides kits comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) artificial analogs of selected ANPs provided herein (e.g., one or more substantially pure artificial analogs of selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7) and / or nucleic acids encoding one or more artificial analogs of selected ANPs provided herein (e.g., nucleic acids encoding one or more artificial analogs of selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7). In one aspect, the kits provided herein comprise one or more artificial analogs of selected ANPs provided herein (e.g., one or more substantially pure artificial analogs of selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7). In one embodiment, the kits provided herein include nucleic acids encoding artificial analogs of one or more selected ANPs provided herein (e.g., nucleic acids encoding artificial analogs of one or more selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7). In one embodiment, the kits provided herein include artificial analogs of one or more selected ANPs provided herein (e.g., artificial analogs of one or more substantially pure selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7) and nucleic acids encoding artificial analogs of one or more selected ANPs provided herein (e.g., nucleic acids encoding artificial analogs of one or more selected ANPs that comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 3-7).

[0069] The examples described herein illustrate one or more aspects of the present disclosure, but should not be construed as limiting the scope of the disclosure in any way. [Example]

[0070] Example 1 Synthesis of CRRL 191: an activator of GC-A CRRL 191 is a novel 48-amino acid (AA) designer natriuretic peptide that activates GC-A (Figure 1B). CRRL 191 is synthesized by Bachem Ltd. (Torrance, CA) using solid-phase peptide synthesis (see Table 2). After synthesis, the peptide is purified by high-performance liquid chromatography (HPLC) and oxidized to form a cyclized ring between cysteine ​​amino acid residues 15 and 31. The structure, as shown in Figure 1B, is confirmed by MALDI mass spectrometry, and the purity is determined to be 97.9% by HPLC analysis. The average mass of the cyclized CRRL 191 is 5557.27 daltons. [Table 2]

[0071] Example 2 Measurement of GC-A activation and cyclic GMP production in HEK293 cells HEK293 cells were stably transfected with human GC-A (cDNA clone from Origene, Rockville, MD) using Lipofectamine (Invitrogen, Grand Island, NY). Receptor overexpression was confirmed by immunofluorescence and Western blotting. GC-A-transfected HEK293 (HEK / GCA) cells 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.

[0072] Two in vitro cGMP assays are performed. Briefly, HEK / GCA cells are seeded into 48-well plates and cultured overnight to reach 80-90% confluency. The treatment buffer used in all experiments contains Hank's balanced salt solution (HBSS), 0.1% bovine serum albumin (BSA), 2 mM HEPES, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor) (Sigma, St. Louis, MO).

[0073] In the first experiment, cells were cultured at different concentrations (10 -14 From 10 -6 The cells were stimulated with 0.1 M ANP or CRRL 191 for 10 min. After treatment, all cells were washed once with phosphate-buffered saline (PBS) and lysed with 0.1 M HCl. Intracellular cGMP in the lysates was measured using a commercially available cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions.

[0074] As shown in Table 3, the EC of CRRL 191 was significantly higher than that of ANP after 10 minutes of stimulation. 50 showed higher activity. [Table 3]

[0075] In the second experiment, ANP or CRRL 191 (10 -8 M) is added to the treatment buffer and the cells are incubated for 6 hours. After treatment, all cells are washed once with phosphate-buffered saline (PBS) and lysed with 0.1 M HCl. Intracellular cGMP in the lysates is measured using a commercially available cGMP ELISA kit (EnzoLifeSciences, Farmingdale, NY) according to the manufacturer's instructions.

[0076] As shown in Figure 2, both ANP and CRRL 191 activate the GC-A receptor and increase cGMP production at similar rates for up to approximately 30 minutes. After 30 minutes, GC-A receptor activation by CRRL 191, as indicated by increased cGMP production, continues to increase for at least 6 hours, while GC-A receptor activation by ANP reaches a steady state and declines.

[0077] Example 3 In vitro neprilysin degradation assay In vitro degradation of ANP and CRRL 191 peptides by recombinant human neprilysin (NEP) (R&D systems, Minneapolis, MN) was measured by cGMP production in HEK / GCA cells. -5 Five microliters of peptide at 100 M was incubated with 5 μl (10 ng / μl) of recombinant NEP in 90 μl of Tris / 0.1% BSA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% BSA) for various times ranging from 15 minutes to 24 hours at 37°C. The peptide at 0 minutes was incubated without NEP (buffer only). After the required incubation time (0 minutes to 24 hours), 100 μl of 0.5 N perchloric acid was added to the reaction solution to inactivate NEP and stop degradation, and 20 μl of 2.5 N NaOH was added to neutralize the solution. The neutralized reaction aliquot was added to HEK / GCA cells, and the undegraded peptide (final concentration 5.5X10 -8 The cGMP production capacity of each of the 191- and 192-fold diluted 1M cGMP-producing antibodies (diluted to 1M) is measured using the same commercially available cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY) described in Example 2 according to the manufacturer's instructions. The degradation of ANP and CRRL 191 is calculated by the decrease in cGMP production capacity in HEK / GCA cells compared to the basal cGMP production capacity at 0 min.

[0078] As shown in Figure 3, CRRL 191 is highly resistant to degradation by neprilysin compared to ANP, as measured by this in vitro assay. After 30 minutes of degradation by neprilysin, CRRL 191 retains approximately 90% of its original biological activity, while ANP retains less than 5% of its original biological activity. After 6 hours of degradation by neprilysin, CRRL 191 retains 84% ​​of its original biological activity, demonstrating significant resistance and stability compared to ANP.

[0079] Example 4 cGMP analysis of human cardiomyocytes (HCM) Human cardiomyocytes (HCM) were purchased from ScienCell (catalog no. 6200) and maintained in cardiomyocyte medium (catalog no. 6101, ScienCell) containing 10% FBS. In vitro cGMP assays were performed. Briefly, HCM cells were seeded into 6-well plates and cultured overnight to reach 80-90% confluency. The treatment buffer used in all experiments contained Hank's balanced salt solution (HBSS), 0.1% BSA, 2 mM HEPES, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor) (Sigma, St. Louis, MO). For test samples, HCM were cultured in a 100% ethanol-free medium containing either ANP or CRRL 191 (100%). -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 treatment buffer containing 0.1 M HCl for 10 min. As a negative control, HCMs were incubated in treatment buffer (vehicle) alone. After 10 min of treatment, all cells were washed once with phosphate-buffered saline (PBS), lysed with 0.1 M HCl, and sonicated for 10 min. Intracellular cGMP in the lysates was measured using a commercially available cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions.

[0080] As shown in Figure 4, CRRL 191 induces higher levels of cGMP production in human primary cells. -12 M to 10 -11 Up to M, CRRL 191 and ANP induce similar levels of cGMP production. -10 M to 10 -6 Up to M, CRRL 191 induces higher cGMP production than ANP.

[0081] As shown in Figure 5, CRRL 191 induces higher levels of cGMP production in human primary cells compared to two other GC-A activators, MANP and CRRL 101. -9 M to 10 -6 Up to M, CRRL 191 induced higher levels of cGMP production than MANP or CRRL 101.

[0082] Example 5 Real-time apoptosis analysis of human cardiomyocytes (HCM) HCM cells were seeded into 96-well plates at approximately 80% confluency. The cells were treated with various concentrations of staurosporine (SP) to induce apoptosis. Based on a dose-response curve, 0.05 μM SP was selected to induce apoptosis in HCM.

[0083] On the day of the experiment, HCMs were incubated in cell growth medium containing 0.05 μM SP, various concentrations of ANP or CRRL 191, and 5 μmol / L IncuCyte Caspase-3 / 7 Green Reagent (Essen Bioscience, Ann Arbor, MI). Plates were then transferred to a time-lapse live imaging system, IncuCyte S3 (Essen Bioscience, Ann Arbor, MI), and apoptosis was monitored every 2 hours for a total of 90 hours. All treatment groups were run in quintuplicate and replicated twice. Images were collected using IncuCyte S3 Live-Cell Analysis System Software (Essen Bioscience, Ann Arbor, MI), and data were analyzed for the green object area of ​​apoptotic bodies in each well.

[0084] As shown in Figure 6, 10 -10 and 10 -8 CRRL 191 at a dose of 10 m attenuated SP-induced cardiomyocyte apoptosis, whereas CRRL 191 at a dose of 10 m attenuated SP-induced cardiomyocyte apoptosis. -6 The M dose of CRRL 191 was most effective in attenuating SP-induced cardiomyocyte apoptosis.

[0085] Example 6 GC-A binding study Surface plasmon resonance (SPR) measurements were performed at 25°C using a BI-4500SPR instrument (Biosensing Instrument Inc., Tempe, AZ). The extracellular domain of GC-A recombinant protein (MyBioSource, Inc., San Diego, CA) containing a C-terminal His tag was immobilized on a Ni-NTA sensor chip (Biosensing Instrument Inc., Tempe, AZ) using 400 μM nickel sulfate in deionized water as a linker, according to the instrument manual. Next, 40 μg / ml of recombinant GC-A was immobilized on the 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 serially diluted CRRL 191 or ANP (0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM) was injected at a flow 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).

[0086] As shown in the binding curves, CRRL 191 (Fig. 7C) has stronger binding affinity to the GC-A receptor than ANP (Fig. 7A) and MANP (Fig. 7B). D The K value is 128 pM, and the K values ​​of ANP and MANP are D The values ​​are 280 pM and 233 pM, respectively. These data indicate that CRRL 191 binds to human GC-A better than ANP or MANP.

[0087] Example 7 In vivo rat experiments All rat experiments were conducted in accordance with the Animal Welfare Act, and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Mei-Oh Clinic. A total of 19 spontaneously hypertensive rats (SHR, male, 250-300 g) were randomly assigned to receive one of the following injections: (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 only accepted for experiments if they were deemed healthy by the institution's veterinary department. The investigator conducting the in vivo study was not blinded to the treatments, but biochemical analyses were performed by a separate investigator blinded to the treatments.

[0088] On the day of the experiment, SHR rats were anesthetized with Inactin (120 mg / kg, intraperitoneal injection, Sigma, St. Louis, MO). Rats were then placed on a 38°C heating pad to maintain normal body temperature throughout the experiment. Vascular and bladder catheterization procedures were performed. Briefly, a polyethylene-50 (PE-50) tubing catheter was inserted into one jugular vein for peptide infusion, and another PE-50 tubing was inserted into the carotid artery for blood pressure (BP) monitoring (Sonometrics, London, ON, Canada) and blood sampling. After instrumentation, a 15-minute equilibration period was performed, followed by a 30-minute pre-infusion period for baseline urine sample collection. Following the pre-infusion period, CRRL 191, ANP, or vehicle was continuously infused for 45 minutes (a 15-minute drug infusion lead-in period and a 30-minute clearance period during drug infusion).

[0089] Immediately after the peptide infusion is stopped, another 30-minute washout clearance period is initiated before termination and sacrifice. Four blood samples are taken to determine circulating cGMP levels: at baseline, before the end of the drug infusion, midway through the washout clearance period, and before the end of the washout clearance period. The rats' blood loss is replaced with an equal volume of saline. After the start of the peptide infusion, blood pressure is monitored and recorded at 15-minute intervals. All blood samples are stored at -80°C until analysis.

[0090] For biochemical analysis, plasma cGMP levels are measured using a cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY).

[0091] CRRL 191 is a more effective antihypertensive agent compared to ANP and vehicle. As shown, CRRL 191 at doses of 100 pmol / kg and 300 pmol / kg is more effective than 100 pmol / kg or 300 pmol / kg ANP in lowering systolic blood pressure (FIG. 8A), diastolic blood pressure (FIG. 8B), and mean arterial pressure (FIG. 8C) in spontaneously hypertensive rats, demonstrating a more sustained effect over the time course following peptide administration.

[0092] Finally, administration of ANP or CRRL 191 increased plasma cGMP levels in spontaneously hypertensive rats (Figure 9A). However, CRRL 191 at doses of 100 pmol / kg and 300 pmol / kg was more effective at activating plasma cGMP than 100 pmol / kg or 300 pmol / kg ANP, and the activation after peptide administration was more sustained (Figure 9B).

[0093] Other embodiments While the present disclosure has been described in connection with its detailed description, it should be understood that the foregoing description is intended to illustrate, but not limit, the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. A list of exemplary embodiments is provided below:

[0094] Embodiment 1 A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0095] Embodiment 2 A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, wherein the polypeptide further comprises one, two, or three amino acid substitutions, additions, or deletions.

[0096] Embodiment 3 A polypeptide comprising, consisting essentially of, or consisting of a sequence comprising at least 90% homology to any one of SEQ ID NOs: 3 to 7.

[0097] Embodiment 4 4. The polypeptide of embodiment 3, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3 to 7.

[0098] Embodiment 5 4. The polypeptide of embodiment 3, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3 to 7.

[0099] Embodiment 6 A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO:7.

[0100] Embodiment 7 A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0101] 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 to 7.

[0102] Embodiment 9 9. The substantially pure polypeptide of embodiment 8, wherein the sequence has at least 95% homology to any one of SEQ ID NOs: 3-7.

[0103] Embodiment 10 9. The substantially pure polypeptide of embodiment 8, wherein the sequence has at least 97% homology to any one of SEQ ID NOs: 3-7.

[0104] Embodiment 11 A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, wherein the polypeptide contains one, two, or three amino acid substitutions, additions, or deletions.

[0105] Embodiment 12 A substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO:7.

[0106] Embodiment 13 A composition comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0107] Embodiment 14 A composition comprising a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, wherein the polypeptide further comprises one, two, or three amino acid substitutions, additions, or deletions.

[0108] Embodiment 15 A composition comprising at least two polypeptides, each of which is a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0109] Embodiment 16 A composition comprising, consisting essentially of, or consisting of the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO:7.

[0110] Embodiment 17 17. The composition of any one of embodiments 13-16, further comprising one or more pharmaceutically acceptable excipients.

[0111] Embodiment 18 18. The composition of any one of embodiments 13-17, further comprising an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

[0112] Embodiment 19 19. The composition of any one of embodiments 13-18, further comprising furosemide.

[0113] Embodiment 20 A nucleic acid encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0114] Embodiment 21 A composition comprising a nucleic acid encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0115] Embodiment 22 A composition comprising a nucleic acid encoding at least two polypeptides, each of which is a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0116] Embodiment 23 23. The composition of any one of embodiments 21-22, further comprising an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

[0117] Embodiment 24 24. The composition of any one of embodiments 21-23, further comprising furosemide.

[0118] Embodiment 25 The composition of any one of embodiments 21 to 24, wherein the nucleic acid is in the form of a non-viral vector.

[0119] Embodiment 26 26. The composition of embodiment 25, wherein the non-viral vector is an expression plasmid.

[0120] Embodiment 27 25. The composition of any one of embodiments 21 to 24, wherein the nucleic acid is in the form of a viral vector.

[0121] Embodiment 28 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, the method comprising administering to the mammal a composition comprising a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, or a nucleic acid encoding the polypeptide.

[0122] Embodiment 29 29. The method of embodiment 28, wherein the mammal is a human.

[0123] Embodiment 30 The method of any one of embodiments 28-29, wherein the cardiovascular, cardiorenal, or metabolic disease is hypertension.

[0124] Embodiment 31 The method of any one of embodiments 28-30, wherein the cardiovascular, cardiorenal, or metabolic disease is resistant hypertension.

[0125] Embodiment 32 The method of any one of embodiments 28-31, wherein the composition comprises an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

[0126] Embodiment 33 The method of any one of embodiments 28-32, wherein the composition comprises furosemide.

[0127] Embodiment 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), the method comprising administering to the mammal a composition comprising a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, or a nucleic acid encoding the polypeptide.

[0128] Embodiment 35 35. The method of embodiment 34, wherein the mammal is a human.

[0129] Embodiment 36 The method of any one of embodiments 34-35, wherein the cardiovascular, cardiorenal, or metabolic disease is hypertension.

[0130] Embodiment 37 The method of any one of embodiments 34-36, wherein the cardiovascular, cardiorenal, or metabolic disease is resistant hypertension.

[0131] Embodiment 38 The method of any one of embodiments 34-37, wherein the composition comprises an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

[0132] Embodiment 39 The method of any one of embodiments 34-38, wherein the composition comprises furosemide.

[0133] Embodiment 40 A method for alleviating 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, the method comprising administering to the mammal a composition comprising a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, or a nucleic acid encoding the polypeptide.

[0134] Embodiment 41 41. The method of embodiment 40, wherein the mammal is a human.

[0135] Embodiment 42 The method of any one of embodiments 40-41, wherein the cardiovascular, cardiorenal, or metabolic disease is hypertension.

[0136] Embodiment 43 The method of any one of embodiments 40-42, wherein the cardiovascular, cardiorenal, or metabolic disease is resistant hypertension.

[0137] Embodiment 44 The method of any one of embodiments 40-43, wherein the composition comprises an agent selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), and a calcium channel blocker (CCB).

[0138] Embodiment 45 The method of any one of embodiments 40-44, wherein the composition comprises furosemide.

Claims

1. A polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

2. A polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 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 to 7.

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

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

6. A polypeptide comprising the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO:

7.

7. A substantially pure polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 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. 9. The substantially pure polypeptide of claim 8, wherein the sequence has at least 95% homology with any one of SEQ ID NOs: 3-7.

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

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

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

7.

13. A composition comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

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

15. A composition comprising at least two polypeptides, each of which comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

16. A composition comprising the amino acid sequence set forth in amino acids 1 to 8 of SEQ ID NO:

7.

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

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

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

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

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

22. A composition comprising a nucleic acid encoding at least two polypeptides, each of the at least two polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7.

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

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

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

28. A method 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, non-alcoholic steatohepatitis (NASH)) in a mammal, the method comprising administering to the mammal a composition comprising a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7 or a nucleic acid encoding the polypeptide.

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

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

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

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

33. 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 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, non-alcoholic steatohepatitis (NASH)), the method comprising administering to the mammal a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7, or a composition comprising a nucleic acid encoding the polypeptide.

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

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

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

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

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

40. A method for alleviating 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, non-alcoholic steatohepatitis (NASH)) in a mammal, the method comprising administering to the mammal a composition comprising a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 7 or a nucleic acid encoding the polypeptide.

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

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

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

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

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

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