Long-acting natriuretic peptides and uses thereof

Abstract

To provide long-acting atrial natriuretic peptide (ANP) polypeptides that bind to natriuretic peptide receptors such as NPR-A, thereby function as NPR-A agonists, and exhibit improved stability.SOLUTION: Provided are atrial natriuretic peptides having specific structural features. The structural features also result in polypeptides having sufficient activity at NPR-A and also result in polypeptides having many other beneficial attributes associated with their developability as therapeutic treatments, including improved solubility of analogs in aqueous solution, improved chemical and physical formulation stability, extended pharmacokinetic profiles, and minimization of immunogenicity potential.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates generally to biology and medicine, and more particularly to peptides that are natriuretic peptide analogs, particularly long-acting atrial natriuretic peptide (ANP) polypeptides that bind to natriuretic peptide receptors such as NPR-A, thereby functioning as NPR-A agonists, and exhibiting improved stability. The disclosure further relates to compositions comprising such peptides and their use in treating cardiovascular conditions, diseases, or disorders. [Background technology]

[0002] There is an unmet medical need for new and improved treatments for heart failure (HF). Currently available treatments aim to slow disease progression and improve symptoms, relying on hemodynamic changes to reduce the burden on the failing heart. These therapies include (a) drugs intended to reduce heart rate, such as beta-blockers and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers (e.g., ivabradine); (b) drugs intended to reduce blood pressure, such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), mineralocorticoid receptor antagonists (MRAs), and the combination of an ARB and a neprilysin (NEP) inhibitor (sacubitril / valsartan (ENTRESTO®)); and / or (c) drugs intended to treat or prevent volume overload, such as diuretics and MRAs. However, these therapies have practical limitations, such as not directly treating the heart and requiring dose titration and monitoring for hypotension. Furthermore, even with the availability of these existing treatment options, all HF patients, even those with mild symptoms, have a high risk of mortality. See, e.g., Ahmed A, A propensity matched study of New York Heart Association class and natural history end points in heart failure, AM. J. CARDIOL. 2007;99(4):549-553. Therefore, new and improved HF treatments are needed.

[0003] Natriuretic peptides (NPs) are a class of endogenous hormones that confer cardiovascular protection through the regulation of fluid homeostasis. They include four structurally related peptide hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and dendroaspis natriuretic peptide (DNP). Three subtypes of natriuretic peptide receptors (NPRs) have been described: NPR-A, NPR-B, and NPR-C.

[0004] Wild-type human ANP is a 28-amino acid peptide with a 17-amino acid loop formed by an intramolecular disulfide bond between two cysteine ​​residues at positions 7 and 23. It is a cardiac hormone that is part of the body's natural defense against hypoxia and pathological cardiac wall stress. ANP is released in response to myocardial wall stress and induces natriuretic, diuretic, and vasodilatory effects. ANP acts via NPR-A to activate the pGC-cGMP pathway and increase intracellular cGMP levels. NPR-A agonists have direct antihypertrophic and antifibrotic effects in the heart, improve pulmonary function, and may have beneficial effects on glucose and energy metabolism. ANP treatment can translate into improved cardiac filling pressures, promote beneficial cardiac remodeling, improve diastolic function, and exert cardioprotective effects in the heart, vasculature, lungs, and kidneys.

[0005] However, wild-type ANP undergoes rapid circulatory clearance, which may be due to its binding to the natriuretic peptide receptor C (NPR-C) followed by internalization and lysosomal proteolysis, proteolytic cleavage by endopeptidases, and renal secretion. Human ANP has an in vivo half-life of only a few minutes. A naturally occurring amino-terminally extended form of ANP, urodilatin, is more resistant to enzymatic degradation but also has an in vivo half-life of only approximately 6 minutes. Polypeptides with such short half-lives typically require administration by continuous intravenous infusion in a hospital or other medical facility. This is often inconvenient for the individual receiving the polypeptide, but it often results in short-term efficacy, typically in a hospital or other medical facility. Short-term intravenous infusion of recombinant ANP (carperitide) has been approved in Japan and has demonstrated some rapid benefits. However, short-term infusions of approximately 48 hours have not demonstrated any benefit in long-term outcomes.

[0006] Several techniques exist to extend peptide half-life, including peptide conjugation to fatty acid moieties, to recombinant human serum albumin (rHSA) or bovine serum albumin (BSA), to pharmaceutically acceptable polymers such as polymeric sequences of amino acids (XTEN), to non-sulfated heparin-like carbohydrate polymers (HEP) or hydroxyl ethyl starch (HES), to llama heavy chain antibody fragments (VHH), PEGylation, and Fc conjugation (see, e.g., Sleep, D. Epert Opin Drug Del (2015) 12, 793-812; Podust VN et. al. J Control. Release, 2015; ePUB; Hey, T. et. al. in: R. Kontermann (Ed.), Therapeutic Proteins: Strategies to Modulate their Plasma See Half-Lives, Wiley-VCH Verlag Gmbh & Co. KGaA, Weinheim, Germany, 2012, pp117-140; DeAngelis, PL, Drug Dev Delivery (2013) January, 12 / 31 / 2012).

[0007] Efforts have been made to prepare ANP analogues and derivatives that mimic the biological activity of natural ANP and / or have improved stability.For example, European Patent No. 465097; U.S. Patent No. 4,607,023; U.S. Patent No. 5,212,286; U.S. Patent No. 5,434,133; U.S. Patent No. 6,525,022; U.S. Patent No. 8,058,242; U.S. Patent No. 9,193,777; U.S. Patent No. 10,947,289; U.S. Patent No. 11,312,758; International Publication No. 1988 / 03537; International Publication No. 1998 / 45329; International Publication No. 2004 / 011498; and International Publication No. 2018 / 175534 describe various ANP analogues and derivatives that have higher stability. U.S. Patent No. 5,204,328 describes ANP analogs containing N-alkylated amino acids to protect the peptide from enzymatic degradation. U.S. Patent No. 6,525,022 describes ANP analogs with equal binding affinity to NPR-A but reduced affinity to NPR-C. WO 1998 / 45329 describes ANP derivatives in which a lipophilic substituent is linked to a peptide. WO 2004 / 011498 describes ANP derivatives containing a reactive element that can be linked to a peptide to make it capable of forming a peptide-blood component complex. U.S. Patent No. 9,193,777 describes ANP analogs containing a 12-amino acid C-terminal extension based on a familial ANP gene frameshift mutation. U.S. Patent No. 10,947,289 describes sugar-modified ANP derivatives in which a sugar moiety is linked to a peptide. WO 2008 / 154226 describes an ANP fusion protein linked to an antibody Fc fragment.

[0008] Nevertheless, there remains a need for alternative treatment options. Therapies that improve long-term outcomes, such as increased survival and reduced hospitalization rates, are needed. Therapies that improve cardiac function, potentially correcting or reversing the disease, are also needed. Therapies that improve quality of life (QoL) for patients with progressive disease are also needed. There is also a need for therapeutic agents available for use with a sufficiently long duration of action to allow for less frequent administration, such as once daily, three times a week, twice a week, or once a week. The present invention seeks to fulfill one or more of these important unmet needs. Summary of the Invention

[0009] Provided herein are ANP polypeptides that bind to and agonize NPR-A and have natriuretic, diuretic, and vasorelaxant activity. The ANP polypeptides described herein also have a long duration of action on NPR-A, allowing for less frequent administration, such as once daily, three times a week, twice a week, or once a week. The ANP polypeptides described herein also exhibit a desirable developability profile, making them suitable for use in therapeutic applications. Thus, the ANP polypeptides described herein may be useful in long-term treatments for lowering blood pressure, reducing pathological wall stress, and ameliorating adverse cardiac remodeling, and may also have beneficial effects on pulmonary congestion.

[0010] Accordingly, the present disclosure also provides methods of using ANP polypeptides for treating or preventing cardiovascular disease (CVD) and related disorders, particularly heart failure (HF). Preferred ANP polypeptides and methods of the invention reduce the risk of CV-related death or HF-related hospitalization, reduce the risk of myocardial infarction (MI) or stroke, reduce the likelihood of needing a left ventricular assist device (LVAD) or heart transplant, improve cardiac function and structure, and / or improve HF-related symptoms and physical limitations, leading to improved quality of life.

[0011] In one embodiment, provided herein is a compound of formula I: X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (SEQ ID NO: 3), (In the formula, X1 is absent, S or E; X2 is absent or is L, K, 4-Pal, H, or E; X3 is absent or is R, β-Ala, P, K, E, or G; X9 is G, 4-Pal, or H; X 10 is G, K, R, or Dap; X 11 is R, K, G, or Dap; X 13 is D or G, X 17 is A, H, Dap, K, R, or Orn, X 18 is Q, Y, or 4-Pal, X 26 is F or L, X 28is Y, H, or 4-Pal, and X 29 does not exist, or GGP, SGAPPPE (SEQ ID NO: 4), KITAKEDE (SEQ ID NO: 5), GPSSGAPPPE (SEQ ID NO: 6), GPSSGAPPPS (SEQ ID NO: 7), GGSSGAPPPS (SEQ ID NO: 8), GGPSSGAPPPS (SEQ ID NO: 9), KGPSSGAPPPS (SEQ ID NO: 10), GGKSSGAPPPS (SEQ ID NO: 11), GGPPS-Aib-KPPPK (SEQ ID NO: 12), GSPSSGAPPPS (SEQ ID NO: 13), RITAREDKQGYA (SEQ ID NO: 14), RITAREDKQGEA (SEQ ID NO: 15), GSPSSGAPPPS-PEG24-G (SEQ ID NO: 16), SGSPSSGAPPPSG (SEQ ID NO: 17), GGESSGEPPPSEE (SEQ ID NO: 18), GSGSPSSGAPPPSG (SEQ ID NO: 19), and SGSPSSGAPPPSEEEG (SEQ ID NO: 20), The C-terminal amino acid is optionally amidated or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the polypeptide contains a disulfide bond between the cysteine ​​at position 7 and the cysteine ​​at position 23 (between C7 and C23). In some embodiments, the polypeptide contains a thioacetal bond between the cysteine ​​at position 7 and the cysteine ​​at position 23 (between C7 and C23).

[0013] In another embodiment, the polypeptide of Formula I or a pharmaceutically acceptable salt thereof is conjugated to a fatty acid. For example, in some embodiments, the polypeptide of Formula I or a pharmaceutically acceptable salt thereof further comprises a fatty acid conjugated to an amino acid present at the N-terminus of the polypeptide, and has the formula II: Fatty acids-Z1-Z2-Z3-X1X2X3RSSCFX9X 10 X 11 IDRIGX 17 X 18 SGLGCX 24 SX 26 RX 28 X 29 (SEQ ID NO: 21), wherein the fatty acid is C 16 ~C 26 a fatty acid conjugated to an amino acid present at the N-terminus of a polypeptide via the structure Z1-Z2-Z3, wherein: Z1 comprises an amino acid selected from γGlu, E, and β-Ala; Z2 comprises a sequence of 4 to 10 amino acids that is absent or contains amino acids independently selected from E, K, G, P, A, and S; Z3 is absent or contains a polyethylene glycol (PEG) or (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety It includes the basic structure from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).

[0014] In some embodiments, Z1 is an amino acid selected from γGlu, E, and β-Ala.

[0015] In some embodiments, Z2 is APPSG, (EK) b G, (EP) b G, K (EK) c G, and (EK) cE, b is 2, 3, or 4, and c is 1, 2, 3, or 4. For example, in some embodiments, Z2 is EKEKEKG (SEQ ID NO: 22), EPEPEPG (SEQ ID NO: 23), APPSG (SEQ ID NO: 24), KEKEKG (SEQ ID NO: 25), or EKEKEKE (SEQ ID NO: 26).

[0016] In some embodiments, Z3 is (polyethylene glycol) m (wherein m is an integer selected from 10 to 30) and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) n (wherein n is an integer selected from 2 to 10). For example, in some embodiments, Z3 is (polyethylene glycol) 12 , (polyethylene glycol) 24 , ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))4, (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))6, or (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))8.

[0017] In another embodiment, there is provided a pharmaceutical composition comprising a polypeptide described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] In another embodiment, provided herein are methods of using a polypeptide described herein or a pharmaceutically acceptable salt thereof for treating or preventing cardiovascular disease (CVD) and related disorders. Such methods may comprise at least the step of administering to an individual in need thereof an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof. In some examples, the CVD is heart failure (HF), particularly heart failure with preserved ejection fraction (HfpEF).

[0019] In another embodiment, the polypeptides described herein or pharmaceutically acceptable salts thereof are provided for use in therapy.

[0020] In another embodiment, the polypeptides described herein or pharmaceutically acceptable salts thereof are provided for use in treating or preventing CVD. In some instances, the CVD is HF, particularly HfpEF.

[0021] In another embodiment, a polypeptide described herein or a pharmaceutically acceptable salt thereof is provided for use in the manufacture of a medicament for treating or preventing CVD. In some instances, the CVD is HF, particularly HfpEF. DETAILED DESCRIPTION OF THE INVENTION

[0022] 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the ANP polypeptides, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0023] Also, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that there is a plurality of elements, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0024] As used herein, "about" means within a statistically significant range of a value(s), such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0025] As used herein, with respect to one or more of the ANP receptors, "activity," "activate," "activating," and the like refer to the ability of a compound, such as the ANP polypeptides described herein, to bind to the receptor and induce a response thereat, as measured using assays known in the art, such as the in vitro assays described below.

[0026] As used herein, "ANP polypeptide" refers to an ANP polypeptide that has structural similarity to naturally occurring ANP (particularly rat ANP (SEQ ID NO: 1) or human ANP (SEQ ID NO: 2)), but has some differences. The ANP polypeptides described herein include amino acid sequences that result in the polypeptide having affinity for and activity at the NPR-A receptor. The term "ANP polypeptide" also includes acylated or otherwise derivatized ANP polypeptides.

[0027] As used herein, "conservative substitution" refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for one or more conservative amino acid substitutions in its amino acid sequence. Generally, conservatively modified variants contain an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference amino acid sequence. More specifically, a conservative substitution refers to the substitution of an amino acid with an amino acid that has similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, and rigidity) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution of functionally similar amino acids is well known in the art and need not be described exhaustively herein.

[0028] As used herein, "C 16 ~C 26 "Fatty acid" means a carboxylic acid having 16 to 26 carbon atoms. 16 ~C 26 The fatty acids may be straight-chain or branched-chain fatty acids. 16 ~C 26 Fatty acids can be saturated monoacids or saturated diacids. As used herein, "saturated" means that the fatty acid does not contain any carbon-carbon double or triple bonds.

[0029] As used herein, "effective amount" refers to an amount, concentration, or dosage of one or more ANP polypeptides described herein, or pharmaceutically acceptable salts thereof, that provides a desired effect in an individual under diagnosis or treatment after single or multiple administration to such individual in need thereof. An effective amount is also an amount in which any toxic or detrimental effects of the polypeptide are outweighed by the therapeutically beneficial effects. An effective amount can be determined by one skilled in the art by using known techniques and observing results obtained under analogous circumstances. In determining an effective amount for a subject, several factors are taken into consideration, including, but not limited to, the species of mammal; its size, age, and general health; the particular disease or disorder involved; the extent or involvement or severity of the disease or disorder; the individual patient's response; the particular ANP polypeptide administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0030] As used herein, "extended duration of action" means that the binding affinity and activity for the ANP polypeptide continues for a longer period than native human ANP polypeptide, allowing administration at least once daily, three times a week, twice a week, once a week, or less than once a week, e.g., every other week (once every two weeks) or once a month. The time-action profile of the ANP polypeptide may be measured using known pharmacokinetic testing methods, such as those utilized in the Examples below.

[0031] As used herein, "half-life" or "t1 / 2" refers to the time it takes for half of the amount of a compound, such as native ANP or an ANP polypeptide herein, to be removed by biological processes from a fluid, such as serum or plasma, or other physiological space of an individual. Alternatively, t1 / 2 can refer to the time it takes for an amount of such a compound to lose half of its pharmacological, physiological, or radiological activity.

[0032] As used herein, "50% effective concentration" or "EC 50" means the concentration of a polypeptide that results in 50% activation / stimulation of an assay endpoint, such as a dose-response curve (e.g., the cGMP signaling pathway).

[0033] As used herein, "in combination with" means administration of at least one of the ANP polypeptides herein simultaneously, sequentially, or in a single combined formulation (combination) with one or more additional therapeutic agents.

[0034] As used herein, "individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein.

[0035] As used herein, "long-acting" means that the binding affinity and activity of the ANP polypeptide herein lasts for a longer period of time than native human ANP (SEQ ID NO: 2), allowing it to be administered at least once daily, or even three times a week, twice a week, or once a week. The time-action profile of the ANP polypeptide may be measured using known pharmacokinetic testing methods, such as those described in the Examples below.

[0036] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the polypeptide of the present invention, wherein the polypeptide of the present invention is modified by making an acid salt or base salt thereof. Pharmaceutically acceptable salts and processes for making them are well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, LV Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the polypeptides described herein, formed, for example, from non-toxic inorganic or organic acids. Such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isethionic acid. Pharmaceutically acceptable refers to a form of the polypeptides described herein that is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salt forms of the polypeptides herein can be synthesized to contain a basic or acidic moiety by conventional chemical methods. Generally, such salts are prepared, for example, by reacting the free acid or free base forms of these polypeptides with a stoichiometric amount of a suitable base or acid in water or in an organic solvent, or in a mixture of the two.Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred (see, e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (Wiley-VCH 2nd ed. 2011)).

[0037] The term "pharmaceutical composition" as used herein refers to a composition having an effective amount of one or more peptides herein in combination with other chemical components such as binders, carriers, diluents, lubricants, pharmaceutical flow agents, and / or other excipients, particularly pharmaceutically acceptable carriers.

[0038] As used herein, "polypeptide" or "peptide" refers to a polymer of amino acid residues, generally comprising two or more amino acids and / or amino acid derivatives linked via peptide bonds. The term applies to polymers comprising naturally occurring amino acids as well as polymers comprising one or more non-naturally occurring amino acids. Embodiments may include modified or amino acid derivatives, including post-translational modifications such as phosphorylation, hydroxylation, sulfonation, palmitoylation, glycosylation, and disulfide formation.

[0039] As used herein, "treat," "treating," "to treat," and the like refer to the management or care of an individual having an abnormality, disease, disorder, or condition for which administration of an ANP polypeptide is indicated for the purpose of reducing, inhibiting, slowing, halting, or reversing the progression of the abnormality, disease, disorder, or condition or its severity. Treating includes administering an ANP polypeptide herein or a composition comprising an ANP polypeptide herein to an individual to prevent the onset of the condition or complications, reduce the symptoms or complications, or eliminate the abnormality, disease, disorder, or condition. Treating includes administering an ANP polypeptide herein or a composition comprising an ANP polypeptide herein to an individual to result in, for example, increased angiogenesis, increased vascular compliance, increased glomerular filtration rate, lowered blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) fibrosis in the heart, kidney, liver, or lung.

[0040] In one embodiment, provided herein is a compound of formula I: X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (SEQ ID NO: 3), (In the formula, X1 is absent, S or E; X2 is absent or is L, K, 4-Pal, H, or E; X3 is absent or is R, β-Ala, P, K, E, or G; X9 is G, 4-Pal, or H; X 10 is G, K, R, or Dap; X 11 is R, K, G, or Dap; X 13 is D or G, X 17is A, H, Dap, K, R, or Orn, X 18 is Q, Y, or 4-Pal, X 26 is F or L, X 28 is Y, H, or 4-Pal, and X 29 does not exist, or GGP, SGAPPPE (SEQ ID NO: 4), KITAKEDE (SEQ ID NO: 5), GPSSGAPPPE (SEQ ID NO: 6), GPSSGAPPPS (SEQ ID NO: 7), GGSSGAPPPS (SEQ ID NO: 8), GGPSSGAPPPS (SEQ ID NO: 9), KGPSSGAPPPS (SEQ ID NO: 10), GGKSSGAPPPS (SEQ ID NO: 11), GGPPS-Aib-KPPPK (SEQ ID NO: 12), GSPSSGAPPPS (SEQ ID NO: 13), RITAREDKQGYA (SEQ ID NO: 14), RITAREDKQGEA (SEQ ID NO: 15), GSPSSGAPPPS-PEG24-G (SEQ ID NO: 16), SGSPSSGAPPPSG (SEQ ID NO: 17), GGESSGEPPPSEE (SEQ ID NO: 18), GSGSPSSGAPPPSG (SEQ ID NO: 19), and SGSPSSGAPPPSEEEG (SEQ ID NO: 20), and the C-terminal amino acid is optionally amidated) or a pharmaceutically acceptable salt thereof.

[0041] The structural features described herein also result in polypeptides with sufficient activity at NPR-A, as well as many other beneficial attributes related to their potential development as therapeutic treatments, including improved solubility of the analogs in aqueous solution, improved chemical and physical formulation stability, an enhanced pharmacokinetic profile, and minimized potential immunogenicity.

[0042] In some embodiments, X1 is selected from S and E. In some embodiments, X2 is selected from K and 4-Pal. In some embodiments, X3 is selected from R, β-Ala, P, and K. In some embodiments, X9 is G, 4-Pal, or H. In some embodiments, X 10 is selected from G, K, R, and Dap. 11 is selected from R and K. In some embodiments, X 13 is selected from D and G. In some embodiments, X is H, K, R, Dap, or Orn. 18 is selected from Q and Y. In some embodiments, X 26 is F or L. In some embodiments, X 28 is H or 4-Pal. In some embodiments, X 29 is absent or selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11) and GSPSSGAPPPS (SEQ ID NO: 13).

[0043] In some embodiments, X1 is selected from S and E; X2 is selected from K and 4-Pal; X3 is selected from R, β-Ala, P, and K; X9 is G, 4-Pal, or H; and X 10 is selected from G, K, R, and Dap; X 11 is selected from R and K; X 13 is selected from D and G; X 17 is H, K, R, Dap, or Orn; X 18 is selected from Q and Y; X26 is F or L; X 28 is H or 4-Pal; and X 29 is absent or selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13).

[0044] In some embodiments, X1 is selected from S and E. In some embodiments, X2 is selected from K and 4-Pal. In some embodiments, X3 is selected from R, β-Ala, and K. In some embodiments, X9 is G. In some embodiments, X 10 is selected from G and K. In some embodiments, X 11 is selected from R and K. In some embodiments, X 13 is selected from D and G. In some embodiments, X 17 is H. In some embodiments, X 18 is selected from Q and Y. In some embodiments, X 26 is F. In some embodiments, X 28 is H. In some embodiments, X 29 is absent or selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13).

[0045] In some embodiments, X1 is selected from S and E, X2 is selected from K and 4-Pal, X3 is selected from R, β-Ala, and K, X9 is G, and X 10 is selected from G and K, and X 11 is selected from R and K, and X 13 is selected from D and G, and X 17 is H and X 18 is selected from Q and Y, and X 26 is F and X 28 is H and X 29is absent or selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13).

[0046] In some embodiments, the polypeptide contains a disulfide bond between the cysteine ​​at position 7 and the cysteine ​​at position 23 (between C7 and C23) of SEQ ID NO: 3. In some embodiments, the polypeptide contains a thioacetal bond between the cysteine ​​at position 7 and the cysteine ​​at position 23 (between C7 and C23).

[0047] In some embodiments, the polypeptides described herein are conjugated to a fatty acid.

[0048] In another embodiment, the polypeptide of Formula I or a pharmaceutically acceptable salt thereof is conjugated to a fatty acid. For example, in some embodiments, it further comprises a fatty acid conjugated to an amino acid present at the N-terminus of the polypeptide, and has the formula II: Fatty acids-Z1-Z2-Z3-X1X2X3RSSCFX9X 10 X 11 IDRIGX 17 X 18 SGLGCX 24 SX 26 RX 28 X 29 (SEQ ID NO: 21), (Wherein, the fatty acid is C 16 ~C 26 a fatty acid conjugated to an amino acid at the N-terminus of the polypeptide via the structure Z1-Z2-Z3; Z1 comprises an amino acid selected from γGlu, E, and β-Ala; Z2 comprises a sequence of 4 to 10 amino acids that is absent or contains amino acids independently selected from E, K, G, P, A, and S; Z3 is absent or comprises a basic structure from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) of polyethylene glycol or (containing a 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety).

[0049] Polypeptides of Formula II described herein include, for example, a fatty acid moiety conjugated to the amino acid present at the N-terminus of SEQ ID NO:3 by a linker comprising the structure Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3. Such conjugation is sometimes referred to as acylation. In embodiments, when X1 is absent, the fatty acid is conjugated to the amino acid present at position X2 of SEQ ID NO:3 by a linker comprising the structure Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3. In embodiments, when both X1 and X2 are absent, the fatty acid is conjugated to the amino acid present at position X3 of SEQ ID NO:3 (e.g., by a linker comprising the structure Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3). In embodiments, when all of X1, X2, and X3 are absent, the fatty acid is conjugated to the amino acid present at position X4 of SEQ ID NO: 3 (e.g., by a linker comprising the structure Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3). The fatty acid, and in certain embodiments the linker, acts as an albumin binder, offering the possibility of generating long-acting polypeptides.

[0050] The polypeptides described herein include C -s that can be chemically conjugated to the functional groups of amino acids by direct bond or by linker. 16 ~C 26 The length and composition of fatty acids influence the half-life of polypeptides, their efficacy in in vivo animal models, and their solubility and stability. 16 ~C 26 Conjugation to fatty acids results in ANP polypeptides that exhibit desirable half-lives, desirable potency in in vivo animal models, and desirable solubility and stability properties.

[0051] In some embodiments, the fatty acid is C 16 ~C 22 A saturated fatty acid mono- or di-acid. 16 ~C 22 Examples of fatty acids include, but are not limited to, palmitic acid (hexadecanoic acid) (C 16 monoacid), hexadecanedioic acid (C 16 diacid), margaric acid (heptadecanoic acid) (C 17 monoacid), heptadecanedioic acid (C 17 diacid), stearic acid (C 18 monoacid), octadecanedioic acid (C 18 diacid), nonadecylic acid (nonadecanoic acid) (C 19 monoacid), nonadecanedioic acid (C 19 diacid), alachadic acid (eicosanoic acid) (C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosylic acid (heneicosanoic acid) (C 21 monoacid), heneicosanoic acid (C 21 diacid), behenic acid (docosanoic acid) (C 22 monoacid), docosanedioic acid (C 22 diacids), including branched and substituted derivatives thereof.

[0052] In one particular example, C 16 ~C 22 Fatty acids are saturated C 16 monoacid, saturated C 16 Diacid, saturated C 18 monoacid, saturated C 18 Diacid, saturated C 20 monoacid, saturated C 20 It can be a diacid, as well as branched and substituted derivatives thereof.

[0053] In some embodiments, the linker can have the structure Z1-Z2-Z3, where Z1 comprises an amino acid selected from γGlu, E, and β-Ala, Z2 is either absent or comprises a 4-10 amino acid sequence comprising amino acids independently selected from E, K, G, P, A, and S; and Z3 is absent or comprises a polyethylene glycol or (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety as shown below.

[0054] [ka] Polyethylene glycol (PEG) (wherein n=10 to 30.) (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) n (AEEA)

[0055] [ka] (wherein n is 1 to 10.)

[0056] Thus, in some embodiments, the fatty acid is attached to Z1, which is attached to the peptide of Formula I directly, through Z2, through Z3, or through Z2-Z3.

[0057] In some examples, Z1 is an amino acid selected from γGlu, E, and β-Ala, or a dipeptide such as γGlu-γGlu or E-γGlu, or a tripeptide such as γGlu-γGlu-γGlu. In some embodiments, Z1 is γGlu or β-Ala. In some embodiments, Z1 is γGlu.

[0058] In some embodiments, the fatty acid is attached to Z1, Z1 is attached to Z2, and Z2 is attached to the peptide of Formula I, either directly or through Z3. In some embodiments, Z2 is APPSG, (EK) b G, (EP)b G, K (EK) c G, and (EK) c E, b is 2, 3, or 4, and c is 1, 2, 3, or 4. For example, Z2 can be (EK)3G, i.e., EKEKEKG, (EP)3G, i.e., EPEPEPG, K(EK)2G, i.e., KEKEKG, or (EK)3E, i.e., EKEKEKE. In some embodiments, Z2 is EKEKEKG.

[0059] In some embodiments, a fatty acid is attached to Z1, Z1 is attached to Z2, Z2 is attached to Z3, and Z3 is attached to a peptide of Formula I. In some embodiments, Z3 is (polyethylene glycol) m (wherein m is an integer selected from 10 to 30), and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) n (wherein n is selected from 2 to 10). For example, in some embodiments, Z3 is selected from (polyethylene glycol) 12 , (polyethylene glycol) 24 , ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))4, (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))6, or (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl))8.

[0060] In some embodiments, the fatty acid is a branched C25 triacid (also referred to herein as a branched fatty acid or "BFA") having the following structure:

[0061] [ka]

[0062] BFA exists in two enantiomeric forms.

[0063] [ka]

[0064] Surprisingly, it was discovered that the purified enantiomer of BFA (EN2) provides stronger binding to albumin compared to the other enantiomer (EN1) or the racemic mixture, resulting in a more favorable PK profile in rats. The isolation of purified EN2 (Preparation Example 8B) from the racemic mixture (Preparation Example 8) is described below. Furthermore, it was discovered that to preserve the stability of enantiomerically pure BFA during the coupling step to the peptide, it is essential to conjugate it to Z1 containing β-Ala, γGlu, or E.

[0065] [ka]

[0066] Thus, in one aspect, the invention includes a purified enantiomer of BFA, EN2, linked to β-Ala, γGlu, or E. Thus, in one embodiment, included herein is a structure in which a purified enantiomer of BFA, EN2, is linked to β-Ala. In another embodiment, included herein is a structure in which a purified enantiomer of BFA, EN2, is linked to γGlu. In another embodiment, included herein is a structure in which a purified enantiomer of BFA, EN2, is linked to E.

[0067] In some embodiments, a polypeptide of Formula II comprises a purified enantiomer EN2 of BFA linked to β-Ala. In some embodiments, a polypeptide of Formula II comprises a purified enantiomer EN2 of BFA linked to γGlu. In some embodiments, a polypeptide of Formula II comprises a purified enantiomer EN2 of BFA linked to E, E-γGlu, γGlu-γGlu, or γGlu-γGlu-γGlu. In some embodiments, Z2 is selected from EKEKEKG, KEKEKG, and EKEKEKE. In some embodiments, Z3 is (polyethylene glycol) 12 and (polyethylene glycol) 24 is selected from.

[0068] The amino acid sequences of the ANP polypeptides described herein typically incorporate naturally occurring amino acids, which are shown herein using the standard single-letter code, e.g., L=leucine, as well as certain other unnatural amino acids, such as 3-(4-pyridyl)-L-alanine (4 Pal), L-ornithine (Orn), L-2,3-diaminopropionic acid (Dap), and β-Ala. The structures of these unnatural amino acids are shown below:

[0069] [ka]

[0070] As described above, the ANP polypeptides described herein have structural similarities to any of the natural human natriuretic peptides, but also have many structural differences. For example, when compared to natural human ANP (SEQ ID NO: 2), the ANP polypeptides described herein contain modifications at one or more of positions 1, 2, 3, 9, 10, 11, 12, 13, 17, 18, 24, 26, 28, and 29. In some examples, the ANP polypeptides described herein contain modifications at each of positions 1, 2, 3, 9, 10, 11, 12, 13, 17, 18, 24, 26, 28, and 29. In some embodiments, the ANP polypeptides contain a thioacetal (S-CH2-S) bond between the cysteine ​​at position 7 and the cysteine ​​at position 23.

[0071] In some embodiments, the ANP polypeptides described herein comprise the following amino acid modifications: S or E at position 1; K or 4-Pal at position 2; R, β-Ala, P, or K at position 3; G, 4-Pal, or H at position 9; G, K, R, or Dap at position 10; R or K at position 11; D or G at position 13; H, K, R, Dap, or Orn at position 17; Q or Y at position 18; F or L at position 26; H or 4-Pal at position 28; and a linkage at positions 29-39 having an amino acid sequence selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13); and a C at amino acid position 1. 16 ~C 22 Conjugation of fatty acids, optionally using a linker comprising the structure Z1-Z2-Z3.

[0072] In particular examples, the ANP polypeptides described herein include the following amino acid modifications: S or E at position 1; K or 4-Pal at position 2; R, β-Ala, or K at position 3; G at position 9; G or K at position 10; R or K at position 11; I at position 12; D or G at position 13; H at position 17; Q or Y at position 18; P at position 24; F at position 26; H at position 28; a bond at positions 29-39 having an amino acid sequence selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13); and C at amino acid position 1. 16 ~C 22 Conjugation of fatty acids, optionally using a linker comprising the structure Z1-Z2-Z3.

[0073] In some embodiments, the ANP polypeptide described herein comprises a sequence selected from any one of SEQ ID NOs: 28-167. In some embodiments, the ANP polypeptide described herein comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 28-167.

[0074] In some embodiments, the ANP polypeptide described herein comprises a sequence selected from any one of SEQ ID NOs: 168-172. In some embodiments, the ANP polypeptide described herein comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 168-172.

[0075] In some embodiments, the ANP polypeptides described herein comprise a sequence selected from SEQ ID NOs: 28, 45, 50, 51, 78, 83, 84, 97, 98, 144, 158, and 159. In some embodiments, the ANP polypeptides described herein comprise a sequence selected from the group consisting of SEQ ID NOs: 28, 45, 50, 51, 78, 83, 84, 97, 98, 144, 158, and 159. For example, in one embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 28. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 45. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 50. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 51. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 78. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 83. In another embodiment, the ANP polypeptides described herein comprise SEQ ID NO: 84. In another embodiment, an ANP polypeptide described herein comprises SEQ ID NO: 97. In another embodiment, an ANP polypeptide described herein comprises SEQ ID NO: 98. In another embodiment, an ANP polypeptide described herein comprises SEQ ID NO: 144. In another embodiment, an ANP polypeptide described herein comprises SEQ ID NO: 158. In another embodiment, an ANP polypeptide described herein comprises SEQ ID NO: 159.

[0076] In certain examples, the ANP polypeptides described herein are amidated. In some embodiments, the ANP polypeptides are NPR-A agonists. In addition to the modifications described herein, the ANP polypeptides described herein may contain one or more additional amino acid modifications, as long as the polypeptides can bind to and activate the NPR-A receptor.

[0077] The affinity of the ANP polypeptides described herein for each of the NPR-A receptors can be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described in the Examples below, and is generally expressed as an inhibition constant (Ki) value. The activity of the polypeptides described herein at the NPR-A receptor may also be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and is generally expressed as an EC 50 Expressed as a value, this is the concentration of polypeptide that produces half-maximal simulation of the dose-response curve.

[0078] In further embodiments, pharmaceutically acceptable salt forms of ANP polypeptides are provided herein. For example, pharmaceutically acceptable salts for use herein include, but are not limited to, sodium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts.

[0079] In a further embodiment, provided herein is a pharmaceutical composition comprising an ANP polypeptide or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0080] The ANP polypeptides described herein can be used to treat various abnormalities, disorders, diseases, or conditions. In particular, methods for treating cardiovascular abnormalities, disorders, or conditions in an individual are provided, comprising at least administering to an individual in need of such treatment an effective amount of an ANP polypeptide described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. Exemplary cardiovascular abnormalities, diseases, and disorders include, but are not limited to, acute heart failure, chronic heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), atherosclerosis, coronary artery disease, diabetes, stroke, hypercholesterolemia, hypertension, ischemia, vascular stenosis, and ventricular hypertrophy, as well as other cardiac-related disorders or abnormalities, such as stroke, hypertension, congestive heart failure, diabetic heart disease, cardiomyopathy, diastolic dysfunction, vascular stenosis, and ventricular hypertrophy. In some embodiments, the cardiac disease is cardiac aging and / or diastolic dysfunction due to aging, or an abnormality associated therewith. In some embodiments, the ANP polypeptides described herein are used to treat HFpEF.

[0081] Another use of the ANP polypeptides herein is for treating pulmonary conditions, diseases, and / or disorders. Exemplary pulmonary conditions, diseases, and disorders include, but are not limited to, pulmonary hypertension and chronic obstructive pulmonary disease (COPD).

[0082] Another use of the ANP polypeptides herein is to treat kidney abnormalities, diseases, and / or disorders. Exemplary kidney abnormalities, diseases, and disorders include, but are not limited to, chronic kidney disease and diabetic nephropathy.

[0083] Thus, such methods may include selecting an individual having or susceptible to a cardiovascular condition, disease, or disorder. Alternatively, the methods may include selecting an individual having or susceptible to a pulmonary condition, disease, or disorder. Alternatively, the methods may include selecting an individual having or susceptible to a renal condition, disease, or disorder. In certain cases, the methods may include selecting an individual who has diabetes, hypertension with renal dysfunction, and / or obesity.

[0084] Thus, in some embodiments, provided herein are methods of treating CVD, comprising administering to a patient in need thereof an effective amount of an ANP polypeptide described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the CVD is heart failure. In one embodiment, the CVD is HFpEF.

[0085] In some embodiments, there is provided an ANP polypeptide or a pharmaceutically acceptable salt thereof for use in therapy.

[0086] In some embodiments, provided herein is the use of an ANP polypeptide or a pharmaceutically acceptable salt thereof in the treatment of CVD. In some embodiments, the CVD is heart failure. In one embodiment, the CVD is HFpEF.

[0087] In some embodiments, provided herein is the use of an ANP polypeptide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating CVD. In some embodiments, the CVD is heart failure. In one embodiment, the CVD is HFpEF.

[0088] Treatment of heart failure or HFpEF according to the present invention may be reflected in one or more of a variety of measures associated with heart failure, including, for example, reduced left ventricular end-diastolic pressure (LVEDP), reduced risk of CV death and / or heart failure hospitalization, reduced risk of all-cause mortality, reduced risk of myocardial infarction (MI), reduced risk of stroke, reduced risk of need for left ventricular assist device (LVAD) implantation and / or heart transplant, improvement in heart failure symptoms and physical limitations, and / or improved quality of life (QoL). Particular benefits of treatment according to embodiments of the present invention may be achieved after at least one month of treatment. Particular benefits of treatment according to embodiments of the present invention may be achieved after at least six months of treatment. Particular benefits of treatment according to embodiments of the present invention may be achieved after at least one year of treatment.

[0089] In certain embodiments, administration of an ANP polypeptide according to the present invention results in a significant reduction in LVEDP after one year of treatment. In certain embodiments, administration of an ANP polypeptide according to the present invention results in a significant reduction in global longitudinal strain (GLS). In certain embodiments, administration of an ANP polypeptide according to the present invention results in at least a 3.5% reduction in GLS. In certain embodiments, administration of an ANP polypeptide according to the present invention results in at least a 15% reduction in the risk of CV death and / or HF hospitalization. In certain embodiments, administration of an ANP polypeptide according to the present invention results in a significant reduction in the risk of one or more of all-cause mortality, MI, stroke, LVAD transplantation, or heart transplantation. In certain embodiments, administration of an ANP polypeptide according to the present invention results in a significant improvement in heart failure symptoms and physical limitations and / or QoL.

[0090] Furthermore, as noted above, administration of an ANP polypeptide according to certain embodiments of the present disclosure can provide such improvements in heart failure-related outcomes without increasing safety risks. Thus, in some embodiments, administration of an ANP polypeptide according to the present invention does not result in increased safety risks, such as increased hypotension, worsening renal function, electrolyte imbalance, liver dysfunction, tumor development, or persistent spermatogenesis.

[0091] The term "therapeutically effective amount" refers to an amount or dose of an ANP polypeptide that provides a desired effect in a patient. In the case of an ANP polypeptide with an extended pharmacokinetic profile, such a dose may be given in single or multiple administrations. An effective amount can be readily determined by one skilled in the art by the use of known techniques and by observing results obtained under analogous circumstances.

[0092] With regard to the route of administration, the ANP polypeptide or a pharmaceutical composition comprising it can be administered according to known methods (e.g., orally; by injection (i.e., intraarterially, intravenously, intraperitoneally, intracerebrally, intraventricularly, intramuscularly, intraocularly, intraportally, or intralesionally); by sustained release system; or by implantation device, etc. Administration of the ANP polypeptide according to the present invention is typically parenteral, e.g., intravenously (IV), subcutaneously (SC or SQ), or intraperitoneally (IP). Thus, in certain embodiments of the present invention, the ANP polypeptide is administered intravenously. In other embodiments of the present invention, the ANP polypeptide is administered intraperitoneally. In other embodiments, the ANP polypeptide is administered subcutaneously. In certain cases, the ANP polypeptide or a pharmaceutical composition comprising it can be administered by bolus injection or continuously SQ.

[0093] The present invention also includes intermediates and processes useful for producing the ANP polypeptides of the present invention. The intermediates and ANP polypeptides of the present invention can be prepared by a variety of procedures known in the art, including processes using chemical synthesis or biological expression, as described in the Examples below.

[0094] For chemical synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, CEM (Charlotte, North Carolina), CSBio (Menlo Park, California), and Gyros Protein Technologies Inc. (Tucson, Arizona). Reagents for solid-phase synthesis are readily available from commercial suppliers. Solid-phase synthesis equipment can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during the reaction, coupling, deprotecting, and capping unreacted amino acids.

[0095] For biological expression, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence encoding all or part of the amino acid sequence of an ANP polypeptide, incorporate the polynucleotide into a recombinant expression vector, and introduce the vector into host cells such as bacteria, yeast, and mammalian cells to produce the ANP polypeptide. See, for example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4 th See, e.g., J. Immunol., 2012. The polypeptides can be readily produced in mammalian cells such as CHO cells, NS0 cells, HEK293 cells, BHK cells, or COS cells; in bacterial cells such as E. coli, Bacillus subtilis, or Pseudomonas fluorescens; in insect cells; or in fungal or yeast cells, which are cultured using techniques known in the art. Vectors containing the polynucleotide sequence of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. Various methods of protein purification can be used, and such methods are known in the art.

[0096] As noted above, all HF patients, even those with mild symptoms, are at increased risk of death. Thus, as used herein, reference to a "patient in need" of treatment for heart failure (HF) can refer to a wide range of individuals with HF, including those with a wide range of disease severity, as described below. The New York Heart Association (NYHA) provides a classification scheme for the degree or severity of HF, as summarized below.

[0097] [Table 1]

[0098] In certain embodiments, the patient in need is in NYHA class II-IV heart failure. In certain embodiments, the patient in need is in NYHA class II heart failure. In certain embodiments, the patient in need is in NYHA class III heart failure. In certain embodiments, the patient in need is in NYHA class IV heart failure. In certain embodiments, the patient in need is in NYHA class II-III heart failure.

[0099] As described above, existing treatment options for heart failure, including the current standard of care, improve symptoms, slow disease progression, and reduce the workload of heart failure through hemodynamic mechanisms, such as reducing blood pressure, heart rate, and / or plasma volume. In contrast, the ANP polypeptides of the present invention achieve their effects through a different mechanism of action, namely, selective NPR-A binding and resulting activity to provide improvements in biomarkers (cGMP, NT-proBNP), hemodynamics (LVEDP), structure (LA volume), and symptoms (pulmonary congestion, dyspnea), thus improving outcomes and QoL in HFpEF patients. Due to these different mechanisms of action, the ANP polypeptides of the present invention can be administered on an existing SoC without titration or monitoring. Therefore, in certain embodiments, the ANP polypeptides of the present invention can be administered in combination with one or more additional therapies for heart failure. In certain embodiments, the one or more additional therapies for heart failure are selected from the administration of therapeutic agents such as anticoagulants, beta-blockers, ACE inhibitors, ARBs, ARNIs, MRAs, diuretics, digitalis, digoxin, hydralazine / isosorbide dinitrate, ivabradine, ARB and NEP inhibitor combinations (sacubitril / valsartan (ENTRESTO®)), statins and / or hypoglycemic agents, and other therapeutic agents to control comorbidities, including, but not limited to, high cholesterol, high blood pressure, atrial fibrillation, and diabetes. In certain embodiments, the ANP polypeptides of the present invention may be administered in combination with an SGLT2 inhibitor or an sGC activator.

[0100] The additional therapeutic agent may be administered simultaneously, separately, or sequentially with the ANP polypeptide or pharmaceutical composition comprising same. Moreover, the additional therapeutic agent may be administered at the same frequency as the ANP polypeptide or pharmaceutical composition comprising same (i.e., every other day, twice a week, or weekly). Alternatively, the additional therapeutic agent may be administered at a different frequency than the ANP polypeptide or pharmaceutical composition comprising same. In other cases, the additional therapeutic agent may be administered SQ. In other cases, the additional therapeutic agent may be administered IV. In still other cases, the additional therapeutic agent may be administered orally.

[0101] It is further contemplated that these methods may be combined with diet and exercise, and / or may be combined with additional therapeutic agents other than those discussed above.

[0102] The ANP polypeptides described herein can be formulated as pharmaceutical compositions that can be administered parenterally (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally). Such pharmaceutical compositions and techniques for preparing them are well known in the art. See, for example, Remington, "The Science and Practice of Pharmacy" (DB Troy ed., 21st Ed., Lippincott, Williams & Wilkins, 2006). In certain cases, the ANP polypeptides are administered SQ or IV. However, if this is not the case, the ANP polypeptides can be formulated in forms for other pharmaceutically acceptable routes, such as tablets or other solid forms for oral administration; sustained-release capsules; and any other form currently used, including creams, lotions, inhalants, etc.

[0103] As mentioned above, to improve their in vivo compatibility and effectiveness, the ANP polypeptides herein can react with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and common techniques for preparing them are well known in the art (see, for example, Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (2nd Revised Ed. Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetate salts, hydrochloride salts, and acetate salts.

[0104] The ANP polypeptides of the present invention can be administered by a physician or self-administered using an injection. It is understood that the gauge size and injection volume can be easily determined by those skilled in the art. However, the injection volume can be about 2 mL or less, or even about 1 mL or less, and the needle gauge can be about 27 G or more, or about 29 G or more.

[0105] The ANP polypeptides herein can be provided as part of a kit. In some cases, the kit includes a device for administering at least one ANP polypeptide (and optionally at least one additional therapeutic agent) to an individual. In certain cases, the kit includes a syringe and needle for administering at least one ANP polypeptide (and optionally at least one additional therapeutic agent). In certain cases, the ANP polypeptide (and optionally at least one additional therapeutic agent) is pre-formulated in an aqueous solution in the syringe.

[0106] This invention is further illustrated by the following examples which should not be construed as limiting. [Example]

[0107] preparation Abbreviations: acetonitrile (ACN); aqueous (aq); octadecylsilane (C18); dichloromethane (DCM); N,N-dimethylformamide (DMF); dimethyl sulfoxide (DMSO); ethyl acetate (EtOAc); hexafluorophosphate azabenzotriazole tetramethyluranium (HATU); high-performance liquid chromatography (HPLC); isopropanol (IPA); liquid chromatography-mass spectrometry (LCMS); methanol (MeOH); minutes (min); mass spectrometry (MS); methyl tert-butyl ether (MTBE); mass-to-charge ratio (m / z); polyethylene glycol (PEG); reversed-phase high-performance liquid chromatography (RP-HPLC); reversed-phase liquid chromatography-mass spectrometry (RP-LCMS); room temperature (rt); saturated (satd); strong cation exchange (SCX); tris(2-carboxyethyl)phosphine (TCEP); trifluoroacetic acid (TFA); tetrahydrofuran (THF); tris(hydroxymethyl)aminomethane (Tris).

[0108] Preparation 1 tert-Butyl 11-bromoundecanoate

[0109] [ka] Under a nitrogen atmosphere, a pressure vessel was charged with a mixture of 11-bromoundecanoic acid (8.00 g, 30.2 mmol), dichloromagnesium hexahydrate (613 mg, 3.01 mmol) in di-tert-butyl dicarbonate (8.65 g, 39.2 mmol) and tert-butanol (60 mmol). The vessel was sealed and then heated to 40 °C for 24 h. The solution was diluted with dichloromethane (100 mL) and washed with saturated ammonium chloride (3 × 50 mL). The organic phase was then dried over sodium sulfate, concentrated to dryness in vacuo, and purified by flash column chromatography (120 g silica column, gradient from 100% hexane to 100% EtOAc in hexane over 20 min). The desired product was isolated as a colorless oil (6.05 g); mz = 265,267 (M-tBu).

[0110] Preparation 2 O1-benzyl O3-tert-butyl 2-undecylpropanedioate

[0111] [ka] Under a nitrogen atmosphere, sodium hydride (60% by weight, 400 mg, 10.0 mmol) in mineral oil was added portionwise to an ice-cooled solution of O1-benzyl O3-tert-butylpropanedioate (2.50 g, 9.99 mmol) in N,N-dimethylformamide (15 mL). After stirring for 1 h, 1-bromoundecane (2.35 g, 9.99 mmol) in 2 mL of DMF was added and mixed at room temperature for 15 h. The mixture was diluted with 60 mL of ether, and the organic layer was washed with 1% aqueous citric acid (50 mL), brine, and water. The organic layer was dried over sodium sulfate, the volatiles removed in vacuo, and purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 40% EtOAc over 25 min). The desired product was isolated as an oil (3.50 g); mz = 403 (M-1).

[0112] Preparation 3 O11-Benzyl O1,O11-ditert-butyldocosane-1,11,11-tricarboxylate

[0113] [ka] Under a nitrogen atmosphere, sodium hydride in mineral oil (60% by weight, 960 mg, 24.0 mmol) was added portionwise to an ice-cooled mixture of O1-benzyl O3-tert-butyl 2-undecylpropanedioate (8.50 g, 20.0 mmol) in N,N-dimethylformamide (40 mL). The mixture was stirred at room temperature for 40 minutes. tert-Butyl 11-bromoundecanoate (7.50 g, 22.2 mmol) in 10 mL of DMF was added. The mixture was mixed at room temperature for 20 hours. The mixture was diluted with 150 mL of ether, and the organic phase was washed with 1% aqueous citric acid (50 mL), brine, and water. The organic layer was dried over sodium sulfate, and the volatiles were removed in vacuo. The mixture was purified by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% DCM over 15 minutes, held for an additional 10 minutes). The desired product was isolated as an oil (13.00 g); mz=533 (M-2x tBU).

[0114] Preparation 4 13-tert-butoxy-2-tert-butoxycarbonyl-13-oxo-2-undecyl-tridecanoic acid

[0115] [ka] A 2250 mL Parr shaker was charged with 10% Pd / C (1.25 g) and purged with nitrogen. Tetrahydrofuran (125 mL) was added, followed by a solution of O11-benzyl O1,O11-ditert-butyldocosane-1,11,11-tricarboxylate (13.00 g, 19.15 mmol) in 125 mL of tetrahydrofuran. The bottle was sealed, purged with nitrogen, and pressurized to 10 psi with hydrogen gas. The mixture was shaken at room temperature for 2 hours. The system was evacuated with nitrogen gas and then filtered through Celite. The solvent was removed from the mixture under reduced pressure, and the racemic product was isolated as a white solid (11.0 g); mz = 443 (M-2xt-Butyl).

[0116] Preparation 5 Benzyl 11-bromoundecanoate

[0117] [ka] 11-Bromoundecanoic acid (10.00 g, 37.71 mmol), benzyl alcohol (4.5 g, 42 mmol), and 4-dimethylaminopyridine (0.4 g, 3 mmol) were dissolved in dichloromethane (150 mL). Dicyclohexylcarbodiimide (9.40 g, 45.6 mmol, 100% by weight) was added to the solution in one portion. The mixture was stirred at room temperature for 8 hours. The white solid was removed by filtration, and the solid was washed with dichloromethane (3 x 10 mL). The organic components were removed under reduced pressure. The mixture was purified by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% dichloromethane over 20 minutes, continued for an additional 5 minutes). Product-containing fractions were combined, and the product was isolated as an oil (11.60 g). 1 H NMR(400MHz, CDCl3):7.39~7.36(m,5H),5.14(s,2H),3.43(t,J=6.9Hz,2H),2.38(t,J=7.6Hz,2 H),1.91~1.84(m,2H),1.67(quintet,J=7.3Hz,2H),1.43(dd,J=7.0,14.4Hz,2H),1.30(s,10H).

[0118] Preparation 6 O1-benzyl O3-tert-butyl 2-undecylpropanedioate

[0119] [ka] Under a nitrogen atmosphere, sodium hydride (60% by weight, 400 mg, 10.0 mmol) in mineral oil was added portionwise to an ice-cold solution of O1-benzyl O3-tert-butylpropanedioate (2.50 g, 9.99 mmol) in N,N-dimethylformamide (15 mL). After stirring for 1 h, 1-bromoundecane (2.35 g, 9.99 mmol) in 2 mL of DMF was added. The mixture was mixed at room temperature for 15 h. The mixture was diluted with 60 mL of ether, and the organic layer was washed with 1% aqueous citric acid (50 mL), brine, and water. The organic layer was dried over sodium sulfate, and the volatiles were removed in vacuo. Purification was performed by flash column chromatography (80 g silica column, gradient from 100% hexane to 40% EtOAc over 25 min). The desired product was isolated as an oil (3.50 g); mz = 403 (M-1).

[0120] Preparation 7 O1,O11-Dibenzyl O11-tert-butyldocosane-1,11,11-tricarboxylate

[0121] [ka] To an ice-cold solution of O1-benzyl O3-tert-butyl 2-undecylpropanedioate (6.2 g, 14.6 mmol) in N,N-dimethylformamide (30 mL) was added sodium hydride in mineral oil (60% by weight, 700 mg, 17.5 mmol) in small portions. After 40 min, benzyl 11-bromoundecanoate (6.00 g, 16.0 mmol) in 8 mL of DMF was added. The mixture was mixed at room temperature for 15 h. The mixture was diluted with 150 mL of ether. The mixture was washed with citric acid (1% in water, 50 mL), brine, and water. The organic layer was dried over sodium sulfate, and the volatiles were removed in vacuo. Purification was carried out by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% DCM over 20 min, held for an additional 10 min). The desired product was isolated as an oil (8.00 g); mz=624 (M-tBu), 702 (M+ Na).

[0122] Preparation 8 13-Benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoic acid

[0123] [ka] O1,O11-Dibenzyl O11-tert-butyldocosane-1,11,11-tricarboxylate (11.0 g, 15.4 mmol) was treated with trifluoroacetic acid (40 mL) at room temperature for 3 hours. Volatiles were removed to a residue, which was purified by flash column chromatography (120 g silica column, gradient from 100% hexane to 100% EtOAc in hexane over 20 minutes). The desired product was isolated as an oil (9.5 g); mz=623 (M+1); 1 H NMR (400MHz, CDCl3):8.77~8.75(m,1H),7.39~7.38(m,10H),5.26(s,2H),5.14(s,2H),2. 38(t,J=7.5Hz,2H),2.02~1.84(m,4H),1.66(quintet,J=7.4Hz,2H),1.31~0.89(m,37H).

[0124] Chiral separation of racemic 13-(benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridecanoic acid (Preparation 8) into enantiomer 1 (Preparation 8A, EN1) and enantiomer 2 (Preparation 8B, EN2).

[0125] [ka] Preparation method: Column used: Chiralpak AD-H, 21 x 150 mm Mobile phase: 20% EtOH:80% CO2 Flow rate: 80mL / min BPR set point: 100 bar BPR temperature: 20℃ Column temperature: 40℃ Detection: 225nm

[0126] Analysis conditions: Chiralpak AD-H, 4.6 x 150mm, 25% EtOH / CO2, 5mL / min, 225nm From 1300 mg of the racemate, enantiomer 1 (EN1; 564 mg, 99% ee, retention time = 2.64 min) and enantiomer 2 (EN2; 511.2 mg, 98% ee, retention time = 3.21 min) were isolated using the conditions of the preparative method.

[0127] Preparation 9 β-ALA linker O1,O11-Dibenzyl O11-(2,5-dioxopyrrolidin-1-yl)docosane-1,11,11-tricarboxylate;

[0128] [ka] N-Hydroxysuccinimide (0.500 g, 4.25 mmol) was added to 13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoic acid (2.45 g, 3.54 mmol) in dichloromethane (20 mL) and THF (5 mL). After stirring for 5 minutes, N,N'-dicyclohexylcarbodiimide (0.880 g, 4.22 mmol) was added in one portion. The mixture was stirred at room temperature under a nitrogen atmosphere for 7 hours. The reaction mixture was stored in a refrigerator at -20 °C for 2 days. The solids were removed by filtration and washed with DCM (3 × 5 mL). The filtrate was stripped of solvent and purified by flash column chromatography (80 g silica column, gradient from 100% hexanes to 50% EtOAc in hexanes over 20 minutes, then increasing to 100% EtOAc over 5 minutes). The desired product was isolated as an oil (2.10 g); 1H NMR(400MHz, CDCl3):7.43~7.34(m,10H),5.25(s,2H),5.14(s,2H),4.15(q,J=7.2Hz,1H),2.84(d,J=3.1Hz, 4H), 2.37(t,J=7.6Hz,2H),2.02~1.97(m,4H),1.69~1.59(m,3H),1.34~1.25(m,38H),0.90(t,J=6.8Hz,3H).

[0129] Preparation 10 3-[(13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl)amino]propanoic acid

[0130] [ka] A suspension of β-alanine (250 mg, 2.80605 mmol) in 1 mL of DMF was added to a room-temperature solution of O1,O11-dibenzyl O11-(2,5-dioxopyrrolidin-1-yl)docosane-1,11,11-tricarboxylate (1.50 g, 2.08 mmol) in tetrahydrofuran (20 mL), followed by triethylamine (0.80 mL, 5.7 mmol). Water (3 mL), acetonitrile (6 mL, 100% by weight), and 4 mL of DMF were added to solubilize the precipitate that formed. The mixture was mixed at room temperature for 15 hours. The mixture was diluted with chloroform / isopropanol (3 / 1, 100 mL) and washed with 10% aqueous citric acid, water, and brine (50 mL). The organics were dried over sodium sulfate and concentrated to dryness in vacuo. Purification by flash column chromatography (80 g silica column, UV 254 nm, gradient from 100% hexane to 100% EtOAc over 15 min, hold for an additional 5 min) gave the desired product, isolated as an oil (1.00 g, 66% yield); mz=694 (M+).

[0131] Preparation 11 NHS ester of β-alanine BFA Dibenzyl 2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioate

[0132] [ka] N-Hydroxysuccinimide (193 mg, 1.64 mmol) was added to a solution of 3-[(13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl)amino]propanoic acid (1.00 g, 1.37 mmol) in dichloromethane (15 mL) and THF (2 mL). After 5 min, N,N'-dicyclohexylcarbodiimide (342 mg, 1.64 mmol) was added in one portion. Stirred at room temperature for 15 h. The solid was removed by filtration and washed with DCM (3 x 5 mL). Concentrated to dryness in vacuo and purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 100% EtOAc within 20 min). The desired product was isolated as an oil (1.00 g); mz = 793 (M+2); 1 H NMR(400MHz, CDCl3):8.27(t,J=6.0Hz,1H),7.38~7.35(m,10H),5.19(s,2H),5.13(s,2H),3.68(q,J=6.2Hz,2H),2.89~2. 84(m,6H),2.36(t,J=7.6Hz,2H),2.02~1.94(m,2H),1.82~1.75(m,2H),1.65(quintet,J=7.4Hz,2H),1.32~1.00(m,34H).

[0133] Preparation 12 Deprotection of benzyl on β-alanine-BFA 2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioic acid

[0134] [ka] A 100 mL Parr shaker was charged with 10% Pd / C (0.193 g) and purged with nitrogen. Tetrahydrofuran (20 mL) was added, followed by a solution of dibenzyl 2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioate (1.93 g, 2.68 mmol) in 20 mL of tetrahydrofuran. The bottle was sealed, purged with nitrogen, and pressurized to 10 psi with hydrogen gas. The mixture was shaken at room temperature for 2 hours. The system was evacuated with nitrogen gas and then filtered through Celite. The solvent was removed from the mixture under reduced pressure, and the product was isolated as a solid (670 mg); mz = 611 (M+).

[0135] Preparation 13 Alternative chemistry for amide formation on quaternary acids β-Ala EN2 3-[[rel-(2R)-13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl]amino]propanoic acid

[0136] Activated esters: O1,O11-Dibenzyl O11-(triazolo[4,5-b]pyridin-3-yl)rel-(11S)-docosane-1,11,11-tricarboxylate

[0137] [ka] Under a nitrogen atmosphere, [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium hexafluorophosphate (2.40 g, 6.12 mmol) was added to a solution of rel-(EN2)-13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoic acid (2.50 g, 4.01 mmol) in THF (10 mL, 100% by weight) and DMF (10 mL). After mixing at room temperature for 3 minutes and cooling on an ice bath, N,N-diisopropylethylamine (1.50 mL, 8.60 mmol) was added. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with DCM (60 mL) and washed with saturated aqueous ammonium chloride (2 × 30 mL). The organic layer was separated and dried over sodium sulfate. The crude material was purified by normal phase flash chromatography (80 g silica gold column, 100% hexanes over 3 min, then a gradient to 60% EtOAc in hexanes over 17 min, then switched to 100% EtOAc and held for an additional 5 min). The activated ester product was isolated and used directly in the next step.

[0138] Coupling: β-Alanine (1.10 g, 12.3 mmol) and N,N-diisopropylethylamine (1.40 mL, 8.03 mmol) were mixed in 10 mL of acetonitrile and 9 mL of water. The activated ester from above was dissolved in acetonitrile (10 mL) and then added dropwise to the β-alanine solution via syringe over 2 minutes. Stirring was continued at room temperature for 1 hour. The reaction mixture was diluted with DCM (100 mL) and washed with saturated ammonium chloride (2 × 30 mL). The organic layer was separated and dried over sodium sulfate. Concentration under reduced pressure gave a residue that was used directly in the next step (2.60 g).

[0139] Preparation 14 EN2-β-Ala-BFA via activation / deprotection

[0140] [ka]

[0141] Activation with NHS esters Dibenzyl rel-(2R)-2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioate N-Hydroxysuccinimide (240 mg, 2.04 mmol) was added to a mixture of 3-[[rel-(2R)-13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl]amino]propanoic acid (1.06 g, 1.53 mmol) in THF (5 mL) and dichloromethane (5 mL) at room temperature. After stirring for 3 minutes, N,N'-dicyclohexylcarbodiimide (420 mg, 2.01 mmol) was added in one portion, followed by 1 mg of DMAP. The mixture was mixed at room temperature for 3 hours and then stored in the refrigerator for 12 hours. The solid was removed by filtration and washed with DCM (3 × 5 mL). The mixture was concentrated under reduced pressure and purified by flash column chromatography (40 g silica / gold column, 100% hexanes for 5 minutes, then gradient to 100% EtOAc over 15 minutes). A second purification was performed by flash chromatography (40 g column, 100% hexanes in 3 min, then gradient to 100% MTBE over 17 min). The desired product was isolated as a thick oil (1.0 g).

[0142] Deprotection A 100 mL Parr shaker was charged with 10% Pd / C (0.152 mg) and purged with nitrogen. Tetrahydrofuran (10 mL) was added, followed by a solution of dibenzyl rel-(2R)-2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioate (1.0 g, 1.20 mmol) in 10 mL of tetrahydrofuran. The bottle was sealed, purged with nitrogen, and pressurized to 10 psi with hydrogen gas. The mixture was shaken at room temperature for 2 hours. The system was evacuated with nitrogen gas and then filtered through Celite. The solvent was removed from the mixture under reduced pressure, and the product was isolated as a solid (750 mg); mz = 611 (M+).

[0143] Preparation 15 Synthesis of γ-Glu-EN2

[0144] [ka]

[0145] HBTU ester O11-(benzotriazol-1-yl) O1,O11-dibenzyl rel-(11R)-docosane-1,11,11-tricarboxylate Under a nitrogen atmosphere, EN2-(2S)-13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoic acid (1.40 g, 2.25 mmol) was mixed with [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium hexafluorophosphate (1.43 g, 3.65 mmol) in DMF (5 mL) and THF (5 mL). The mixture was cooled to 10 °C in an ice-water bath, and then N,N-diisopropylethylamine (0.85 mL, 4.9 mmol) was added. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with DCM (60 mL) and washed with saturated ammonium chloride (2 × 30 mL). The organic layer was separated, dried over sodium sulfate, and concentrated to dryness in vacuo. Purification by normal phase flash chromatography (80 g silica gold column, 100% hexanes over 3 min, then gradient to 60% EtOAc in hexanes over 17 min, then switch to 100% EtOAc and hold for an additional 5 min) isolated the desired product as an oil (1.15 g); 1 H NMR(400MHz,CDCl3):8.68(dd,J=1.3,4.5Hz,1H),8.40(dd,J=1.4,8.4Hz,1H),7.52~7.50(m,2H),7.43~7.36(m,10H),5.38(s,2H), 5.14(s,2H),2.38(t,J=7.5Hz,2H),2.18(t,J=7.8Hz,4H),1.68(quintet,J=7.2Hz,2H),1.36~1.30(m,35H),0.91(t,J=6.8Hz,3H).

[0146] Preparation 16 γ-Glu coupling to EN2 activated ester (4S)-4-[[(2S * )-13-Benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl]amino]-5-tert-butoxy-5-oxo-pentanoic acid

[0147] [ka] N,N-Diisopropylethylamine (0.55 mL, 3.2 mmol) was added to a solution of (4S)-4-amino-5-tert-butoxy-5-oxo-pentanoic acid (650 mg, 3.13 mmol) dissolved in acetonitrile (4 mL) and water (4 mL). Then, O11-(benzotriazol-1-yl)O1,O11-dibenzyl rel-(11R)-docosane-1,11,11-tricarboxylate (1.15 g, 1.55 mmol) in acetonitrile (3 mL) was added. Stirring was continued for 12 hours at room temperature. The mixture was diluted with 50 mL of DCM and washed with 50 mL of aqueous ammonium chloride (2x). The organic phase was separated and dried over sodium sulfate. The mixture was concentrated to dryness in vacuo. Purification by normal phase flash chromatography (40 g silica gold column, 100% hexanes over 5 min, then gradient to 100% EtOAc in hexanes over 15 min, hold for an additional 5 min) isolated the desired product as an oil (900 mg); mz=806 (M-2); 1 H NMR(400MHz,CDCl3):8.57(d,J=7.5Hz,1H),7.37~7.35(m,10H),5.21(s,2H),5.13(s,2H),4.56(td,J=7.7,5.2Hz,1H),4.14(q,J=7.2Hz,1H),2.4 4~2.35(m,4H),2.27~2.20(m,1H),2.02~1.93(m,3H),1.85~1.77(m,2H), 1.65 (quintet, J=7.3Hz, 2H), 1.32~1.18 (m, 34H), 0.90 (t, J=6.9Hz, 3H).

[0148] Preparation 17 Two-step procedure for γ-Glu-EN2-BFA (2S * )-2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecanedioic acid

[0149] [ka]

[0150] Step 1: Activated ester Dibenzyl (2S * )-2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecanedioate Under a nitrogen atmosphere, N-hydroxysuccinimide (170 mg, 1.44 mmol) was dissolved in tetrahydrofuran (3 mL) and dichloromethane (6 mL) in (4S)-4-[[(2S * To a solution of N,N'-dicyclohexylcarbodiimide (300 mg, 1.43 mmol) as a solid and 1 mg of DMAP were added. The mixture was stirred at room temperature for 3 minutes, followed by stirring at room temperature for 3 hours. The white precipitate was removed by filtration and washed with DCM (3 x 5 mL). Concentration to dryness in vacuo afforded the crude activated ester. Purification was performed by flash column chromatography (40 g silica column, 100% hexanes over 5 minutes, gradient to 100% EtOAc over 15 minutes, hold for another 5 minutes). The activated ester was isolated (850 mg); mz = 905 (M+).

[0151] Second step: Hydrogenation A 250 mL Parr shaker was charged with 10% Pd / C (0.150 mg) and purged with nitrogen. Tetrahydrofuran (10 mL) was added, followed by dibenzyl (2S) in 15 mL of tetrahydrofuran. * A solution of 2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecanedioate (0.800 g, 0.883 mmol) was added. The bottle was sealed, purged with nitrogen, and pressurized to 20 psi with hydrogen gas. Shaking was performed at room temperature for 2 hours. The system was evacuated with nitrogen gas and then filtered through Celite. The solvent was removed from the mixture under reduced pressure, and the product was isolated as a solid (770 mg); mz=725 (M+1).

[0152] Preparation of Exemplary Polypeptides Example 1 Example 1 is a polypeptide represented by the following description (SEQ ID NO: 45). HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKGS-4Pal-RRSS[CFGGRIDRIGHQSGLGC]PSFRHGGPSSGAPPPS-NH2 (disulfide bond)

[0153] Below is a depiction of the structure of Example 1 using the standard single letter code for L-amino acids, with the exception of the γ-glutamic acid and 4-Pal residues, the structures of these residues being expanded.

[0154] [ka]

[0155] The primary peptide sequence of Example 1 was synthesized on a 0.1 mmol scale on a Symphony-X, 24-channel multiple peptide synthesizer (Gyros Protein Technologies, Inc.) using standard 9-fluorenyl-methyloxycarbonyl (Fmoc) tert-butyl (t-Bu) solid-phase peptide chemical synthesis protocols.

[0156] The solid support used consisted of low-loading 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucyl-4-methylbenzhydrylamine resin (Fmoc-Rink-MBHA low-loading resin, EMD Millipore) (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g. Standard side-chain protecting groups were used for all Fmoc-L-amino acids used. The non-standard amino acids used in the synthesis of Example 1 were N-α-Fmoc-L-glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.) and N-Fmoc-3-(4-pyridyl)-L-alanine (Fmoc-4Pal-OH, Combi-Blocks Inc.). Fmoc deprotection before each coupling step was performed with 20% piperidine (PIP; Sigma-Aldrich) in dimethylformamide (DMF; Fisher Chemicals) for 2 × 7 min under nitrogen, followed by eight DMF wash cycles. All amino acid couplings were performed with Fmoc Amino Acid (0.3 M in DMF), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU; Ambeed; 0.9 M in DMF), and N,N-diisopropylethylamine (DIPEA; Sigma-Aldrich; 1.2 M in DMF) for 1 h, using a 9-fold molar excess of AA / HBTU and a 12-fold molar excess of DIPEA relative to the theoretical resin loading level. After synthesis of the primary peptide sequence, final Fmoc deprotection, and DMF washes were completed, attachment of the fatty acid (FA) moiety was achieved by manual addition of a threefold excess of 20-tert-butoxy-20-oxo-icosanoic acid (OtBu-C20-OH) preactivated (2 min) with a solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; Alfa Aesar) and DIPEA (1:1:3; FA:HATU:DIPEA) in DMF (3 mL).This solution was added directly to the Symphony-X reaction vessel containing the peptidyl resin via a transfer pipette. The reaction time for FA coupling was 3 h, after which the resin was washed three times with DMF and a Kaiser test was performed to confirm completion of the coupling. If the Kaiser test was positive, the FA coupling process was repeated as necessary. After FA acylation was complete, the peptidyl resin was transferred as a DCM slurry to a disposable fritted plastic syringe with a Teflon stopper, washed further with DCM, and finally dried completely under vacuum. The dried resin was then treated with 10 mL of a cleavage cocktail consisting of trifluoroacetic acid (TFA), water, 3,6-dioxa-1,8-octanedithiol (DODT), and triisopropylsilane (TIPS) (TFA:water:DODT:TIPS = 92.5:2.5:2.5:2.5 v / v) for 2 h at room temperature. After 2 hours of incubation, the resin was filtered off and washed twice with 2 mL of neat TFA. The combined filtrate / wash was collected in a 50 mL conical disposable tube, and the solution was then treated with 35 mL of cold diethyl ether (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 minutes to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated twice more with fresh ether and dried in vacuo.

[0157] Disulfide bond formation The crude peptide was solubilized in a suitable glass vessel with 25% aqueous acetic acid to a relatively low concentration (0.2-0.5 mg per mL of crude peptide). The solution was then placed on a magnetic stirrer equipped with the required spinning blade, mixed vigorously, and titrated with a few drops of saturated iodine in methanol solution until a pale yellow endpoint was achieved. After reaching the endpoint, the reaction was incubated at room temperature for 15 minutes, at which point the excess iodine was quenched by the addition of a few drops of 0.1 M aqueous ascorbic acid.

[0158] HPLC purification The crude oxidized solution was directly loaded onto a preparative HPLC system (Waters 2545 Binary Systems) equipped with a column heater and using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc., 5 μm, 100 Å; 250 × 21.2 mm). The running buffers used were A: 0.1% TFA / HO and B: 0.1% TFA / acetonitrile (ACN). The initial load was 20% B, followed by a 5-minute isocratic wash and then equilibration to 25% B. The column heating was set to 60°C, and the sample was eluted using a linear gradient of 25 to 45% B over 60 minutes at a flow rate of 15 mL / min. Fractions determined to contain the desired product (as determined by LC-MS analysis) were pooled, frozen, and lyophilized to yield the amorphous solid product as the TFA salt of Example 1. The purity, as assessed by RP-HPLC, was found to be greater than 95% and the observed molecular weight was 5293.4 daltons, in agreement with the theoretically calculated molecular weight of 5293.9 daltons.

[0159] Example 2 Example 2 is a polypeptide represented by the following description (SEQ ID NO: 60): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKGS-4Pal-RRSS[CFGGRIDRIGHQSGLGC]PSFRHGGPSSGAPPPS-NH2 (thioacetal bond)

[0160] Below is a depiction of the structure of Example 2 using the standard single-letter code for L-amino acids, with the exception of the γ-glutamic acid and 4-Pal residues, and the cysteine ​​residue, the structures of the above residues are expanded.

[0161] [ka]

[0162] The primary peptide sequence of Example 2 is the same as that of Example 1. The disulfide bond of Example 1 was replaced with a thioacetal bond in Example 2. The synthesis, disulfide bond formation, and purification of the acylated polypeptide of Example 2 were carried out in the same manner as in Example 1.

[0163] Thioacetal bond formation in Example 2 After purification of the disulfide-bridged polypeptide form (as in Example 1), the appropriate pooled fractions containing the peptide were not lyophilized but instead diluted with water and ACN to obtain an approximately 50 / 50 mixture of water / ACN (approximately 400 mL total volume) with a low concentration of peptide (approximately 0.2 mg / mL). The solution was then adjusted to pH 8 with approximately 10 equivalents of triethylamine (TEA), and the peptide's disulfide bridges were reduced by adding 2–4 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) reducing agent. Following disulfide bridge reduction, thioacetal bonds were formed by adding 7–10 equivalents of diiodomethane (CHI). The thioacetal-forming reaction was carried out by incubating the solution at room temperature with magnetic stirring for 18 hours. The reaction progress was monitored using analytical LC-MS by observing a mass shift of +12 daltons from the starting reduced peptide molecular weight.

[0164] HPLC purification The crude thioacetal reaction solution was diluted to 1000 mL with water and then loaded directly via an infusion pump onto a preparative HPLC system (Shimadzu LC-8A Binary Systems) using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc.; 5 μm, 100 Å; 250 × 21.2 mm). The running buffers used were A: 0.1% TFA / HO and B: 0.1% TFA / ACN. The initial load was 20% B, followed by a 5-minute isocratic wash and then equilibration to 25% B. The column heating was set to 50 °C, and the sample was eluted using a linear gradient of 25–45% B over 60 minutes at a flow rate of 25 mL / min. Fractions determined to contain the desired product (as determined by LC-MS analysis) were pooled, frozen, and lyophilized to yield the white amorphous solid product as the TFA salt of Example 2. The purity, as assessed by RP-HPLC 1, was found to be greater than 95% and the observed molecular weight was 5308.6 daltons, in agreement with the theoretically calculated molecular weight of 5307.9 daltons.

[0165] Example 3 Example 3 is a polypeptide represented by the following description (SEQ ID NO: 107): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKG-PEG24-EK-βAla-RSS[CFGKRIDRIGHQSGLGC]PSFRHGGKSSGAPPPS-NH2 (thioacetal bond)

[0166] Below is a depiction of the structure of Example 3 using the standard single-letter code for L-amino acids, with the exception of the γ-glutamic acid residue, the glycine residue at position 8, the β-alanine residue, and the cysteine ​​residue, which are expanded.

[0167] [ka]

[0168] The primary peptide sequence of Example 3 was synthesized in a manner substantially similar to that of Examples 1 and 2, with the unnatural β-alanine (βAla) residue incorporated using Fmoc-βAla-OH (ChemImpex International Inc.). The exception to the synthesis method was the coupling of Fmoc-N-amido-PEG24-OH, which required a temporary pause in the automated synthesis protocol. Coupling of the PEG24 residue was achieved by manual addition of a 1.5-fold excess of Fmoc-N-amido-PEG24-OH (BroadPharm) solution, preactivated (2 min) with diisopropylcarbodiimide (DIC) and ethyl-cyano(hydroxyamino)acetate (Oxyma) (1:1.2:1; PEG24:DIC:Oxyma) in 3 mL of DMF. This solution was added directly via transfer pipette to the Symphony-X reaction vessel containing the peptidyl resin. The reaction time for PEG24 coupling was 18 hours, after which the resin was washed three times with DMF and a Kaiser test was performed to confirm complete coupling. If the Kaiser test was positive, the PEG24 coupling process was repeated as necessary. The automated method was resumed to complete the synthesis of the remaining sequence, and FA was coupled as described in Example 1. Cleavage, disulfide bond formation, thioacetal bond formation, and purification were performed as previously described in Examples 1 and 2. Purity, as assessed by RP-HPLC, was found to be greater than 95%, and the observed molecular weight was 6475.0 daltons, consistent with the theoretically calculated molecular weight of 6475.4 daltons.

[0169] Example 4 Example 4 is a polypeptide represented by the following description (SEQ ID NO: 144): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKG-PEG24-EK-βAla-RSS[CFGKRIDRIGHQSGLGC]PSFRHGSPSSGAPPPS-NH2 (thioacetal bond)

[0170] Below is a depiction of the structure of Example 4 using the standard single-letter code for L-amino acids, with the exception of the γ-glutamic acid residue, the glycine residue at position 8, the β-alanine residue, and the cysteine ​​residue, with the structures of the above residues expanded.

[0171] [ka]

[0172] Example 4 was synthesized in a manner substantially similar to that of Example 3. The purity of Example 4, as assessed by RP-HPLC, was found to be greater than 95%, and the observed molecular weight was 6474.6 daltons, in agreement with the theoretically calculated molecular weight of 6474.4 daltons.

[0173] Example 5 Example 5 is a polypeptide represented by the following description (SEQ ID NO: 146): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKGEKPRSS[CFGKRIDRIGHYSGLGC]PSFRHGSPSSGAPPPS-NH2 (thioacetal bond)

[0174] Below is a depiction of the structure of Example 5 using the standard single letter code for L-amino acids, with the exception of the γ-glutamic acid and cysteine ​​residues, the structures of the above residues being expanded.

[0175] [ka]

[0176] Example 5 was synthesized in a manner substantially similar to that described for Example 2. The purity of Example 5, as assessed by RP-HPLC, was found to be greater than 95%, and the observed molecular weight was 5406.8 daltons, in agreement with the theoretically calculated molecular weight of 5407.1 daltons.

[0177] Example 6 Example 6 is a polypeptide represented by the following description (SEQ ID NO: 158): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKG-PEG24-EK-βAla-RSS[CFGGKIDRIGHYSGLGC]PSFRHGSP-SSGAPPPS-NH2 (thioacetal bond)

[0178] [ka]

[0179] Example 6 was synthesized in a manner substantially similar to that described for Example 3. The purity of Example 6, as assessed by RP-HPLC, was found to be greater than 95%, and the observed molecular weight was 6409.6 daltons, in agreement with the theoretically calculated molecular weight of 6410.3 daltons.

[0180] Example 7 Example 7 is a polypeptide represented by the following description (SEQ ID NO: 159): HOOC-(CH2) 18 -CO-(γGlu)-EKEKEKG-PEG24-EK-βAla-RSS[CFGGKIDRIGHQSGLGC]PSFRHGSP-SSGAPPPS-NH2 (thioacetal bond)

[0181] [ka]

[0182] Example 7 was synthesized in a manner substantially similar to that described for Example 3. The purity of Example 7, as assessed by RP-HPLC, was found to be greater than 95%, and the observed molecular weight was 6375.2 daltons, in agreement with the theoretically calculated molecular weight of 6375.2 daltons.

[0183] The polypeptides according to Examples 8 to 140 listed in Table 1 (SEQ ID NOS: 28-44, 46-59, 61-106, 108-143, 145, 147-157, 160-167) are prepared substantially using the procedures described in Examples 1 to 3. For example, Examples 8-16 and 18-55 (SEQ ID NOS: 28-36, 38-59, and 61-77) contain disulfide bonds and are prepared substantially as described by the procedure of Example 1. Examples 17 and 56-140 (SEQ ID NOS: 37, 78-106, 108-143, 145, 147-157, and 160-167) contain thioacetal bonds and are prepared substantially as described by the procedure of Example 2. Additionally, Examples 61-62, 75, 77, 78, 82, 84, 95, 102, 106, 109, 110, 115, 121, 124, 125, 129, 130, 132, and 133 (SEQ ID NOs: 83, 84, 97, 99, 100, 104, 106, 118, 125, 129, 132, 133, 138, 145, 149, 150, 154, 155, 157, 160) contain PEG24 or PEG12 introduced substantially as described by the procedure of Example 3. and Examples 83, 86-94, 96-100, 103-105, 107, 108, 111, 113, 116-120, 122, 123, 126 (SEQ ID NOs: 105, 109-117, 119-123, 126-128, 130-131, 134, 136, 139-143, 147-148, 151) contain (AEEA)4, (AEEA)6, or (AEEA)8 introduced using standard amino acid coupling methods substantially as described in Example 2.

[0184] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0185] Example 141 Example 141 is a polypeptide represented by the following description (SEQ ID NO: 168). BFA-EKEKEKGEKGRSS[CFGGKIDRIGHYSGLGC]PSFRHGGPSSGAPPPS-NH2(Disulfide Linkage)

[0186] BFA means branched fatty acid. Below is a depiction of the structure of Example 141 using the standard single letter L-amino acid code.

[0187] [ka]

[0188] The peptide backbone of Example 141 was synthesized using fluorenylmethyloxycarbonyl (Fmoc) / tert-butyl (t-Bu) chemistry on a Symphony-X, 24-channel multiplex peptide synthesizer (Gyros Protein Technologies, Inc.). The solid support used consisted of low-loading 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucyl-4-methylbenzhydrylamine resin (Fmoc-Rink-MBHA low-loading resin, EMD Millipore) (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g. Standard side-chain protecting groups were used for all Fmoc-L-amino acids used. Fmoc deprotection before each coupling step was performed with 20% piperidine in DMF (4 min once and 10 min once) under nitrogen bubbling, followed by six DMF wash cycles. All amino couplings were performed using Fmoc Amino Acid (0.3 M in DMF), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, Ambeed, 0.9 M in DMF), and N,N-diisopropylethylamine (DIPEA; 1.2 M in DMF) for 1 h with a 9-fold molar excess of AA / HBTU and a 12-fold molar excess of DIPEA relative to the theoretical resin loading level. Final Fmoc deprotection and washes were completed after synthesis of the primary peptide sequence.

[0189] Conjugation of the fatty acid (BFA) moiety was achieved by manually adding a 2-3x excess of 13-tert-butoxy-2-tert-butoxycarbonyl-13-oxo-2-undecyl-tridecanoic acid dissolved in 5-7 mL of DMF and transferring it to a Symphony-X reactor. This was followed by the addition of a 2-3x excess of diisopropylcarbodiimide (DIC) and a 2-3x excess of ethyl cyano(hydroxyamino)acetate (Oxyma). The reaction time for BFA coupling was approximately 18 h, after which the resin was washed three times with DMF and a Kaiser test was performed to confirm complete coupling. After acylation was complete, the peptidyl resin was transferred as a DCM slurry to a disposable fritted plastic syringe with a Teflon stopper, further washed with DCM, and finally allowed to air-dry completely. The dried resin was then treated with 10 mL of a cleavage cocktail consisting of trifluoroacetic acid (TFA), water, 3,6-dioxa-1,8-octanedithiol (DODT), and triisopropylsilane (TIPS) (TFA:water:DODT:TIPS; 92.5:2.5:2.5:2.5 v / v) for 2 h at room temperature. After the 2-h incubation, the resin was filtered off and collected in a 50 mL conical disposable tube containing 35 mL of cold diethyl ether (-20 °C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 min to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated twice more with ether and dried in vacuo.

[0190] Disulfide bond formation The crude peptide was solubilized in approximately 50 mL of a 10% acetonitrile solution in 0.1% TFA-HO in a 50 mL Falcon tube. The solution was then added to an Erlenmeyer flask placed on a magnetic stirrer equipped with the necessary spin blades, diluted to a total volume of 100 mL with 0.1% TFA-HO (approximately 5 mg / mL crude peptide concentration), and then treated with a few drops of saturated iodine in methanol until a faint yellow color persisted. The reaction was stirred at room temperature for 10 minutes, at which point the excess iodine was quenched with a few drops of 0.1 M aqueous ascorbic acid.

[0191] HPLC purification The crude oxidized solution was then loaded directly onto a Waters semi-preparative HPLC system and purified on a Symmetry C18 (7 μm, 19 × 300 mm; Waters) using a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer system (10–40% over 70 min). Peptide purity was assessed using analytical LC-MS, and pooling criteria were >90%. The major pool of Example 141 was found to be >95.0%. The final major product pool was then lyophilized to yield the lyophilized peptide as the TFA salt. The molecular weight was determined by analytical LC-MS (observed: 5194.2; calculated: 5194.8).

[0192] Example 142 Example 142 is a polypeptide represented by the following description (SEQ ID NO: 169). (BFAEN2-βAla)-EKEKEKG-PEG24-EK-βAla-RSS[CFGGKIDRIGHYSGLGC]PSFRHGGKSSGAPPPS-NH2 (thioacetal bond)

[0193] BFAEN2 means branched fatty acid enantiomer 2. Below is a depiction of the structure of Example 142 using the standard single letter amino acid code, with the exception of βAla, where the structure of the amino acid residues above has been expanded.

[0194] [ka]

[0195] The peptide backbone of Example 142 was synthesized as described for Example 141. The exception to the synthesis method was the coupling of Fmoc-N-amido-PEG24-OH, which required a temporary pause in the automated synthesis protocol. PEG24 residue coupling was achieved by manually adding a three-fold excess of Fmoc-N-amido-PEG24-OH (BroadPharm) dissolved in 5 mL of DMF and transferring it to a Symphony-X reaction vessel, followed by the addition of a two-fold excess of diisopropylcarbodiimide (DIC) and a two-fold excess of ethyl-cyano(hydroxyamino)acetate (Oxyma). The coupling reaction time was approximately 18 hours, after which the resin was washed three times with DMF and a Kaiser test was performed to confirm complete coupling. The automated method was resumed to complete the synthesis of the remaining sequence, and the fatty acids were coupled as described below.

[0196] BFAEN2-βAla binding Fatty acid coupling was achieved by manual addition of a 1.5-fold excess of 2-[[3-(2,5-dioxypyrrolidin-1-yl)oxy-3-oxo-propyl]carbamoyl]-2-undecyl-tridecanedioic acid and a 3-fold excess of N,N-diisopropylethylamine (DIPEA) dissolved in 5-7 mL of DMF and transferred to a Symphony-X reaction vessel. The reaction time for FA coupling was approximately 18 hours, after which the resin was washed three times with DMF. Cleavage was then carried out as described in Example 141, followed by disulfide bond formation as described in Example 141.

[0197] Thioacetal bond formation The crude oxidized solution was then loaded directly onto a Waters semi-preparative HPLC and purified on a Symmetry C18 (7 μm, 19 × 300 mm; Waters) using a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer system (10–40% over 70 min). Fraction purity was assessed using LC-MS, with a pooling standard of >80% for fractions used for thioacetal conversion. These fractions were combined and diluted 1:1 with a 50% mixture of acetonitrile and HO. Disulfide bonds were reduced by first adding 1 mL of a 0.25 M aqueous solution of tris(2-carboxyethyl)phosphine (TCEP, TCI America), followed by the addition of 400–500 μL of pure triethylamine (TEA, Sigma-Aldrich) to bring the solution pH above 7. After 10 minutes, 50-100 μL of diiodomethane (TCI America) was added, followed by an additional 50-100 μL of TEA. The reaction was monitored by LC-MS, and the conversion was complete within approximately 18 hours.

[0198] The crude thioacetal solution was diluted 1:1 with HO and then loaded onto a Waters semi-preparative HPLC system and purified on a Symmetry C18 (7 μm, 19 × 300 mm; Waters) using a linear gradient of 100% acetonitrile and 0.1% formic acid / water buffer system (5–35% over 70 min). LC-MS was used to assess peptide purity, with a pooling standard of >90%. Approximately 100 μL of pure TFA was added to the pooled fractions. The purity of the major pool of Example 142 was found to be >95.0%. Subsequent lyophilization of the final major product pool yielded the lyophilized peptide TFA salt. The molecular weight was determined by analytical LC-MS (observed: 6452.5; calculated: 6453.4).

[0199] Example 143 Example 143 is a polypeptide represented by the following description (SEQ ID NO: 170): (BFAEN2-βAla)-EKEKEKG-PEG24-E-4Pal-KRSS[CFGKKIDRIGHYSGLGC]PSFRHGGKSSGAPPPS-NH2 (thioacetal bond)

[0200] [ka]

[0201] Example 143 was synthesized essentially as described in Example 142. The molecular weight was determined by LC-MS (observed: 6601.6; calculated: 6601.5).

[0202] Example 144 Example 144 is a polypeptide represented by the following description (SEQ ID NO: 171): (BFAEN2-βAla)-EKEKEKG-PEG24-EK-βAla-RSS[CFGKRIDRIGHQSGLGC]PSFRHGSPSSGAPPPS-NH2 (thioacetal bond)

[0203] [ka]

[0204] Example 144 was synthesized essentially as described in Example 142. The molecular weight was determined by LC-MS (observed: 6515.6; calculated: 6516.4).

[0205] Example 145 Example 145 is a polypeptide represented by the following description (SEQ ID NO: 172): (BFAEN2-γGlu)-KEKEKG-PEG24-EK-βAla-RSS[CFGGKIDRIGHYSGLGC]PSFRHGSPSSGAPPPS-NH2 (thioacetal bond)

[0206] [ka]

[0207] Example 145 was synthesized essentially as described in Example 142, except that BFAEN2-γGlu was attached as follows.

[0208] BFAEN2-γGlu binding Fatty acid coupling was achieved by manual addition of a 1.5-fold excess of 2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecanedioic acid and a 3-fold excess of N,N-diisopropylethylamine (DIPEA) dissolved in 5-7 mL of DMF and transferred to a Symphony-X reaction vessel. The reaction time for FA coupling was approximately 18 hours, after which the resin was washed three times with DMF. Cleavage was then carried out as described in Example 141.

[0209] Disulfide bond formation followed by thioacetal bond formation was carried out as described in Example 142.

[0210] The molecular weight was determined by LC-MS (observed: 6381.6; calculated: 6381.2).

[0211] In vitro function Functional activity assay: The functional activity of the ANP polypeptide is determined in an NPR-A-expressing HEK-293 clonal cell line, as described below.

[0212] Full-length cDNA cloning and generation of cell lines overexpressing natriuretic peptide receptors (NPRs) All sequences were verified by full-length sequencing performed by ACGT DNA Sequencing Services (Wheeling, IL). Target cDNAs were cloned into the pJT1 R4 CMV-TO MCS pA vector and then co-transfected with the pJT1R4 Int vector into Jump-in™ T-Rex™ HEK293 cells using the Jump-in™ T-Rex™ HEK293 kit and Lipofectamine LTX and Plus Reagent according to the manufacturer's protocol for inducible mammalian expression, as briefly described below.

[0213] Jump-in™ T-Rex™ HEK293 cells were seeded at 1 million cells / well in 2 mL of culture medium onto BioCOAT® poly-D-lysine-coated 6-well plates (Becton Dickinson, catalog number 354413) and incubated at 37°C for 18 hours in 5% CO2 until 50-70% confluence was reached. A cDNA mixture was prepared in a 50 mL tube by sequentially adding 1.5 μg of target cDNA, 1.5 μg of pJT1R4 Int vector, 3 μL of Plus Reagent, and 300 μL of Opti-MEM I. A reagent mixture was prepared in another 50 mL tube by adding 7.5 μL of Lipofectamine LTX to 300 μL of Opti-MEM I. The mixture was incubated at room temperature for 5 minutes. The cDNA mixture was then transferred to the above reagent mixture, mixed thoroughly, and incubated for an additional 30 minutes at room temperature. 500 μL of the cDNA / Lipofectamine complex was then transferred to a well of a cell plate, where the culture medium was replaced with 2 mL of transfection medium containing DMEM supplemented with 4.5 g / L D-glucose, 10% FBS-HI, and 20 mM HEPES. The transfected cells were cultured for 48 hours in an incubator at 37°C and 5% CO2. Subclones or pools from each overexpressing cell line were maintained in culture medium supplemented with 2 mg / mL G418 sulfate for clonal selection based on their inherent resistance to G418 sulfate, with the medium replaced every 2–3 days, for at least 3 weeks.

[0214] NPR was overexpressed in T-Rex™ HEK293 cells after 48 hours of induction with 300 ng / mL tetracycline in the culture medium. The exponentially growing induced cell line was treated with 0.05% trypsin-EDTA for a few seconds at room temperature, harvested in cell culture medium containing FBS to neutralize the trypsin, counted, and cryopreserved at a density of 2 million cells / mL in a cell storage solution containing FBS-HI with 5% DMSO. The cryopreserved cells were stored at -80°C for several days before being transferred to a liquid nitrogen tank. The induced cell line was then used for suspension assays to measure the activity of the polypeptide in stimulating cGMP production in a cGMP assay, or for preparing cell membranes to measure the binding activity of the polypeptide in a competitive radioligand binding assay, as described below.

[0215] Human and rat NPRA cGMP activity assay Cells overexpressing human or rat NPRA were seeded into 96-well assay plates and stimulated in the presence of assay buffer (normalized as 0% response), human ANP, amidated rat ANP (100 nM, normalized as 100% response), or various concentrations of test polypeptide. Test polypeptides were added at 10 μM starting concentrations and at 10-fold decreasing concentrations to obtain an 8-point concentration-response curve (i.e., 10 μM to 1 pM). The amount of cGMP produced was detected using HTRF® technology and normalized to the maximum amount produced by 100 nM amidated rat ANP and the minimum amount produced by assay buffer. Detailed steps are outlined below.

[0216] A stock solution (2 mM) of the test polypeptide dissolved in DMSO was first prepared by adding Ca 2+ and Mg 2+The polypeptides were diluted 100-fold in assay buffer (pH 7.4) containing HBSS, 5 mM HEPES, 0.5 mM IBMX, and either 0.1% BSA or 0.1% casein. The polypeptides were further serially diluted in 1:10 dilution steps in assay buffer containing 0.1% BSA or 0.1% casein to generate eight 2x working stock solutions ranging from 20 μM to 2 pM.

[0217] 10 μL of cell suspension containing 4000 cells was seeded into a Costar® half-area white opaque 96-well plate (Corning, Cat. No. 3693). Then, 10 μL of assay buffer (basal activity), 200 nM amidated rat ANP (maximum activity), or a 2x working stock solution of the test polypeptide was transferred to the plate. The final concentration of DMSO in each well was 0.5%. The plate was shaken for 15 seconds and then incubated at room temperature for 40 minutes.

[0218] The cGMP produced was measured using a cGMP kit according to the manufacturer's instructions, as described below.

[0219] The cyclic GMP (cGMP) standards provided in the kit were serially diluted 1:3 in assay buffer containing 0.1% BSA + 0.5% DMSO or 0.1% casein + 0.5% DMSO over the range of 1 μM to 0.17 nM. 20 μL of the cGMP standards were then transferred to a separate Costar® 3693 plate.

[0220] cGMP production was stopped, and cGMP content was measured by sequentially adding 10 μL of cGMP-d2 and 10 μL of anti-cGMP-cryptate (prediluted 1:50 in the lysis buffer provided in the kit). The plate was shaken for 15 s, incubated for 2 h at room temperature in the dark, and read on a Pherastar® FSX plate reader (BMG LABTECH, Ortenberg, Germany) at 337 nm for excitation and 665 / 620 nm for emission.

[0221] The 665 nm / 620 nm ratio multiplied by 10,000 was plotted against the logarithmic scale of cGMP standard concentrations to generate a standard curve using an internally generated four-parameter nonlinear regression curve-fitting template. The amount of cGMP produced by cells overexpressing NPRA was interpolated using the cGMP standard curve. A 100% response was determined from wells in the presence of a saturating concentration of amidated rat ANP (100 nM). A 0% response was determined from wells containing assay buffer. Eight-point concentration-response curves for test polypeptides (10 μM to 1 pM) were fitted to a four-parameter model using Prism 9 (GraphPad Software, Inc., San Diego, CA) to determine potency (EC 50 ) values ​​and maximum activation (%Max) were determined.

[0222] Data for exemplary analogs and hANP are shown in Table 2 below.

[0223] [Table 11] [Table 12] [Table 13]

[0224] As can be seen in Table 2, in the presence of BSA, the example ANP polypeptides have lower agonist activity than the natural ligand hANP as determined by the hNPR-A assay. However, when the assay is performed in the presence of casein (in place of serum albumin) as a nonspecific blocker that does not interact with the fatty acid moiety of the molecule being analyzed, the example ANP polypeptides have agonist activity comparable to hANP.

[0225] Human and rat NPRB cGMP activity assay The functional activity of ANP polypeptides is determined in NPR-B-expressing HEK-293 clonal cell lines as described below.

[0226] Cells overexpressing human or rat NPRB were seeded into 96-well assay plates and stimulated in the presence of assay buffer (normalized as 0% response), human CNP-22 (1 μM, normalized as 100% response), or various concentrations of test polypeptide. Test polypeptides were added at 10-fold decreasing concentrations, starting at 10 μM, to obtain a 10-point concentration-response curve (i.e., 10 μM to 0.01 pM). The amount of cGMP produced was detected using HTRF® technology and normalized to the maximum amount produced by 1 μM human CNP-22 and the minimum amount produced by assay buffer. Detailed steps are outlined below.

[0227] A stock solution (2 mM) of the test polypeptide dissolved in DMSO was first prepared by adding Ca 2+ and Mg 2+ The polypeptides were diluted 100-fold in assay buffer (pH 7.4) containing HBSS, 5 mM HEPES, 0.5 mM IBMX, and either 0.1% BSA or 0.1% casein. The polypeptides were further serially diluted in 1:10 dilution steps in assay buffer containing 0.1% BSA or 0.1% casein to generate 10 2x working stock solutions ranging from 20 μM to 0.02 pM.

[0228] 15 μL of assay buffer (basal activity), 1 μM human CNP-22 (maximum activity), or a 2x working stock solution of the test polypeptide was transferred to a Costar® 3693 plate. 15 μL of cell suspension containing 4000 cells was then plated. The final concentration of DMSO in each well was 0.5%. The plate was shaken for 15 seconds and then incubated at room temperature for 40 minutes.

[0229] The cGMP produced was measured using a cGMP kit according to the manufacturer's instructions, as described below.

[0230] The cyclic GMP (cGMP) standards provided in the kit were serially diluted 1:3 in assay buffer containing 0.1% BSA + 0.5% DMSO or 0.1% casein + 0.5% DMSO over the range of 1 μM to 0.17 nM. 30 μL of the cGMP standards were transferred to a separate Costar® 3693 plate.

[0231] cGMP production was stopped, and cGMP content was measured by sequentially adding 15 μL of cGMP-d2 and 15 μL of anti-cGMP-cryptate (prediluted 1:50 in the lysis buffer provided in the kit). The plate was shaken for 15 seconds, incubated in the dark at room temperature for 2 hours, and read on a Pherastar® FSX plate reader at 337 nm for excitation and 665 nm / 620 nm for emission.

[0232] A standard curve was generated using an internally generated four-parameter nonlinear regression curve-fitting template by plotting the 665 nm / 620 nm ratio multiplied by 10,000 against the cGMP standard concentration on a logarithmic scale. The amount of cGMP produced by cells overexpressing NPRB was interpolated using the cGMP standard curve. A 100% response was determined from wells in the absence of test polypeptide and in the presence of a saturating concentration of human CNP-22 (1 μM). A 0% response was determined from wells containing assay buffer. Prism 9 was used to fit a 10-point concentration-response curve for the test polypeptide (10 μM to 0.01 pM) to a four-parameter model to determine potency (EC 50 ) values ​​and maximum activation (%Max) were determined.

[0233] Similar to hANP, none of the example polypeptides exhibited significant agonist activity at NPR-B.

[0234] In vivo studies Pharmacokinetics in male Sprague Dawley rats: The pharmacokinetics of exemplary analogs are evaluated after a single subcutaneous dose of 200 nM / kg in male Sprague Dawley rats. Blood samples are collected over 120 hours, and the resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K3EDTA) concentrations are determined using a qualified LC / MS method that measures the intact mass of the ANP polypeptide. Each peptide and analog is extracted from 100% specific plasma using methanol containing 0.1% formic acid as an internal standard. LC / MS detection is performed using a Thermo Q-Exactive high-resolution instrument coupled with a Thermo Easy Spray PepMap. Mean pharmacokinetic parameters are shown in Table 3.

[0235] [Table 14]

[0236] Studies in the salt-containing drinking water / unilateral nephrectomy / aldosterone (SAUNA) mouse model The effects of the example ANP polypeptides are investigated in the saline drinking water / unilateral nephrectomy / aldosterone (SAUNA) mouse model, a mouse model of heart failure induced by chronic aldosterone infusion. After approximately two weeks of acclimation, heart failure is induced in male C57BL / 6N (Taconic) mice by unilateral nephrectomy, continuous d-aldosterone infusion, and 1.0% sodium chloride in the drinking water for four weeks (see Tanaka et al., 2016; Valero-Munoz, Li, Wilson, Boldbaatar, et al., 2016; Valero-Munoz, Li, Wilson, Hulsmans, et al., 2016; Yang, Kong, Shuai, Zhang, & Huang, 2020; Yoon et al., 2021). Approximately two weeks after induction of the heart failure protocol, mice are divided into groups to provide comparable variance in body weight and blood pressure (measured in conscious mice using a non-invasive tail cuff system (Kent Scientific)); mice are randomized using the Block Randomized Allocation Tool (BRAT, Eli Lilly and Company). Once randomized, mice are treated once daily by subcutaneous (SC) injection of ANP polypeptide (0.4 mg / kg). Blood pressure is monitored weekly throughout the study period. Two weeks after the start of treatment (four weeks after induction of heart failure), mice are anesthetized with isoflurane and intubated via tracheotomy, and the chest is opened to expose the heart, allowing for placement of a pressure-volume catheter (Transonic). The pressure-volume (PV) catheter is introduced into the left ventricle via a tip puncture using a 27G needle. Calibration of the PV catheter is performed according to the manufacturer's instructions. Data are analyzed using LabChart Pro software (AD Instruments). After PV loop measurements, the mice are sacrificed and the ratio of heart weight to tibia length is used as an index of hypertrophy.

[0237] The effect of Example 8 was investigated using the above-mentioned SAUNA mouse model. Administration of Example 8 resulted in a decrease in blood pressure, heart weight, and tibia length, as well as a decrease in left ventricular diastolic pressure.

[0238] In vivo monkey studies - cGMP levels In vivo monkey studies were conducted as described in detail below. A single dose was administered subcutaneously (SC) to young to adult male cynomolgus monkeys. Blood was collected pre-dose and at various time points throughout the study. Aliquots of cynomolgus monkey plasma were received for cyclic GMP (cGMP) measurements and stored at -80°C before use.

[0239] Control plasma Control plasma was generated to determine the recovery rate of the cGMP spike using the Enzo cGMP complete ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY, catalog number ADI-901-164). Briefly, blood from approximately 7-month-old male Sprague-Dawley rats (Inotiv, Indianapolis, IN) was collected via retro-orbital bleeding in a volume of approximately 4 mL into BD Vacutainer EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, catalog number BD-367856). Plasma was prepared by spinning the tubes at 3500 rpm (3000 × g) for 10 minutes at 4°C in an Eppendorf Refrigerated Centrifuge 5810R (Brinkman Instruments, Inc., Westbury, NY). Plasma was collected as a positive control plasma. Cyclic GMP (cGMP standard from the Enzo cGMP complete ELISA kit) was added to the positive control plasma at an additional final concentration of 40 nM. Positive control and spike-in control plasma were aliquoted and stored at -80°C.

[0240] Assay method - Monkey plasma cGMP ELISA cGMP content was measured using the Enzo cGMP complete ELISA kit according to the manufacturer's instructions, but with modifications as described below. The cGMP standards provided in the kit were diluted 1:3 from 50 nM to 0.023 nM in 1x assay buffer diluted with water from the 2x assay buffer provided in the kit. The cGMP standards (150 μL) were transferred to a 96-well polypropylene plate (Thermo Scientific, catalog number 442587).

[0241] Plasma samples were thawed from -80°C and diluted 1:20 with 1x assay buffer in the plate to a final volume of 150 μL. Assay buffer (150 μL) was added to duplicate wells for both the nonspecific binding control (absence of cGMP antibody) and the maximum binding control (absence of competing cGMP). Positive control and spike-in control plasma were diluted 1:20 with 1x assay buffer to a final volume of 150 μL in duplicate wells on the plate. All diluted plasma was mixed by pipetting several times.

[0242] The acetylating agent mixture was prepared by adding 1 part acetic anhydride to 2 parts trimethylamine provided in the kit and mixing thoroughly using a Vortex (Scientific Industries, Inc., Bohemia, NY). All controls, standards, and plasma samples were acetylated by adding 15 μL of the acetylating agent mixture to the plate, one column at a time, and shaking for 1 minute at room temperature on a Titer Plate Shaker (Lab-Line Instrument, Inc., Melrose Park, IL). After acetylating the last column, the plate was shaken for an additional 1 minute to ensure the reaction was complete.

[0243] Acetylated controls, standards, and plasma samples (100 μL) were transferred to an ELISA plate (n=1). Then, 50 μL of cGMP conjugate was added to each well, followed by 50 μL of cGMP antibody, except for two nonspecific binding wells, which received 50 μL of 1x assay buffer. The plate was then sealed and shaken on a plate shaker at room temperature for 2 hours. After this incubation, the plate was washed five times with 200 μL of 1x wash buffer diluted with water from the 5x wash buffer provided in the kit. Then, 200 μL of para-nitrophenyl phosphate (pNpp) was added to each well. The plate was sealed with a new plate sealer and shaken on a plate shaker in the dark at room temperature for 1 hour. 50 μL of stop solution was added to each well to stop the enzyme reaction. Plates were then read at 405 nm using a SpectraMax Plus (Molecular Devices, San Jose, Calif.).

[0244] Number of copies N = 3 monkeys per group.

[0245] Data analysis The average absorbance at 405 nm (OD405nm) for the nonspecific binding control was subtracted from the OD405nm of all samples. The subtracted OD405nm of all samples was then normalized to the average subtracted OD405nm of the maximum binding control, yielding B / B0%. The B / B0% of the cGMP standards was then plotted against the logarithmic scale of cGMP standard concentrations to generate a standard curve using an internally generated four-parameter nonlinear regression curve fitting template. The amount of total cGMP present in the experimental samples was interpolated using this standard curve in the template. The net cGMP change (nM) was calculated by subtracting the cGMP value for each animal measured in plasma before administration of each polypeptide shown in Table 4 (pre-dose, time 0) from the cGMP value for the same animal at each post-dose time point. Microsoft Excel (Microsoft Corp., Redmond, WA) was used to graph cGMP (nM, mean ± standard deviation of the mean SEM) at various time points and the net cGMP change (nM, mean ± SEM). The monkey cGMP data are shown in Table 4.

[0246] [Table 15]

[0247] [Table 16]

[0248] As can be seen from Table 4, when the polypeptides of Examples 1, 2, 3, 4, 6, 7, 29, 56, 61, 81, 89, 106, 127, 132, 136, 144, and 145 were each administered to monkeys, the net cGMP level increased.

[0249] In vivo studies - cGMP levels In vivo studies in dogs were conducted as detailed below. A single dose was administered subcutaneously (SC) to young to adult male purebred beagle dogs from the Labcorp stock colony housed at Labcorp-Madison. Blood was collected pre-dose and at various time points throughout the study. Aliquots of beagle plasma were received for cyclic GMP (cGMP) measurements and stored at -80°C before use.

[0250] Control plasma Control plasma was generated to determine the recovery rate of the cGMP spike using the Enzo cGMP complete ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY, catalog number ADI-901-164). Briefly, blood from approximately 7-month-old male Sprague-Dawley rats (Inotiv, Indianapolis, IN) was collected via retro-orbital bleeding in a volume of approximately 4 mL into BD Vacutainer EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, catalog number BD-367856). Plasma was prepared by spinning the tubes at 3500 rpm (3000 × g) for 10 minutes at 4°C in an Eppendorf Refrigerated Centrifuge 5810R (Brinkman Instruments, Inc., Westbury, NY). Plasma was collected as a positive control plasma. Cyclic GMP (cGMP standard from the Enzo cGMP complete ELISA kit) was added to the positive control plasma at an additional final concentration of 40 nM. Positive control and spike-in control plasma were aliquoted and stored at -80°C.

[0251] Assay Method - Canine Plasma cGMP ELISA cGMP content was measured using the Enzo cGMP complete ELISA kit according to the manufacturer's instructions, but with modifications as described below. The cGMP standards provided in the kit were diluted 1:3 from 50 nM to 0.023 nM in 1x assay buffer diluted with water from the 2x assay buffer provided in the kit. The cGMP standards (150 μL) were transferred to a 96-well polypropylene plate (Thermo Scientific, catalog number 442587).

[0252] Plasma samples were thawed from -80°C and diluted 1:20 and 1:40 with 1x assay buffer in the plate to a final volume of 150 μL. Assay buffer (150 μL) was added to duplicate wells for both the nonspecific binding control (absence of cGMP antibody) and the maximum binding control (absence of competing cGMP). Positive control and spike-in control plasma were diluted 1:20 with 1x assay buffer to a final volume of 150 μL in duplicate wells of the polypropylene plate. All diluted plasmas were mixed by pipetting several times.

[0253] An acetylating agent mixture was prepared by adding 1 part acetic anhydride to 2 parts trimethylamine provided in the kit and mixing thoroughly using a Vortex (Scientific Industries, Inc., Bohemia, NY). All controls, standards, and plasma samples were acetylated by adding 15 μL of the acetylating agent mixture to eight wells of the polypropylene plate, one column at a time, and shaking at room temperature for 1 minute on a Titer Plate Shaker (Lab-Line Instrument, Inc., Melrose Park, IL). After acetylating the last column, the plate was shaken for an additional 1 minute to ensure the reaction was complete.

[0254] Acetylated controls, standards, and plasma samples (100 μL) were transferred to an ELISA plate (n=1). Then, 50 μL of cGMP conjugate was added to each well, followed by 50 μL of cGMP antibody, except for two nonspecific binding wells, which received 50 μL of 1x assay buffer. The plate was then sealed and shaken on a plate shaker at room temperature for 2 hours. After this incubation, the plate was washed five times with 200 μL of 1x wash buffer diluted with water from the 5x wash buffer provided in the kit. Then, 200 μL of para-nitrophenyl phosphate (pNpp) was added to each well. The plate was sealed with a new plate sealer and shaken on a plate shaker in the dark at room temperature for 1 hour. 50 μL of stop solution was added to each well to stop the enzyme reaction. Plates were then read at 405 nm using a SpectraMax Plus (Molecular Devices, San Jose, Calif.).

[0255] Number of copies N=3 dogs per group.

[0256] Data analysis The average absorbance at 405 nm (OD405nm) for the nonspecific binding control was subtracted from the OD405nm of all samples. The subtracted OD405nm of all samples was then normalized to the average subtracted OD405nm of the maximum binding control, yielding B / B0%. The B / B0% of the cGMP standards was then plotted against the logarithmic scale of cGMP standard concentrations to generate a standard curve using an internally generated four-parameter nonlinear regression curve fitting template. The amount of total cGMP present in the experimental samples was interpolated using this standard curve in the template. The net cGMP change (nM) was calculated by subtracting the cGMP value for each animal measured in plasma before administration of each polypeptide shown in Table 5 (pre-dose, time 0) from the cGMP value for the same animal at each post-dose time point. Microsoft Excel (Microsoft Corp., Redmond, WA) was used to graph cGMP (nM, mean ± standard deviation of the mean SEM) at various time points and the net cGMP change (nM, mean ± SEM). Dog cGMP data are shown in Table 5.

[0257] [Table 17]

[0258] [Table 18]

[0259] As can be seen from Table 5, administration of each of the polypeptides of Examples 2, 4 and 7 to dogs increased net cGMP levels.

[0260] array SEQ ID NO: 1: rANP (rat ANP) SLRRSS[CFGGRIDRIGAQSGLGC]NSFRY (disulfide bond between C7 and C23) SEQ ID NO: 2: hANP (human ANP) SLRRSS[CFGGRMDRIGAQSGLGC]NSFRY (disulfide bond between C7 and C23) SEQ ID NO: 3:- Formula I X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (In the formula, X1 is absent, S or E; X2 is absent or is L, K, 4-Pal, H, or E; X3 is absent or is R, β-Ala, P, K, E, or G; X9 is G, 4-Pal, or H; X 10 is G, K, R, or Dap X 11 is R, K, G, or Dap; X 13 is D or G, X 17 is A, H, Dap, K, R, or Orn, X 18 is Q, Y, or 4-Pal, X 26 is F or L, X 28 is Y, H, or 4-Pal, and X 29 is absent, is GGP, or is selected from SEQ ID NOs: 4 to 20; and the C-terminal amino acid is optionally amidated. SEQ ID NO:4 SGAPPPE SEQ ID NO:5 KITAKEDE SEQ ID NO:6 GPSSGAPPPE SEQ ID NO:7 GPSSGAPPPS SEQ ID NO:8 GGSSGAPPPS SEQ ID NO:9 GGPSSGAPPPS SEQ ID NO: 10 KGPSSGAPPPS SEQ ID NO: 11 GGKSSGAPPPS SEQ ID NO: 12 GGPPS-Aib-KPPPK SEQ ID NO: 13 GSPSSGAPPPS SEQ ID NO: 14 RITAREDKQGYA SEQ ID NO: 15 RITAREDKQGEA SEQ ID NO: 16 GSPSSGAPPPS-PEG24-G SEQ ID NO: 17 SGSPSSGAPPPSG SEQ ID NO: 18 GGESSGEPPPSEE SEQ ID NO: 19 GSGSPSSGAPPPSG SEQ ID NO: 20 SGSPSSGAPPPSEEEG SEQ ID NO: 21:- Formula II Fatty acids-Z1-Z2-Z3-X1X2X3RSSCFX9X 10 X 11 IDRIGX 17 X 18 SGLGCX 24 SX 26 RX 28 (In the formula, The fatty acid is a C16 to C26 fatty acid, Z1 comprises an amino acid selected from γGlu, E, and β-Ala; Z2 comprises a sequence of 4 to 10 amino acids that is absent or contains amino acids independently selected from E, K, G, P, A, and S; Z3 is absent or comprises a polyethylene glycol or (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety; The C-terminal amino acid is optionally amidated. SEQ ID NO: 22 EKEKEKG SEQ ID NO: 23 EPEPEPG SEQ ID NO: 24 APPSG SEQ ID NO: 25 KEKEKG SEQ ID NO: 26 EKEKEKE SEQ ID NO: 27 X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (In the formula, X1 is S or E; X2 is K or 4-Pal; X3 is R, β-Ala, or K; X9 is G, X 10 is G or K, X 11 is R or K, X 13 is D or G, X 17 is H, X 18 is Q or Y, X 26 is F, X 28 is H, and X 29 is selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13); The C-terminal amino acid is optionally amidated. SEQ ID NOs: 28 to 167 Examples 1-140, respectively, are listed in Table 1. SEQ ID NOs: 168-172 Examples 141 to 145, respectively. SEQ ID NO: 173 (amidated rat ANP) SLRRSS[CFGGRIDRIGAQSGLGC]NSFRY-NH2 (disulfide bond between C7 and C23)

Claims

1. X １ X ２ X ３ RSSCFX ９ X １０ X １１ IX １３ RIGX １７ X １８ SGLGCPSX ２６ RX ２８ X ２９ (SEQ ID NO: 3), (In the formula, X １ is absent or is S or E, X ２ is absent or is L, K, 4-Pal, H, or E; X ３ is absent or is R, β-Ala, P, K, E, or G; X ９ is G, 4-Pal, or H, X １０ is G, K, R, or Dap; X １１ is R, K, G, or Dap; X １３ is D or G, X １７ is A, H, Dap, K, R, or Orn; X １８ is Q, Y, or 4-Pal; X ２６ is F or L, X ２８ is Y, H, or 4-Pal, and X ２９ does not exist, or GGP, SGAPPPE (SEQ ID NO: 4), KITAKEDE (SEQ ID NO: 5), GPSSGAPPPE (SEQ ID NO: 6), GPSSGAPPPS (SEQ ID NO: 7), GGSSGAPPPS (SEQ ID NO: 8), GGPSSGAPPPS (SEQ ID NO: 9), KGPSSGAPPPS (SEQ ID NO: 10), GGKSSGAPPPS (SEQ ID NO: 11), GGPPS-Aib-KPPPK (SEQ ID NO: 12), GSPSSGAPPPS (SEQ ID NO: 13), RITAREDKQGYA (SEQ ID NO: 14), RITAREDKQGEA (SEQ ID NO: 15), GSPSSGAPPPS-PEG24-G (SEQ ID NO: 16), SGSPSSGAPPPSG (SEQ ID NO: 17), GGESSGEPPPSEE (SEQ ID NO: 18), GSGSPSSGAPPPSG (SEQ ID NO: 19), and SGSPSSGAPPPSEEG (SEQ ID NO: 20), The C-terminal amino acid is optionally amidated. or a pharmaceutically acceptable salt thereof.

2. 2. The polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, comprising a disulfide bond or a thioacetal bond between the cysteine ​​at position 7 and the cysteine ​​at position 23 of SEQ ID NO:

3.

3. The polypeptide further comprises a fatty acid conjugated to an amino acid present at the N-terminus of the polypeptide, the fatty acid having the structure: Fatty acids - Z1 - Z2 - Z3 - X １ X ２ X ３ RSSCFX ９ X １０ X １１ IDRIGX １７ X １８ SGLGCX ２４ SX ２６ RX ２８ X ２９ wherein the fatty acid is C １６ ~C ２６ a fatty acid conjugated to an amino acid present at the N-terminus of said polypeptide via the structure Z1-Z2-Z3, wherein: Z1 is an amino acid selected from γGlu, E, and β-Ala; Z2 is (EK) ｂ G, (EP) ｂ G, K (EK) ｃ G. (E.K.) ｃ E, and APPSG (SEQ ID NO:24), wherein b is 2, 3, or 4, and c is 1, 2, 3, or 4; and Z3 is absent or (polyethylene glycol) ｍ (wherein m is an integer selected from 10 to 30) or ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) ｎ 3. The polypeptide according to claim 1 or 2, comprising: (wherein n is an integer selected from 1 to 10); or a pharmaceutically acceptable salt thereof.

4. X １ is S or E, X ２ is K or 4-Pal, X ３ is R, β-Ala, or K; X ９ is G, X １０ is G or K, X １１ is R or K, X １３ is D or G, X １７ is H, X １８ is Q or Y, X ２６ is F, X ２８ is H, and X ２９ is selected from GGPSSGAPPPS (SEQ ID NO: 9), GGKSSGAPPPS (SEQ ID NO: 11), and GSPSSGAPPPS (SEQ ID NO: 13), or a pharmaceutically acceptable salt thereof.

5. The fatty acid is C １６ ~C ２２ 5. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 3 or 4, which is a fatty acid.

6. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 5, wherein Z1 is γ-Glu.

7. 7. The polypeptide of claim 6, or a pharmaceutically acceptable salt thereof, wherein Z2 comprises a sequence selected from EKEKEKG (SEQ ID NO: 22), EPEPEPG (SEQ ID NO: 23), and APPSG (SEQ ID NO: 24).

8. Z3 is absent or (polyethylene glycol) ｍ (wherein m is 12 or 24), and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) ｎ 8. The polypeptide of claim 7, or a pharmaceutically acceptable salt thereof, wherein n is selected from the group consisting of:

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide is selected from SEQ ID NOs: 28 to 167, or a pharmaceutically acceptable salt thereof.

10. The polypeptide according to any one of claims 1 to 9, wherein the polypeptide is selected from SEQ ID NOs: 28, 45, 50, 51, 78, 83, 84, 97, 98, 144, 158, and 159, or a pharmaceutically acceptable salt thereof.

11. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the C-terminus is amidated.

12. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the polypeptide is an agonist of NPR-A.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

14. 14. The pharmaceutical composition of claim 13, formulated for subcutaneous (SQ) or intravenous (IV) administration.

15. 15. The pharmaceutical composition of claim 14, formulated for SQ administration.

16. A method for treating cardiovascular disease (CVD), comprising administering to a patient in need thereof an effective amount of the polypeptide of any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 13.

17. 17. The method of claim 16, wherein the CVD is heart failure.

18. 18. The method of claim 17, wherein the heart failure is HFpEF.

19. A polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for use in therapy.

20. The polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, for use in treating CVD.

21. The polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, for use in treating heart failure.

22. The polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, for use in treating HFpEF.

23. Use of the polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the manufacture of a drug for treating CVD.

24. Use of the polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the manufacture of a drug for treating heart failure.

25. Use of the polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the manufacture of a drug for treating HFpEF.

Images