Long-acting natriuretic peptides and their use

Abstract

This invention provides atrial natriuretic peptide (ANP) polypeptides and therapeutic methods using ANP polypeptides. [Solution] The present invention relates to the provision of a polypeptide having a specific sequence that is an agonist of NPR-A, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing said polypeptide, and the use of the pharmaceutical composition as a method for treating heart disease.

Description

[Technical Field]

[0001] This disclosure relates generally to biology and medicine, and more particularly to peptides that are natriuretic peptide analogs, in particular to long-acting atrial natriuretic peptide (ANP) polypeptides that bind to natriuretic peptide receptors such as NPR-A and thereby function as NPR-A agonists, exhibiting improved stability. This disclosure further relates to compositions comprising the above peptides, and their use in the treatment of cardiovascular abnormalities, diseases, or disorders. [Background technology]

[0002] There is still 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 dysfunctional heart. These therapies include (a) drugs intended to lower heart rate, e.g., beta-blockers and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers (e.g., ivabradine); (b) drugs intended to lower blood pressure, e.g., angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), mineralocorticoid receptor antagonists (MRAs), and combinations of ARBs and neprilysin (NEP) inhibitors (e.g., sacubitril / valsartan (ENTRESTO®)); and / or (c) drugs intended to treat or prevent volume overload, e.g., diuretics and MRAs. However, these therapies do not directly treat the heart and have practical limitations, such as the need for dose setting and monitoring of hypotension. Furthermore, even with these existing treatment options available, all HF patients, even those with mild symptoms, have a high risk of death. See, for example, 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 provide 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, including 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 located 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 anti-hypertrophic and anti-fibrotic effects in the heart, can improve lung function, and may have beneficial effects on glucose and energy metabolism. ANP therapy can be rephrased as improving cardiac filling pressure, promoting beneficial cardiac remodeling, improving diastolic function, and exerting cardioprotective effects in the heart, vascular system, lungs, and kidneys.

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

[0006] Several techniques exist to extend the half-life of peptides, including peptide conjugation, pegylation, and Fc conjugation to fatty acid moieties, recombinant human serum albumin (rHSA) or bovine serum albumin (BSA), pharmaceutically acceptable polymers such as amino acid polymer sequences (XTEN), non-sulfated heparin-like carbohydrate polymer (HEP) or hydroxyl ethyl starch (HES), and llama heavy chain antibody fragments (VHH). (For example, 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 analogs and derivatives that mimic the biological activity of natural ANP and / or have improved stability. For example, European Patent No. 465097; U.S. Patents No. 4,607,023; U.S. Patents No. 5,212,286; U.S. Patents No. 5,434,133; U.S. Patents No. 6,525,022; U.S. Patents No. 8,058,242; U.S. Patents No. 9,193,777; U.S. Patents No. 10,947,289; U.S. Patents No. 11,312,758; International Publications No. 1988 / 03537; International Publications No. 1998 / 45329; International Publications No. 2004 / 011498; and International Publications No. 2018 / 175534 describe various ANP analogs and derivatives with higher stability. U.S. Patent No. 5,204,328 describes an ANP analog containing N-alkylated amino acids to protect peptides from enzymatic degradation. U.S. Patent No. 6,525,022 describes an ANP analog having equal binding affinity to NPR-A but reduced affinity to NPR-C. International Publication No. 1998 / 45329 describes an ANP derivative in which a lipophilic substituent is linked to a peptide. International Publication No. 2004 / 011498 describes an ANP derivative containing a reactive element that links to a peptide, making that peptide capable of forming a peptide-blood component complex. U.S. Patent No. 9,193,777 describes an ANP analog containing a 12-amino acid C-terminal elongation based on a familial ANP gene frameshift mutation. U.S. Patent No. 10,947,289 describes a glycosylated ANP derivative in which a glycosphing is linked to a peptide. International Publication No. 2008 / 154226 describes an ANP fusion protein linked to an antibody Fc fragment.

[0008] Nevertheless, alternative treatment options are still needed. Treatments that improve long-term outcomes, such as increased survival rates and reduced hospitalization rates, are needed. Treatments that improve cardiac function, potentially correcting or reversing the disease, are also needed. Treatments that improve the quality of life (QoL) of patients with progressive disease are also needed. Furthermore, there is a need for therapeutic agents that can be administered at low frequencies, such as once daily, three times a week, twice a week, or once a week, and that have a sufficiently long duration of action. This invention aims to meet one or more of these important, unmet needs. [Overview of the project]

[0009] Provided herein are ANP polypeptides that bind to and act upon NPR-A, possessing natriuretic, diuretic, and vasodilatory activity. Furthermore, the ANP polypeptides described herein have a long-lasting effect on NPR-A, enabling administration at low frequencies, such as once daily, three times a week, twice a week, or once a week. The ANP polypeptides described herein also exhibit desirable development potential profiles that make them suitable for therapeutic use. Thus, the ANP polypeptides described herein may be useful in long-term treatment to lower blood pressure, reduce pathological wall stress, and improve harmful cardiac remodeling, and may also have beneficial effects against pulmonary congestion.

[0010] Thus, the present disclosure also provides methods of using ANP polypeptides for treating or preventing cardiovascular disease (CVD) and related disorders, such as heart failure (HF) in particular. 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 the need for a left ventricular assist device (LVAD) or heart transplant, improve heart function and structure, and / or lead to an improvement in QoL through improvement of symptoms and physical limitations associated with HF.

[0011] In one embodiment, as used herein, Formula I: X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (SEQ ID NO: 3), (wherein, X1 is absent or is 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 It does not exist, or GGP, SGAPPPE (SEQ ID NO: 4), KITAKEDE (Sequence ID 5), GPSSGAPPPE (Sequence ID 6), GPSSGAPPPS (Sequence ID 7), GGSSGAPPPS (Sequence ID 8), GGPSSGAPPPS (Sequence ID 9), KGPSSGAPPPS (Sequence ID 10), GGKSSGAPPPS (Sequence ID 11), GGPPS-Aib-KPPPK (Sequence ID 12), GSPSSGAPPPS (Sequence ID 13), RITAREDKQGYA (Sequence ID 14), RITAREDKQGEA (Sequence ID 15), GSPSSGAPPPS-PEG24-G (Sequence ID 16), SGSPSSGAPPPSG (Sequence ID 17), GGESSGEPPPSEE (Sequence ID 18), GSGSPSSGAPPPSG (Sequence ID 19), and Selected from SGSPSSGAPPPSEEEG (Sequence ID 20), (The C-terminal amino acid is optionally amidated.) Polypeptides of or pharmaceutically acceptable salts thereof are provided.

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

[0013] In another embodiment, the polypeptide of formula I or a pharmaceutically acceptable salt thereof is conjugated with 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 formula II: Fatty acids-Z1-Z2-Z3-X1X2X3RSSCFX9X 10 X 11 IDRIGX 17 X 18 SGLGCX 24 SX 26 RX 28 X 29 (Sequence ID 21) (In the formula, the fatty acid is C 16 ~C 26 It is a fatty acid, and is conjugated to an amino acid located at the N-terminus of a polypeptide via the structure Z1-Z2-Z3, and in the formula, Z1 contains amino acids selected from γGlu, E, and β-Ala. Z2 is either absent or contains a sequence of 4 to 10 amino acids, including amino acids independently selected from E, K, G, P, A, and S. Z3 is either absent or contains 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 carboxyl 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) cSelected from E, b is 2, 3, or 4, and c is 1, 2, 3, or 4. For example, in some embodiments, Z2 is EKEKEKG (sequence number 22), EPEPEEPG (sequence number 23), APPSG (sequence number 24), KEKEKG (sequence number 25), or EKEKEKE (sequence number 26).

[0016] In some embodiments, Z3 is (polyethylene glycol) m (Note that in the formula, m is an integer selected from 10 to 30) and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) n (Note that in the formula, n is an integer selected from 2 to 10). For example, in some embodiments, Z3 is (polyethylene glycol) 12 (Polyethylene glycol) 24 These are ((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, a pharmaceutical composition is provided comprising a polypeptide described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] In another embodiment, a method is provided herein for using the polypeptides described herein or pharmaceutically acceptable salts thereof to treat or prevent cardiovascular disease (CVD) and related abnormalities. Such a method may include at least the step of administering an effective amount of the polypeptide described herein or a pharmaceutically acceptable salt thereof to an individual in need thereof. In some examples, CVD is heart failure (HF), and in particular heart failure with preserved ejection factor (HfpEF).

[0019] In another embodiment, a polypeptide described herein or a pharmaceutically acceptable salt thereof is provided for therapeutic use.

[0020] In another embodiment, polypeptides described herein or pharmaceutically acceptable salts thereof are provided for use in the treatment or prevention of CVD. In some examples, CVD is HF, and in particular HfpEF.

[0021] In another embodiment, polypeptides described herein or pharmaceutically acceptable salts thereof are provided for use in the manufacture of pharmaceuticals for the treatment or prevention of CVD. In some examples, CVD is HF, and in particular HfpEF. [Modes for carrying out the invention]

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the ANP polypeptides, pharmaceutical compositions, and methods, but preferred methods and materials are described herein.

[0023] Furthermore, the indefinite article "a" or "an" does not rule out the possibility of multiple elements unless the context explicitly requires that there be one or only one element. Therefore, the indefinite article "a" or "an" usually means "at least one."

[0024] As used herein, “about” means a statistically significant range of values ​​such as concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The permissible variation encompassed by “about” depends on the particular system in the study and will be readily apparent to those skilled in the art.

[0025] As used herein, with respect to one or more ANP receptors, “activity,” “activating,” “activating,” etc., means the ability of a compound such as an ANP polypeptide described herein to bind to the receptor and induce a response thereon, as measured using an assay known in the art, such as the in vitro assay described below.

[0026] As used herein, “ANP polypeptide” means an ANP polypeptide that has structural similarities to, but differs from, naturally occurring ANP (in particular, rat ANP (SEQ ID NO: 1) or human ANP (SEQ ID NO: 2)). The ANP polypeptides described herein contain amino acid sequences that result in polypeptides with affinity to 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” means a variant of a reference peptide or polypeptide that is identical to the reference molecule except that it has one or more conservative amino acid substitutions in its amino acid sequence. Generally, a conservatively modified variant includes 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 an amino acid substitution by an amino acid that has similar properties (e.g., charge, side chain size, hydrophobic / hydrophilic, skeletal structure, and stiffness) and has minimal impact on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitutions of functionally similar amino acids are well known in the art and therefore do not need to be described in detail herein.

[0028] When used herein, "C 16 ~C 26 "Fatty acid" refers to a carboxylic acid having 16 to 26 carbon atoms. C is suitable for use in this specification. 16 ~C 26 Fatty acids may be straight-chain or branched-chain fatty acids. Straight-chain fatty acids are preferred for use in this specification. 16 ~C 26 Fatty acids may be saturated monoacids or saturated diacids. As used herein, “saturated” means that the fatty acid does not contain carbon-carbon double or triple bonds.

[0029] As used herein, “effective dose” means the amount, concentration, or dosage of one or more ANP polypeptides described herein, or pharmaceutically acceptable salts thereof, that, after a single or multiple administration to an individual in need, provide the desired effect in such individual under diagnosis or treatment. The effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the polypeptide. The effective dose can be determined by those skilled in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining the effective dose for a subject, several factors are taken into consideration, including, but not limited to, the species of mammal; its size, age, and overall health status; the specific disease or disorder involved; the degree or involvement or severity of the disease or disorder; the individual patient’s response; the specific ANP polypeptide administered; the mode of administration; the bioavailability characteristics of the administered formulation; the administration regimen chosen; the use of concomitant drugs; and other relevant circumstances.

[0030] As used herein, “extended duration of action” means that the binding affinity and activity to the ANP polypeptide persist for a longer period than that of the natural human ANP polypeptide, enabling administration at least once daily, three times a week, twice a week, once a week, or less than once a week, for example, 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 used in the following examples.

[0031] As used herein, “half-life” or “t1 / 2” means the time it takes for half the amount of a compound, such as natural ANP or the ANP polypeptide as used herein, to be removed by biological processes from a fluid or other physiological space, such as the serum or plasma of an individual. Alternatively, t1 / 2 may also mean the time it takes for half of the amount of such a compound to lose half of its pharmacological, physiological, or radioactive activity.

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

[0033] As used herein, “in combination with ~” means administering at least one of the ANP polypeptides specified herein simultaneously with one or more additional therapeutic agents, either sequentially or in a single combination formulation (combination agent) with them.

[0034] As used herein, “individuals in need of it” means mammals such as humans that have an abnormality, disease, disorder, or symptom in need of 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 as used herein persist for a longer period than that of natural human ANP (SEQ ID NO: 2), allowing administration at least once daily, or even three times, twice, or once per week. The time-acting profile of the ANP polypeptide may be measured using known pharmacokinetic testing methods, such as those described in the following examples.

[0036] As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of the polypeptide herein, wherein the polypeptide herein is modified by producing an acidic or basic salt thereof. Pharmaceutically acceptable salts and processes for producing 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 salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic or quaternary ammonium salts of the polypeptides herein, formed 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, and 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. Pharmacopoecitable refers to polypeptides described herein in a form suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, corresponding to a reasonable benefit / risk ratio. The pharmaceutically acceptable salt forms of polypeptides described herein can be synthesized by conventional chemical methods to include a basic or acidic moiety. Generally, such salts are prepared, for example, by reacting the free acid or free base form of these polypeptides with a suitable stoichiometric amount of a base or acid in water, an organic solvent, or a mixture thereof.Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred (see, for example, Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (Wiley-VCH 2nd ed. 2011)).

[0037] As used herein, the term “pharmaceutical composition” refers to a composition having an effective amount of one or more peptides as specified herein, in combination with other chemical components such as binders, carriers, diluents, lubricants, pharmaceutically acceptable fluids, and / or other excipients, in particular pharmaceutically acceptable carriers.

[0038] As used herein, “polypeptide” or “peptide” means 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 containing naturally occurring amino acids and polymers containing one or more unnaturally 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,” and “to treat” mean managing or caring for an individual having an abnormality, disease, disorder, or symptom to which ANP polypeptide administration is indicated for the purpose of reducing, suppressing, slowing, stopping, or reversing the progression or severity of the abnormality, disease, disorder, or symptom. Treatment includes administering ANP polypeptide or a composition containing ANP polypeptide as specified herein to an individual to prevent the onset of symptoms or complications, reduce symptoms or complications, or eliminate an abnormality, disease, disorder, or symptom. Treating includes administering ANP polypeptide or a composition containing ANP polypeptide as specified herein to an individual to produce, for example, increased angiogenesis, increased vascular compliance, increased glomerular filtration rate, decreased blood pressure, decreased (or prevention) of inflammation, and / or decreased (or prevention) of cardiac, renal, hepatic, or pulmonary fibrosis.

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

[0041] The structural features described herein also result in polypeptides with sufficient activity in NPR-A, as well as polypeptides with many other beneficial attributes related to their potential for development as therapeutic treatments, including improved solubility of analogs in aqueous solutions, improved chemical and physical formulation stability, an extended pharmacokinetic profile, and minimization of 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 This is selected from G, K, R, and Dap. In some embodiments, X 11 is selected from R and K. In some embodiments, X 13 X17 is selected from D and G. In some embodiments, X17 is H, K, R, Dap, or Orn. ​​In some embodiments, X 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 Either it does not exist, or it is selected from GGPSSGAPPPS (sequence number 9), GGKSSGAPPPS (sequence number 11), and GSPSSGAPPPS (sequence number 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; X 10 The following are 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 It either does not exist, or it is selected from GGPSSGAPPPS (sequence number 9), GGKSSGAPPPS (sequence number 11), and GSPSSGAPPPS (sequence number 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 In some embodiments, X 28 is H. In some embodiments, X 29 It either does not exist, or it is selected from GGPSSGAPPPS (sequence number 9), GGKSSGAPPPS (sequence number 11), and GSPSSGAPPPS (sequence number 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, and X9 is G, 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, X 17 H is X 18 is selected from Q and Y, and X 26 is F, and X 28 H is X 29is absent or is 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 of SEQ ID NO: 3 between cysteine at position 7 and cysteine at position 23 (C7 and C23). In some embodiments, the polypeptide contains a thioacetal bond between cysteine at position 7 and cysteine at position 23 (C7 and C23).

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

[0048] [[ID=**12**]]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, having the formula II: Fatty acid-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 a C 16 ~C 26 fatty acid and is conjugated to the amino acid present 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 is absent or comprises a 4- to 10-amino acid sequence independently selected from E, K, G, P, A, and S, Z3 is either absent or contains a basic structure from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) of polyethylene glycol or (containing a 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety.

[0049] The polypeptides of formula II described herein include, for example, a fatty acid moiety conjugated to the N-terminal amino acid of SEQ ID NO: 3 by a linker containing the structures Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3. Such conjugation may also be referred to as acylation. In embodiments, if X1 is absent, the fatty acid is conjugated to the amino acid at position X2 of SEQ ID NO: 3 by a linker containing the structures Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3. In embodiments, if both X1 and X2 are absent, the fatty acid is conjugated to the amino acid at position X3 of SEQ ID NO: 3 (by a linker containing the structures Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3). In embodiments, if X1, X2, and X3 are all absent, the fatty acid is conjugated to the amino acid present at position X4 in SEQ ID NO: 3 (by a linker including, for example, the structures Z1, Z1-Z2, Z1-Z3, or Z1-Z2-Z3). The fatty acid, and in certain embodiments, the linker, acts as an albumin binder, providing the potential to generate long-acting polypeptides.

[0050] The polypeptides described herein may be chemically conjugated to the functional groups of amino acids by direct bonding or by linkers. 16 ~C 26 Fatty acids are utilized. The length and composition of fatty acids affect the half-life of polypeptides, their potency in in vivo animal models, and their solubility and stability. 16 ~C 26 Conjugation to fatty acids yields ANP polypeptides exhibiting 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 It is a saturated fatty acid monoacid or diacid. Saturated C for use in this specification 16 ~C 22 Examples of fatty acids include, but are not limited to, palmitic acid (hexadecanoic acid) (C 16 (C1), hexadecanedioic acid (C1) 16 Diacid), margaric acid (heptadecanoic acid) (C 17 (C1 acid), heptadecanedioic acid) 17 Diacid, stearic acid (C 18 (C monoacid), octadecane diacid (C) 18 Diacid), nonadecylic acid (nonadecanoic acid) (C 19 (C1 acid), nonadecanedioic acid) 19 Diacid), arakadic acid (eicosanoic acid) (C 20 (C monoacid), eicosanedioic acid (C) 20 Diacid, Heneicosylic acid (Heneicosanic acid) (C 21 (C1 acid), Heneicosanoic acid (C1) 21 Diacids), behenic acid (docosanoic acid) (C 22 (C monoacid), docosanedioic acid (C) 22 Examples include diacides, and also include their branched and substituted derivatives.

[0052] In a 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 These can be diacids, as well as branched and substituted derivatives thereof.

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

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

[0055] [ka] (In the formula, n is between 1 and 10.)

[0056] Therefore, in some embodiments, the fatty acid is bound to Z1, and Z1 is bound directly to the peptide of formula I, or via Z2, via Z3, or via 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, a fatty acid is bound to Z1, Z1 is bound to Z2, and Z2 is bound to a peptide of formula I, either directly or via Z3. In some embodiments, Z2 is APPSG, (EK) b G, (EP)b G, K (EK) c G, and (EK) c E is selected from, b is 2, 3, or 4, and c is 1, 2, 3, or 4. For example, Z2 could 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 bound to Z1, Z1 is bound to Z2, Z2 is bound to Z3, and Z3 is bound to a peptide of formula I. In some embodiments, Z3 is (polyethylene glycol) m (Note that in the formula, m is an integer selected from 10 to 30), and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) n (Note that in the formula, n is selected from 2 to 10). For example, in some embodiments, Z3 is (polyethylene glycol) 12 (Polyethylene glycol) 24 These are ((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 having the following structure (also referred to herein as a branched fatty acid or "BFA").

[0061] [ka]

[0062] BFA exists in two enantiomer forms.

[0063] [ka]

[0064] Surprisingly, the purified enantiomer (EN2) of BFA was found to offer stronger binding to albumin compared to the other enantiomer (EN1) or the racemic mixture, resulting in a more desirable 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 found that to preserve the stability of enantiomerically pure BFA during the peptide coupling process, it is essential to bind it to Z1 containing β-Ala, or γGlu, or E.

[0065] [ka]

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

[0067] In some embodiments, the polypeptide of formula II comprises a purified enantiomer EN2 of BFA bound to β-Ala. In some embodiments, the polypeptide of formula II comprises a purified enantiomer EN2 of BFA bound to γGlu. In some embodiments, the polypeptide of formula II comprises a purified enantiomer EN2 of BFA bound 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 Selected from.

[0068] The amino acid sequences of the ANP polypeptides described herein typically incorporate naturally occurring amino acids, as shown herein using standard single-letter codes such as L=leucine, as well as certain other non-natural 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 non-natural 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 many structural differences. For example, when compared to natural human ANP (SEQ ID NO: 2), the ANP polypeptides described herein include modifications at one or more of the positions 1, 2, 3, 9, 10, 11, 12, 13, 17, 18, 24, 26, 28, and 29. In some examples, the ANP polypeptides described herein include modifications at each of the positions 1, 2, 3, 9, 10, 11, 12, 13, 17, 18, 24, 26, 28, and 29. In some embodiments, the ANP polypeptide includes a thioacetal (S-CH2-S) bond between the cysteine ​​at position 7 and the cysteine ​​at position 23.

[0071] In some embodiments, the ANP polypeptide described herein includes 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 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 a C at the amino acid at position 1. 16 ~C 22 Fatty acid conjugation, conjugation using linkers containing structures Z1-Z2-Z3 as optional choice.

[0072] In certain 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 a C at the amino acid at position 1. 16 ~C 22 Fatty acid conjugation, conjugation using linkers containing structures Z1-Z2-Z3 as optional choice.

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

[0074] In some embodiments, the ANP polypeptide described herein comprises a sequence selected from any one of sequence numbers 168 to 172.

[0075] In some embodiments, the ANP polypeptide described herein includes 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 polypeptide described herein includes 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 polypeptide described herein includes SEQ ID NOs. 28. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 45. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 50. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 51. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 78. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 83. In another embodiment, the ANP polypeptide described herein includes SEQ ID NOs. 84. In another embodiment, the ANP polypeptide described herein includes SEQ ID NO: 97. In another embodiment, the ANP polypeptide described herein includes SEQ ID NO: 98. In another embodiment, the ANP polypeptide described herein includes SEQ ID NO: 144. In another embodiment, the ANP polypeptide described herein includes SEQ ID NO: 158. In another embodiment, the ANP polypeptide described herein includes SEQ ID NO: 159.

[0076] In certain examples, the ANP polypeptides described herein are amidated. In some embodiments, the ANP polypeptide is an NPR-A agonist. In addition to the modifications described herein, the ANP polypeptides described herein may include one or more further amino acid modifications, insofar as the polypeptide can bind to and activate the NPR-A receptor.

[0077] The affinity of the ANP polypeptides described herein for each NPR-A receptor may be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described below, but generally expressed as an inhibitory constant (Ki) value. The activity of the polypeptides described herein in the NPR-A receptor may further be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and generally EC 50 This value represents the polypeptide concentration that causes a simulation of the maximum half dose in 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 further embodiments, pharmaceutical compositions comprising an ANP polypeptide or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, or excipient are provided herein.

[0080] The ANP polypeptides described herein may be used to treat a variety of abnormalities, disorders, diseases, or symptoms. In particular, methods are provided for treating cardiovascular abnormalities, disorders, or diseases in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of the ANP polypeptides described herein or pharmaceutically acceptable salts thereof, or a pharmaceutical composition containing them. Exemplary cardiovascular abnormalities, diseases, and disorders include, but are not limited to, acute heart failure, chronic heart failure, heart failure with maintained ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), atherosclerosis, coronary artery disease, diabetes, stroke, hypercholesterolemia, hypertension, ischemia, vascular stenosis and ventricular hypertrophy, and 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 age-related cardiac aging and / or diastolic dysfunction, or abnormalities associated therewith. In some embodiments, the ANP polypeptide described herein is used to treat HFpEF.

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

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

[0083] Therefore, such methods may include selecting individuals who have or are susceptible to cardiovascular abnormalities, diseases, or disorders. Alternatively, the methods may include selecting individuals who have or are susceptible to lung abnormalities, diseases, or disorders. Alternatively, the methods may include selecting individuals who have or are susceptible to kidney abnormalities, diseases, or disorders. In certain cases, the methods may include selecting individuals who have diabetes, hypertension with renal impairment, and / or obesity.

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

[0085] In some embodiments, ANP polypeptides or pharmaceutically acceptable salts thereof are provided for use in therapy.

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

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

[0088] Treatment of heart failure or HFpEF according to the present invention may be reflected in one or more of the various measures related to heart failure, including, for example, a reduction in left ventricular end-diastolic pressure (LVEDP), a reduction in the risk of cardiovascular death and / or hospitalization for heart failure, a reduction in the risk of all-cause mortality, a reduction in the risk of myocardial infarction (MI), a reduction in the risk of stroke, a reduction in the risk of needing left ventricular assist device (LVAD) implantation and / or heart transplantation, improvement in the symptoms and physical limitations of heart failure, and / or improvement in quality of life (QoL). Specific benefits of treatment according to embodiments of the present invention may be achieved after at least one month of treatment. Specific benefits of treatment according to embodiments of the present invention may be achieved after at least six months of treatment. Specific 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 the ANP polypeptide according to the present invention results in a significant reduction in LVEDP after one year of treatment. In certain embodiments, administration of the ANP polypeptide according to the present invention results in a significant reduction in global longitudinal strain (GLS). In certain embodiments, administration of the ANP polypeptide according to the present invention results in a reduction of at least 3.5% of GLS. In certain embodiments, administration of the ANP polypeptide according to the present invention results in a reduction of at least 15% of the risk of CV death and / or HF hospitalization. In certain embodiments, administration of the ANP polypeptide according to the present invention results in a significant reduction in the risk of one or more of the following: all-cause mortality, MI, stroke, LVAD transplantation, or heart transplantation. In certain embodiments, administration of the ANP polypeptide according to the present invention results in a significant improvement in the symptoms and physical limitations of heart failure and / or QoL.

[0090] Furthermore, as described above, the administration of ANP polypeptides according to certain embodiments of this disclosure can provide improvements in the heart failure-related measures described above without increasing safety risks. Therefore, in some embodiments, the administration of ANP polypeptides according to the present invention does not result in increased safety risks such as, for example, increased hypotension; worsening of renal function; electrolyte imbalance; hepatic dysfunction; tumor development or persistent sperm deficiency.

[0091] The term "therapeutic effective dose" refers to the amount or dosage of ANP polypeptide that provides the desired effect to the patient. For ANP polypeptides with an extended pharmacokinetic profile, such a dose may be the amount administered in a single or multiple doses. The effective dose can be readily determined by those skilled in the art by the use of known techniques and by observing results obtained under similar circumstances.

[0092] Regarding the route of administration, ANP polypeptides or pharmaceutical compositions containing them can be administered according to known methods (e.g., orally; by injection (i.e., intra-arterial, intra-venous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, portal vein, or intrafocal); by a sustained-release system; or by an implantable device, etc.). Administration of ANP polypeptides according to the present invention is typically parenteral, for example, intravenous (IV), subcutaneous (SC or SQ), or intraperitoneal (IP). Therefore, in certain embodiments of the present invention, ANP polypeptides are administered intravenously. In other embodiments of the present invention, ANP polypeptides are administered intraperitoneally. In other embodiments, ANP polypeptides are administered subcutaneously. In certain cases, ANP polypeptides or pharmaceutical compositions containing them may be administered by bolus injection or sequentially by SQ.

[0093] The present invention also includes intermediates and processes useful for the production of the ANP polypeptide of the present invention. The intermediates and ANP polypeptide 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 following examples.

[0094] For chemical synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from companies such as 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 synthesizers can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during reaction, coupling, deprotection, and capping unreacted amino acids.

[0095] Regarding biological expression, standard recombination techniques can be used to construct polynucleotides containing nucleic acid sequences encoding all or part of the amino acid sequence of the ANP polypeptide. These polynucleotides can then be incorporated into recombinant expression vectors, and the vectors can be introduced into host cells such as bacteria, yeast, and mammalian cells to produce the ANP polypeptide. For example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4 th See ed., 2012). This polypeptide 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 Escherichia coli, Bacillus subtilis, or fluorescent bacteria; in insect cells; or in fungal or yeast cells (which are cultured using techniques known in the art). Vectors containing the target polynucleotide sequence can be transferred into host cells by well-known methods, which vary depending on the type of cell host. Various methods of protein purification can be used, and such methods are known in the art.

[0096] As described above, all HF patients, even those with mild symptoms, are at increased risk of death. Therefore, as used herein, the reference to “patients in need of treatment” for heart failure (HF) may refer to a wide range of individuals with HF, including those with a wide range of disease severities, 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 patients requiring treatment are those with heart failure in NYHA class II-IV. In certain embodiments, the patients requiring treatment are those with heart failure in NYHA class II. In certain embodiments, the patients requiring treatment are those with heart failure in NYHA class III. In certain embodiments, the patients requiring treatment are those with heart failure in NYHA class IV. In certain embodiments, the patients requiring treatment are those with heart failure in NYHA class II-III.

[0099] As described above, existing treatment options for heart failure, including current standard care, improve symptoms and slow disease progression through hemodynamic mechanisms, such as reducing blood pressure, heart rate, and / or plasma volume, thereby reducing the workload of heart failure. In contrast, the ANP polypeptide of the present invention achieves its effects through a different mechanism of action, namely selective NPR-A binding and the resulting activity to provide improvements in biomarkers (cGMP, NT-proBNP), hemodynamics (LVEDP), structure (LA volume), and symptoms (pulmonary congestion, dyspnea), and thus improve outcomes and QoL in HFpEF patients. Due to these different mechanisms of action, the ANP polypeptide of the present invention can be administered on existing SoCs without titration or monitoring. Therefore, in certain embodiments, the ANP polypeptide of the present invention can be administered in combination with one or more additional treatments for heart failure. In certain embodiments, one or more additional therapeutic agents 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, combinations of ARBs and NEP inhibitors (sacubitril / valsartan (ENTRESTO®)), statins and / or hypoglycemic agents, as well as other therapeutic agents to control comorbidities including but not limited to high cholesterol, hypertension, atrial fibrillation and diabetes. In certain embodiments, the ANP polypeptide of the present invention may be administered in combination with an SGLT2 inhibitor or an sGC activator.

[0100] Additional therapeutic agents may be administered simultaneously, separately, or consecutively with the ANP polypeptide or a pharmaceutical composition containing it. Furthermore, additional therapeutic agents may be administered at the same frequency as the ANP polypeptide or the pharmaceutical composition containing it (i.e., every other day, twice a week, or weekly). Alternatively, additional therapeutic agents may be administered at a different frequency than the ANP polypeptide or the pharmaceutical composition containing it. In other cases, additional therapeutic agents may be administered via SQ. In other cases, additional therapeutic agents may be administered via IV. In yet other cases, additional therapeutic agents may be administered orally.

[0101] It is further intended that these methods may be combined with diet and exercise, and / or 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 via parenteral routes (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or transdermal). 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 specific cases, the ANP polypeptide is administered via SQ or IV. However, where not applicable, the ANP polypeptide can be formulated in forms for other pharmaceutically acceptable routes, such as tablets or other solids for oral administration; sustained-release capsules; and any other forms currently in use, including creams, lotions, inhalants, etc.

[0103] As described above, in order to improve their in vivo compatibility and efficacy, the ANP polypeptides herein may react with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmacochemically acceptable salts and general 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)). Pharmacochemically acceptable salts for use herein include sodium salts, trifluoroacetate salts, hydrochloride salts, and acetate salts.

[0104] The ANP polypeptides described herein may 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 may be about 2 mL or less, and even about 1 mL or less, and the needle gauge may be about 27 G or greater or about 29 G or greater.

[0105] The ANP polypeptides described herein may 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 within the syringe.

[0106] The present invention will be further illustrated by the following embodiments, but these embodiments should not be construed as limiting. [Examples]

[0107] preparation Abbreviations: Acetonitrile (ACN); aqueous solution (aq); octadecylsilane (C18); dichloromethane (DCM); N,N-dimethylformamide (DMF); dimethyl sulfoxide (DMSO); ethyl acetate (Â); 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 mixture of 11-bromoundecanoic acid (8.00 g, 30.2 mmol) and dichloromagnesium hexahydrate (613 mg, 3.01 mmol) in di-tert-butyl dicarbonate (8.65 g, 39.2 mmol) and tert-butanol (60 mmol) was placed in a pressure vessel. The vessel was sealed and then heated to 40°C for 24 hours. 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 under vacuum, and purified by flash column chromatography (120 g silica column, gradient from 100% hexane to 100% siRNA in hexane over 20 minutes). 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-undecylpropane dioate

[0111] [ka] Under a nitrogen atmosphere, sodium hydride (60% by mass, 400 mg, 10.0 mmol) in mineral oil was added in small increments to an ice-cold solution of O1-benzyl O3-tert-butylpropanediote (2.50 g, 9.99 mmol) in N,N-dimethylformamide (15 mL). After stirring for 1 hour, 1-bromoundecane (2.35 g, 9.99 mmol) in 2 mL of DMF was added, and the mixture was mixed at room temperature for 15 hours. The mixture was diluted with 60 mL of ether, and the organic layer was washed with 1% aqueous citrate (50 mL), brine, and water. The organic layer was dried over sodium sulfate, volatile substances were removed under vacuum, and the mixture was purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 40% siRNA over 25 minutes). The desired product was isolated as an oily substance (3.50 g); mz=403 (M-1).

[0112] Preparation 3 O11-benzyl O1,O11-ditert-butyldocosanet-1,11,11-tricarboxylate

[0113] [ka] Under a nitrogen atmosphere, a cold mixture of O1-benzyl O3-tert-butyl 2-undecylpropanediote (8.50 g, 20.0 mmol) in N,N-dimethylformamide (40 mL) was gradually mixed with sodium hydride (60% by mass, 960 mg, 24.0 mmol) in mineral oil. 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 citrate solution (50 mL), brine, and water. The organic layer was dried over sodium sulfate, and volatile substances were removed under vacuum. The mixture was purified by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% DCM over 15 minutes, followed by 10 minutes of maintenance). The desired product was isolated as an oily substance (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] 10% Pd / C (1.25 g) was added to a 2250 mL Parr shaker 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. Shaking was performed at room temperature for 2 hours. The system was reduced in pressure 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 Bromoundecanoart

[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 mass) was added to the solution all at once. 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 × 10 mL). Organic components were removed under reduced pressure. The solution was purified by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% dichloromethane over 20 minutes, followed by 5 minutes). The product-containing fractions were combined, and the product was isolated as an oily substance (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-undecylpropane dioate

[0119] [ka] Under a nitrogen atmosphere, sodium hydride (60% by mass, 400 mg, 10.0 mmol) in mineral oil was added in small amounts to an ice-cold solution of O1-benzyl O3-tert-butylpropane dioate (2.50 g, 9.99 mmol) in N,N-dimethylformamide (15 mL). After stirring for 1 hour, 1-bromoundecane (2.35 g, 9.99 mmol) in 2 mL of DMF was added. The mixture was mixed at room temperature for 15 hours. The mixture was diluted with 60 mL of ether, and the organic layer was washed with 1% aqueous citrate solution (50 mL), brine, and water. The organic layer was dried over sodium sulfate, and volatile substances were removed under vacuum. The mixture was purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 40% siRNA over 25 minutes). The desired product was isolated as an oily substance (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-undecylpropanediote (6.2 g, 14.6 mmol) in N,N-dimethylformamide (30 mL), sodium hydride (60% by mass, 700 mg, 17.5 mmol) in mineral oil was added in small increments. After 40 minutes, 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 hours. 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 volatile substances were removed under vacuum. The mixture was purified by flash column chromatography (220 g silica column, gradient from 100% hexane to 100% DCM over 20 minutes, maintained for another 10 minutes). The desired product was isolated as an oily substance (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. The residue was removed and purified by flash column chromatography (120 g silica column, gradient from 100% hexane to 100% phenyl in hexane over 20 minutes). The desired product was isolated as an oily substance (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 the racemic compound 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 setting 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 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 from 1300 mg of the racemic compound using the conditions of the preparation method.

[0127] Preparation 9 β-ALA linker O1,O11-Dibenzyl O11-(2,5-Dioxopyrrolidine-1-yl)docosanate-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 all at once. 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 solid was removed by filtration and washed with DCM (3 × 5 mL). The solvent was removed from the filtrate and purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 50% siRNA in hexane over 20 minutes, then increasing to 100% siRNA over 5 minutes). The desired product was isolated as an oily substance (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-dioxopyrrolidine-1-yl)docosa-1,11,11-tricarboxylate (1.50 g, 2.08 mmol) in tetrahydrofuran (20 mL), followed by the addition of triethylamine (0.80 mL, 5.7 mmol). Water (3 mL), acetonitrile (6 mL, 100% by mass), and 4 mL of DMF were added to solubilize the precipitate that would form. The mixture was mixed at room temperature for 15 hours. The mixture was diluted with chloroform / isopropanol (1 / 3, 100 mL) and washed with 10% aqueous citric acid solution, water, and brine (50 mL). The organic matter was dried over sodium sulfate and concentrated to dryness under vacuum. The product was purified by flash column chromatography (80g silica column, UV 254nm, gradient from 100% hexane to 100% siRNA over 15 minutes, followed by maintenance for 5 minutes). The desired product was isolated as an oily substance (1.00g, 66% yield); mz = 694 (M+).

[0131] Preparation 11 β-alanine BFA NHS ester Dibenzyl 2-[[3-(2,5-dioxopyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecane dioate

[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 minutes, N,N'-dicyclohexylcarbodiimide (342 mg, 1.64 mmol) was added all at once. The mixture was stirred at room temperature for 15 hours. The solid was removed by filtration and washed with DCM (3 × 5 mL). The mixture was concentrated to dryness under vacuum and purified by flash column chromatography (80 g silica column, gradient from 100% hexane to 100% siRNA within 20 minutes). 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-dioxopyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecanediic acid

[0134] [ka] 10% Pd / C (0.193 g) was added to a 100 mL Parr shaker and purged with nitrogen. Tetrahydrofuran (20 mL) was added, followed by a solution of dibenzyl 2-[[3-(2,5-dioxopyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecane dioate (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. Shaking was allowed at room temperature for 2 hours. The system was reduced in pressure 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 chemical reactions 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]pyridine-3-yl)rel-(11S)-docosane-1,11,11-tricarboxylate

[0137] [ka] Under a nitrogen atmosphere, [dimethylamino(triazolo[4,5-b]pyridine-3-yloxy)methylene]-dimethylammonium;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 mass) and DMF (10 mL). The mixture was stirred at room temperature for 3 minutes, then cooled on an ice bath, after which N,N-diisopropylethylamine (1.50 mL, 8.60 mmol) was added. The mixture was stirred at room temperature for 3 hours. Diluted with DCM (60 mL) and washed with a saturated aqueous solution of ammonium chloride (2 × 30 mL). The organic layer was separated and dried over sodium sulfate. The crude product was purified by normal-phase flash chromatography (80 g silica gold column, gradient from 100% hexane for 3 minutes, then from 60% siRNA in hexane for 17 minutes, then from 100% siRNA, and retained for another 5 minutes). The active 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 was dissolved in acetonitrile (10 mL) and then added dropwise to the β-alanine solution via syringe over 2 minutes. The mixture was stirred 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. The residue was concentrated under reduced pressure and used directly in the next step (2.60 g).

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

[0140] [ka]

[0141] Activation by NHS esters Dibenzylrel-(2R)-2-[[3-(2,5-dioxopyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecane dioate N-hydroxysuccinimide (240 mg, 2.04 mmol) was added at room temperature 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). After stirring for 3 minutes, N,N'-dicyclohexylcarbodiimide (420 mg, 2.01 mmol) was added all at once, followed by 1 mg of DMAP. The mixture was mixed at room temperature for 3 hours and then stored in a 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, gradient to 100% hexane for 5 minutes, then to 100% siRNA over 15 minutes). A second purification was performed by flash chromatography (40g column, 100% hexane for 3 minutes, followed by a gradient to 100% MTBE over 17 minutes). The desired product was isolated as a concentrated oily substance (1.0g).

[0142] Deprotection 10% Pd / C (0.152 mg) was placed in a 100 mL Parr shaker and purged with nitrogen. Tetrahydrofuran (10 mL) was added, followed by a solution of dibenzylrel-(2R)-2-[[3-(2,5-dioxopyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecane dioate (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. Shaking was allowed at room temperature for 2 hours. The system was reduced in pressure 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-dibenzylrel-(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, and hexafluorophosphate (1.43 g, 3.65 mmol) in [dimethylamino(triazolo[4,5-b]pyridine-3-yloxy)methylene]dimethylammonium;DMF (5 mL) and THF (5 mL) was added. The mixture was cooled to 10°C on an ice 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 under vacuum. The product was purified by normal-phase flash chromatography (80 g silica gold column, gradient from 100% hexane for 3 minutes, then from 60% siRNA in hexane for 17 minutes, then from 100% siRNA, maintained for another 5 minutes). The desired product was isolated as an oily substance (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-oxopentanoic 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-oxopentanoic acid (650 mg, 3.13 mmol) dissolved in acetonitrile (4 mL) and water (4 mL). Then, O11-(benzotriazol-1-yl)O1,O11-dibenzylrel-(11R)-docosane-1,11,11-tricarboxylate (1.15 g, 1.55 mmol) in acetonitrile (3 mL) was added. The mixture was stirred at room temperature for 12 hours. It was diluted in 50 mL of DCM and washed with 50 mL of aqueous ammonium chloride (2 ×). The organic phase was separated and dried over sodium sulfate. It was concentrated to dryness under vacuum. The product was purified by normal-phase flash chromatography (40g silica gold column, gradient from 100% hexane over 5 minutes, followed by a gradient from 100% siRNA in hexane over 15 minutes, maintained for another 5 minutes). The desired product was isolated as an oily substance (900mg); 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-dioxopyrrolidine-1-yl)oxy-4-oxobutyl]carbamoyl]-2-undecyl-tridecanediic acid

[0149] [ka]

[0150] Step 1: Activated ester Dibenzyl (2S) * )-2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidine-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecane dioate Under a nitrogen atmosphere, N-hydroxysuccinimide (170 mg, 1.44 mmol) was dissolved in tetrahydrofuran (3 mL) and dichloromethane (6 mL) (4S)-4-[[(2S * The solution was added to )-13-benzyloxy-2-benzyloxycarbonyl-13-oxo-2-undecyl-tridecanoyl]amino]-5-tert-butoxy-5-oxopentanoic acid (900 mg, 1.11 mmol). The mixture was stirred at room temperature for 3 minutes, and then N,N'-dicyclohexylcarbodiimide (300 mg, 1.43 mmol) and 1 mg of DMAP were added as solids. The mixture was stirred at room temperature for 3 hours. The white precipitate was removed by filtration and washed with DCM (3 × 5 mL). The mixture was concentrated to dryness under vacuum to obtain the crude activated ester. The mixture was purified by flash column chromatography (40 g silica column, gradient from 100% hexane over 5 minutes to 100% siRNA over 15 minutes, and maintained for another 5 minutes). The activated ester was isolated (850 mg); mz = 905 (M+).

[0151] Step 2: 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 a solution of dibenzyl (2S * )-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) in 15 mL of tetrahydrofuran. The bottle was sealed, purged with nitrogen, and pressurized to 20 psi with hydrogen gas. It was shaken at room temperature for 2 h. The system was depressurized with nitrogen gas and then filtered through celite. The solvent was removed from the mixture under reduced pressure to isolate the product 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] The following depicts the structure of Example 1 using the standard one-letter code for L-amino acids, except for γ-glutamic acid residues and 4-Pal residues, and the structures of the above residues are expanded.

[0154]

Chemical Structure

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

[0156] The solid support used consisted of a 1% DVB cross-linked polystyrene core and a 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 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 prior to each coupling step was performed using 20% ​​piperidine (PIP; Sigma Aldrich) in dimethylformamide (DMF; Fisher Chemicals) with nitrogen mixing for 2 x 7 minutes, followed by 8 DMF washing cycles. All amino acid coupling was performed for 1 hour using Fmoc Amino Acid (0.3 M in DMF), N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate (HBTU; Ambeed; 0.9 M in DMF), and N,N-diisopropylethylamine (DIPEA; Sigma Aldrich; 1.2 M in DMF) with a molar excess of AA / HBTU (9 times the theoretical resin loading level) and a molar excess of DIPEA (12 times the theoretical resin loading level). After synthesizing the primary sequence of the peptide and completing the final Fmoc deprotection and DMF washing, the fatty acid (FA) moiety was attached by manual addition of a 3-fold excess solution of 20-tert-butoxy-20-oxo-icosanoic acid (OtBu-C20-OH), which had been pre-activated (for 2 minutes) with a DMF (3 mL) solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; Alfa Aesar) and DIPEA (1:1:3; FA:HATU:DIPEA).This solution was added directly to the Symphony-X reaction vessel containing the peptidyl resin via a transfer pipette. The FA coupling reaction time was 3 hours, after which the resin was washed three times with DMF, and the completion of the coupling was confirmed by the Kaiser test. If the Kaiser test was positive, the FA coupling process was repeated as needed. After the acylation of FA was complete, the peptidyl resin was transferred as a DCM slurry to a disposable frit plastic syringe equipped with a Teflon stopper, washed further with DCM, and finally the resin was completely dried in a vacuum. The dried resin was then treated with a 10 mL 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) at room temperature for 2 hours. After a 2-hour incubation, the resin was filtered and washed twice with 2 mL of raw TFA. The combined filtrate / wash solution 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 drained, and the solid pellet was further ground twice with fresh, unused ether. The solid pellet was then dried in a vacuum.

[0157] Formation of disulfide bonds The crude peptide was solubilized in a suitable glass container with a relatively low concentration (0.2–0.5 mg per 1 mL of crude peptide) in a 25% aqueous acetic acid solution. The solution was then placed on a magnetic stirrer equipped with the necessary spin blades, 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 mixture was incubated at room temperature for 15 minutes, at which point the excess iodine was quenched by adding a few drops of 0.1 M aqueous ascorbic acid solution.

[0158] HPLC purification The crude oxidation solution was directly loaded into 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 / H2O and B: 0.1% TFA / acetonitrile (ACN). The initial loading was performed with 20% B, followed by a 5-minute constant composition wash, and then the buffer was set to 25% B for equilibration. The column heating was set to 60°C, and the sample was eluted using a linear gradient of 25–45% B over 60 minutes at a flow rate of 15 mL / min. The fraction determined to contain the desired product (by analysis by LC-MS) was pooled, frozen, and lyophilized to obtain the amorphous solid product as the TFA salt of Example 1. The purity, as assessed by RP-HPLC, was found to be over 95%, and the observed molecular weight was 5293.4 daltons, which matched 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] The following diagram illustrates the structure of Example 2, which uses standard single-letter codes for L-amino acids, excluding γ-glutamic acid residues, 4-Pal residues, and cysteine ​​residues, with the structures of the above residues enlarged.

[0161] [ka]

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

[0163] Thioacetal bond formation in Example 2 After purification of the disulfide-crosslinked polypeptide form (same as in Example 1), the appropriate pooled fraction containing the peptide was diluted with water and ACN instead of lyophilizing to obtain a water / ACN mixture of approximately 50 / 50 (total volume approximately 400 mL) 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 disulfide crosslinks of the peptide were reduced by the addition of 2-4 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) reducing agent. Following the reduction of the disulfide crosslinks, thioacetal bonds were formed by the addition of 7-10 equivalents of diiodomethane (CH2I2). The thioacetal formation reaction was carried out by incubation of the solution at room temperature for 18 hours with magnetic stirring. The progress of the reaction was monitored by observing the mass change of +12 Daltons from the starting molecular weight of the reduced peptide using analytical LC-MS.

[0164] HPLC purification The crude thioacetal reaction solution was diluted to 1000 mL with water and then directly loaded via an injection pump into a preparative HPLC system (LC-8A Binary Systems manufactured by Shimadzu Corporation) 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 / H2O and B: 0.1% TFA / ACN. The initial loading was performed with 20% B, followed by a constant composition wash for 5 minutes, and then set to 25% B for equilibration. The column heating was set to 50 °C, and the sample was eluted using a linear gradient of 25 - 45% B at a flow rate of 25 mL / min over 60 minutes. Fractions determined to contain the desired product (by analysis using LC-MS) were pooled, frozen, and lyophilized to obtain a white amorphous solid product as the TFA salt of Example 2. The purity evaluated by RP-HPLC 1 was found to be over 95%, and the observed molecular weight was 5308.6 Daltons, which matched 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] The following depicts the structure of Example 3 using the standard one-letter codes for L-amino acids, excluding the γ-glutamic acid residue, the glycine residue at position 8, the β-alanine residue, and the cysteine residue, and the structures of the above residues are expanded.

[0167]

Chemical Structure

[0168] The primary peptide sequence of Example 3 was synthesized in substantially the same manner as in Examples 1 and 2, using a non-natural β-alanine (βAla) residue incorporated with Fmoc-βAla-OH (ChemImpex International Inc.). The exception to the synthesis method was the coupling of Fmoc-N-amide-PEG24-OH, which required a temporary pause in the automated synthesis protocol. The coupling of the PEG24 residue was achieved by manually adding a 1.5-fold excess of Fmoc-N-amido-PEG24-OH (BroadPharm) solution pre-activated (for 2 minutes) with diisopropylcarbodiimide (DIC) and ethyl-cyano(hydroxyamino)acetate (Oxyma) (1:1.2:1; PEG24:DIC:Oxyma) to 3 mL of DMF. This solution was added directly to a Symphony-X reaction vessel containing peptidyl resin via a transfer pipette. The PEG24 coupling reaction time was 18 hours, after which the resin was washed three times with DMF, and a Kaiser test was performed to confirm the completion of coupling. If a positive result was obtained in the Kaiser test, the PEG24 coupling process was repeated as necessary. The automated method was restarted to complete the synthesis of the remaining sequences, and FA was coupled as described in Example 1. Cleavage, disulfide bond formation, thioacetal bond formation, and purification were carried out as previously described in Examples 1 and 2. The purity, as evaluated by RP-HPLC, was found to be over 95%, and the observed molecular weight was 6475.0 daltons, which matched 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] The following diagram illustrates the structure of Example 4, which uses standard single-letter codes for L-amino acids, excluding 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 enlarged.

[0171] [ka]

[0172] Example 4 was synthesized in substantially the same manner as Example 3. The purity of Example 4, as evaluated by RP-HPLC, was found to be over 95%, and the observed molecular weight was 6474.6 daltons, which matched 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] The following diagram shows the structure of Example 5 using standard single-letter codes for L-amino acids, excluding the γ-glutamic acid and cysteine ​​residues, and the structures of the above residues are enlarged.

[0175] [ka]

[0176] Example 5 was synthesized in substantially the same manner as described for Example 2. The purity of Example 5, as evaluated by RP-HPLC, was found to be over 95%, and the observed molecular weight was 5406.8 daltons, which matched 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-SSGAPPS-NH2 (thioacetal bond)

[0178] [ka]

[0179] Example 6 was synthesized in substantially the same manner as described for Example 3. The purity of Example 6, as evaluated by RP-HPLC, was found to be over 95%, and the observed molecular weight was 6409.6 daltons, which matched 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 substantially the same manner as described for Example 3. The purity of Example 7, as evaluated by RP-HPLC, was found to be over 95%, and the observed molecular weight was 6375.2 daltons, which matched the theoretically calculated molecular weight of 6375.2 daltons.

[0183] The polypeptides (SEQ ID NOs: 28-44, 46-59, 61-106, 108-143, 145, 147-157, 160-167) listed in Table 1 are prepared substantially using the procedures described in Examples 1-3. 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 in the procedure of Example 1. Examples 17 and 56-140 (SEQ ID NOs: 37, 78-106, 108-143, 145, 147-157, 160-167) contain thioacetal bonds and are prepared substantially as described in the procedure of Example 2. Furthermore, 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) include PEG24 or PEG12 introduced substantially as described by the procedure of Example 3. 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) include (AEEA)4, (AEEA)6, or (AEEA)8, which are introduced using the standard amino acid coupling method 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 stands for branched fatty acid. The following is a diagram of the structure of Example 141, which uses a 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) chemical synthesis in a Symphony-X, 24-channel multiplex peptide synthesizer (Gyros Protein Technologies, Inc.). The solid support used consisted of a 1% DVB cross-linked polystyrene core and a 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 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 by treating with 20% piperidine in DMF with nitrogen mixing (once every 4 minutes and once every 10 minutes), followed by six DMF washing cycles. All amino couplings were performed for 1 hour using Fmoc Amino Acid (0.3 M in DMF), N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate (HBTU, Ambeed, 0.9 M in DMF), and N,N-diisopropylethylamine (DIPEA; 1.2 M in DMF) at a 9-fold molar excess of AA / HBTU and a 12-fold molar excess of DIPEA relative to the theoretical resin loading level. After synthesizing the primary peptide sequences, the final Fmoc deprotection and washing were completed.

[0189] 13-tert-butoxy-2-tert-butoxycarbonyl-13-oxo-2-undecyl-tridecanoic acid, dissolved in 5-7 mL of DMF, was manually added and transferred to a Symphony-X reactor. Subsequently, 2-3 times excess diisopropylcarbodiimide (DIC) and 2-3 times excess ethyl-cyano(hydroxyamino)acetate (Oxyma) were added to achieve the bonding of the fatty acid (BFA) moiety. The BFA coupling reaction time was approximately 18 hours. After that, the resin was washed three times with DMF, and the completion of the coupling was confirmed by a Kaiser test. After acylation was complete, the peptidyl resin was transferred as a DCM slurry to a disposable frit plastic syringe equipped with a Teflon stopper, further washed with DCM, and finally the resin was completely air-dried. Next, the dried resin was treated with a 10 mL 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) at room temperature for 2 hours. After 2 hours of 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 minutes to form a solid pellet, the supernatant was drained, and the solid pellet was further pulverized twice with ether and dried in a vacuum.

[0190] Formation of disulfide bonds Crude peptide was solubilized in a 50 mL Falcon tube with approximately 50 mL of 10% acetonitrile solution in 0.1% TFA-H2O. 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-H2O (crude peptide concentration of approximately 5 mg / mL), and then treated with a few drops of methanol solution of saturated iodine until a faint yellow color persisted. The reaction mixture 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 solution.

[0191] HPLC purification Next, the crude oxidation solution was directly loaded into a Waters half-portion HPLC system and purified using a Symmetry C18 (7 μm, 19 × 300 mm; Waters) with a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer system (10–40% over 70 minutes). Peptide purity was evaluated using analytical LC-MS, and the pooling standard was greater than 90%. The main pool of Example 141 was found to be greater than 95.0%. The final main product pool was then lyophilized to obtain the lyophilized peptide as the TFA salt. The molecular weight was determined by analytical LC-MS (measured value was 5194.2; calculated value was 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 stands for branched fatty acid enantiomer 2. The following diagram illustrates the structure of Example 142, using standard single-letter amino acid codes except for βAla, and the structure of the above amino acid residues has been extended.

[0194] [ka]

[0195] The peptide skeleton of Example 142 was synthesized in the same manner as described for Example 141. The only exception to the synthesis method was the coupling of Fmoc-N-amide-PEG24-OH, which required a temporary pause in the automated synthesis protocol. PEG24 residue coupling was achieved by manually adding a 3-fold excess of Fmoc-N-amide-PEG24-OH (BroadPharm) dissolved in 5 mL of DMF, transferring it to a Symphony-X reaction vessel, and subsequently adding a 2-fold excess of diisopropylcarbodiimide (DIC) and a 2-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 the completion of coupling was confirmed by a Kaiser test. The automated method was restarted to complete the synthesis of the remaining sequence, and the fatty acids were coupled as described below.

[0196] BFAEN2-βAla binding Fatty acid bonding was achieved by manually adding 1.5-fold excess 2-[[3-(2,5-dioxypyrrolidine-1-yl)oxy-3-oxopropyl]carbamoyl]-2-undecyl-tridecanedioic acid and 3-fold excess N,N-diisopropylethylamine (DIPEA) to a Symphony-X reaction vessel after dissolution in 5-7 mL of DMF. The FA coupling reaction time was approximately 18 hours, after which the resin was washed three times with DMF. Next, cleavage was performed as described in Example 141, followed by the formation of disulfide bonds as described in Example 141.

[0197] Formation of thioacetal bonds Next, the crude oxidation solution was directly loaded into a Waters HPLC (half-portion) and purified using a Symmetry C18 (7 μm, 19 × 300 mm; Waters) with a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer system (10–40% over 70 minutes). The purity of the fractions was evaluated using LC-MS, and the pool standard for the fraction used for thioacetal conversion was over 80%. These fractions were combined and diluted 1:1 with a 50% acetonitrile-H2O mixture. First, the disulfide bond was reduced by adding 1 mL of a 0.25 M aqueous solution of tris(2-carboxyethyl)phosphine (TCEP, TCI America), and then 400–500 μL of pure triethylamine (TEA, Sigma Aldrich) was added to raise the pH of the solution 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 completed within approximately 18 hours.

[0198] The crude thioacetal solution was diluted 1:1 with H2O and then loaded onto a Waters half-portion HPLC system. Purification was performed using a Symmetry C18 (7 μm, 19 × 300 mm; Waters) with a linear gradient of 100% acetonitrile and 0.1% formic acid / water buffer system (5–35% over 70 minutes). Peptide purity was assessed using LC-MS, and the pool standard was greater than 90%. Approximately 100 μL of pure TFA was added to the pooled fraction. The main pool purity of Example 142 was found to be greater than 95.0%. Subsequent lyophilization of the final main product pool yielded the lyophilized peptide TFA salt. Molecular weight was determined by analytical LC-MS (measured value was 6452.5; calculated value was 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 substantially as described in Example 142. The molecular weight was determined by LC-MS (measured value was 6601.6; calculated value was 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 substantially as described in Example 142. The molecular weight was determined by LC-MS (measured value was 6515.6; calculated value was 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 involved the attachment of BFAEN2-γGlu as described below, but was essentially synthesized in the same manner as described in Example 142.

[0208] Binding of BFAEN2-γGlu Fatty acid bonding was achieved by manually adding 1.5-fold excess 2-[[(1S)-1-tert-butoxycarbonyl-4-(2,5-dioxopyrrolidine-1-yl)oxy-4-oxo-butyl]carbamoyl]-2-undecyl-tridecanedioic acid and 3-fold excess N,N-diisopropylethylamine (DIPEA), which were dissolved in 5-7 mL of DMF and transferred to a Symphony-X reaction vessel. The FA coupling reaction time was approximately 18 hours, after which the resin was washed three times with DMF. Next, cutting was performed 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 (measured value was 6381.6; calculated value was 6381.2).

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

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

[0213] Jump-in® T-Rex® HEK293 cells were seeded at a rate of 1 million cells / well in 2 mL of culture medium in BioCOAT® poly-D-lysine coated 6-well plates (Becton Dickinson, catalog no. 354413), and incubated at 37°C for 18 hours and in 5% CO2 to reach 50-70% confluence. A cDNA mixture was prepared in a 50 mL test 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 to a test tube. A reagent mixture was prepared in another 50 mL test tube by adding 7.5 μL of lipofectamine LTX to 300 μL of Opti-MEM I. The mixtures were incubated at room temperature for 5 minutes. Next, the cDNA mixture was transferred to the reagent mixture described above, thoroughly mixed, and incubated at room temperature for a further 30 minutes. Then, 500 μL of the cDNA / lipofectamine complex was transferred to the wells 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 overexpression cell line were maintained for at least 3 weeks in culture medium supplemented with 2 mg / mL of G418 sulfate, changing the medium every 2-3 days, for clonal selection based on their intrinsic resistance to G418 sulfate.

[0214] NPR was overexpressed in T-Rex® HEK293 cells after induction with 300 ng / mL tetracycline in culture medium for 48 hours. The induced cell lines in the exponential growth phase were treated with 0.05% trypsin-EDTA for several seconds at room temperature, collected in cell medium containing FBS to neutralize the trypsin, counted, and cryopreserved at a density of 2 million cells / mL in a cell preservation 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 lines were then used for suspension assays to measure the activity of polypeptides that stimulate cGMP production in cGMP assays, or for cell membrane preparations to measure polypeptide binding activity in competitive radioligand binding assays, as described below.

[0215] Human and rat NPRA cGMP activity assays Cells overexpressing human or rat NPRA were seeded in 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 the test polypeptide. The test polypeptide was added starting at a concentration of 10 μM and decreasing tenfold 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. The detailed procedure is outlined below.

[0216] First, prepare the stock solution (2 mM) of the test polypeptide dissolved in DMSO, and then add Ca 2+ and Mg 2+The polypeptide was diluted 100-fold in an assay buffer (pH 7.4) containing HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% BSA or 0.1% casein. The polypeptide was further serially diluted in an assay buffer containing 0.1% BSA or 0.1% casein in a 1:10 dilution step to produce 2x working stock solutions at 8 different concentrations ranging from 20 μM to 2 pM.

[0217] A 10 μL cell suspension containing 4000 cells was seeded into a Costar® half-area white opaque 96-well plate (Corning, catalog no. 3693). Then, 10 μL of assay buffer (basic activity), 200 nM amidated rat ANP (maximal activity), or twice the 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 generated cGMP was measured using a cGMP kit according to the manufacturer's instructions, as described below.

[0219] The cyclic GMP (cGMP) standard provided in the kit was serially diluted 1:3 in an assay buffer containing 0.1% BSA + 0.5% DMSO or 0.1% casein + 0.5% DMSO, ranging from 1 μM to 0.17 nM. The cGMP standard (20 μL) was then transferred to another Costar® 3693 plate.

[0220] cGMP production was stopped, and the cGMP content was measured by sequentially adding 10 μL of cGMP-d2 and 10 μL of anti-cGMP-cryptate (these were pre-diluted 1:50 in the lysis buffer provided in the kit). The plate was shaken for 15 seconds and incubated in the dark at room temperature for 2 hours. Excitation readings were taken at 337 nm and emission readings at 665 nm / 620 nm using a Pherastar® FSX plate reader (BMG LABTECH, Ortenberg, Germany).

[0221] The ratio of 665 nm / 620 nm multiplied by 10000 was plotted on a logarithmic scale against the cGMP standard concentration, and a standard curve was created using an internally developed 4-parameter nonlinear regression curve fitting template. The amount of cGMP produced by cells overexpressing NPRA was interpolated using the cGMP standard curve. 100% response was determined from wells in the presence of saturated amidated rat ANP (100 nM). 0% response was determined from wells containing assay buffer. Using Prism 9 (GraphPad Software, Inc., San Diego, California), eight concentration-response curves for test polypeptides (10 μM to 1 pM) were fitted to the 4-parameter model to determine potency (EC2). 50 The ) value and maximum activation (%Max) were determined.

[0222] The data for exemplary analogues and hANP are shown in Table 2 below.

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

[0224] As shown in Table 2, in the presence of BSA, the ANP polypeptide of the examples exhibits 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 (instead of serum albumin) as a nonspecific blocker that does not interact with the fatty acid portion of the molecule being analyzed, the ANP polypeptide of the examples exhibits agonist activity comparable to hANP.

[0225] Human and rat NPRB cGMP activity assays The functional activity of the ANP polypeptide is determined in the NPR-B expressing HEK-293 clone cell line, as described below.

[0226] Cells overexpressing human or rat NPRB were seeded in 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 the test polypeptide. The test polypeptide was added starting at a concentration of 10 μM and decreasing tenfold 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. The detailed procedure is outlined below.

[0227] First, prepare the stock solution (2 mM) of the test polypeptide dissolved in DMSO, and then add Ca 2+ and Mg 2+ The polypeptide was diluted 100-fold in an assay buffer (pH 7.4) containing HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% BSA or 0.1% casein. The polypeptide was further serially diluted in an assay buffer containing 0.1% BSA or 0.1% casein in a 1:10 dilution step to produce 2x working stock solutions at 10 points, ranging from 20 μM to 0.02 pM.

[0228] 15 μL of assay buffer (basic activity), 1 μM human CNP-22 (maximum activity), or twice the working stock solution of the test polypeptide were transferred to a Costar® 3693 plate. Then, 15 μL of cell suspension containing 4000 cells was 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 generated cGMP was measured using a cGMP kit according to the manufacturer's instructions, as described below.

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

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

[0232] The ratio of 665 nm / 620 nm multiplied by 10000 was plotted on a logarithmic scale against the cGMP standard concentration, and a standard curve was created using an internally developed 4-parameter nonlinear regression curve fitting template. The amount of cGMP produced by cells overexpressing NPRB was interpolated using the cGMP standard curve. 100% response was determined from wells in the absence of the test polypeptide and in the presence of saturated human CNP-22 (1 μM). 0% response was determined from wells containing assay buffer. Using Prism 9, 10 concentration-response curves for the test polypeptide (10 μM to 0.01 pM) were fitted to the 4-parameter model to determine potency (EC2). 50 The ) value and maximum activation (%Max) were determined.

[0233] Similar to hANP, none of the polypeptides in the examples showed significant agonist activity on NPR-B.

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

[0235] [Table 14]

[0236] Research using a mouse model with sodium chloride-containing drinking water, unilateral nephrectomy, and aldosterone (SAUNA). The effects of the ANP polypeptide in the example will be investigated in a mouse model of saline-containing drinking water / unilateral nephrectomy / aldosterone (SAUNA) and a mouse model of heart failure induced by chronic aldosterone infusion. After approximately two weeks of adaptation, male C57BL / 6N (Taconic) mice will be induced to have heart failure over four weeks by unilateral nephrectomy, continuous d-aldosterone infusion, and 1.0% sodium chloride in drinking water (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 and provided with comparable variances of body weight and blood pressure (measured in conscious mice using a non-invasive tail cuff system (Kent Scientific)); and mice are randomized using the Block Randomized Allocation Tool (BRAT, Eli Lilly and Company). Once randomized, mice are treated once daily with 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, intubated via tracheostomy, and the chest is opened to expose the heart, allowing placement of a pressure-volume catheter (Transonic). The pressure-volume (PV) catheter is introduced into the left ventricle via 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 measuring the PV loop, the mice were sacrificed, and the ratio of heart weight to tibial length was used as an indicator of hypertrophy.

[0237] The effects of Example 8 were investigated using the SAUNA mouse model described above. Administration of Example 8 resulted in a decrease in blood pressure, cardiac weight, and tibia length, as well as a decrease in left ventricular diastolic blood pressure.

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

[0239] control plasma Using the Enzo cGMP Complete ELISA Kit (Enzo Life Sciences, Inc., Farmingdale, New York, catalog number ADI-901-164), control plasma was prepared to determine the recovery rate of rapidly elevated cGMP. Briefly, blood was collected in approximately 4 mL volumes from approximately 7-month-old male Sprague Dawley rats (Inotiv, Indianapolis, Indiana) via posterior orbital hemorrhage into BD Vacutainer EDTA tubes (Becton Dickinson, Franklin Lakes, New Jersey, catalog number BD-367856). Plasma was prepared by rotating the tubes in an Eppendorf Refrigerated Centrifuge 5810R (Brinkman Instruments, Inc., Westbury, New York) at 3500 rpm (3000 × g) for 10 minutes at 4°C. Plasma was collected as positive control plasma. Cyclic GMP (cGMP standard from the Enzo cGMP complete ELISA kit) was added to the positive control plasma at a final concentration of 40 nM. The positive control and spike-in control plasmas were divided equally and stored at -80°C.

[0240] Assay method - Monkey plasma cGMP ELISA cGMP content was measured using the Enzo cGMP complete ELISA kit, following the manufacturer's instructions but with modifications as described below. The cGMP standard provided in the kit was diluted 1:3 in a 1x assay buffer, which was obtained by diluting the 2x assay buffer provided in the kit with water, to a concentration ranging from 50 nM to 0.023 nM. 150 μL of the cGMP standard was 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. For both the non-specific binding control (in the absence of cGMP antibody) and the maximum binding control (in the absence of competitive cGMP), 150 μL of assay buffer was added to each pair of wells. The positive control and spike-in control plasmas were diluted 1:20 with 1x assay buffer to a final volume of 150 μL in each pair of wells on the plate. All diluted plasmas were mixed by pipetting several times.

[0242] An acetylating agent mixture was prepared by adding 1 part anhydride acetate to 2 parts trimethylamine provided in the kit and mixing thoroughly using a Vortex (Scientific Industries, Inc., Bohemia, New York). 15 μL of the acetylating agent mixture was added to the plate one column at a time, and all control, standard, and plasma samples were acetylated by shaking the plate on a Titer Plate Shaker (Lab-Line Instrument, Inc., Melrose Park, Illinois) at room temperature for 1 minute. After acetylating the last column, the plate was shaken for an additional 1 minute to confirm that the reaction was complete.

[0243] Acetylated control, standard, and plasma sample (100 μL) were transferred to an ELISA plate (n=1). 50 μL of cGMP conjugate was then added to each well, followed by 50 μL of cGMP antibody in each well, except for two nonspecific binding wells to which 50 μL of 1x assay buffer was added. 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 using 200 μL of 1x wash buffer diluted with water from the 5x wash buffer provided in the kit. 200 μL of p-nitrophenyl phosphate (pNpp) was then 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 enzymatic reaction. Next, the plate was read at 405nm using SpectraMax Plus (manufactured by Molecular Devices, Inc., San Jose, California).

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

[0245] Data Analysis The mean absorbance at 405 nm (OD405nm) for the nonspecific binding control was subtracted from the OD at 405 nm for all samples. The subtracted OD at 405 nm for all samples was then normalized by the subtracted mean OD at 405 nm of the maximum binding control to obtain B / B 0%. The B / B 0% of the cGMP standard was then plotted on a logarithmic scale against the cGMP standard concentration, and a standard curve was created using an internally developed 4-parameter nonlinear regression curve fitting template. The total amount of 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 of each animal measured in plasma before administration (before administration, time 0) of each polypeptide, as shown in Table 4, from the cGMP value of the same animal at each time point after administration. Using Microsoft Excel (manufactured by Microsoft Corporation, Redmond, Washington), cGMP (nM, mean ± standard deviation of mean SEM) and net cGMP change (nM, mean ± SEM) at various time points were graphed. The cGMP data for monkeys is 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 administered to monkeys, the net cGMP levels increased.

[0249] In vivo dog research - cGMP levels An in vivo study using dogs was conducted as described in detail below. A single dose was administered subcutaneously (SC) to young adult to adult purebred beagle dogs from the Labcorp stock colony housed at Labcorp-Madison. Blood was collected pre-administration and at various predetermined time points throughout the study period. Aliquots of beagle canine plasma were received for cyclic GMP (cGMP) analysis and stored at -80°C before use.

[0250] control plasma Using the Enzo cGMP Complete ELISA Kit (Enzo Life Sciences, Inc., Farmingdale, New York, catalog number ADI-901-164), control plasma was prepared to determine the recovery rate of rapidly elevated cGMP. Briefly, blood was collected in approximately 4 mL volumes from approximately 7-month-old male Sprague Dawley rats (Inotiv, Indianapolis, Indiana) via posterior orbital hemorrhage into BD Vacutainer EDTA tubes (Becton Dickinson, Franklin Lakes, New Jersey, catalog number BD-367856). Plasma was prepared by rotating the tubes in an Eppendorf Refrigerated Centrifuge 5810R (Brinkman Instruments, Inc., Westbury, New York) at 3500 rpm (3000 × g) for 10 minutes at 4°C. Plasma was collected as positive control plasma. Cyclic GMP (cGMP standard from the Enzo cGMP complete ELISA kit) was added to the positive control plasma at a final concentration of 40 nM. The positive control and spike-in control plasmas were divided equally and stored at -80°C.

[0251] Assay method - Canine plasma cGMP ELISA cGMP content was measured using the Enzo cGMP complete ELISA kit, following the manufacturer's instructions but with modifications as described below. The cGMP standard provided in the kit was diluted 1:3 in a 1x assay buffer, which was obtained by diluting the 2x assay buffer provided in the kit with water, to a concentration ranging from 50 nM to 0.023 nM. 150 μL of the cGMP standard was 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. For both the non-specific binding control (in the absence of cGMP antibody) and the maximum binding control (in the absence of competitive cGMP), 150 μL of assay buffer was added to each pair of wells. The positive control and spike-in control plasmas were diluted 1:20 with 1x assay buffer to a final volume of 150 μL in each pair of wells on the polypropylene plate. All diluted plasmas were mixed by pipetting several times.

[0253] An acetylating agent mixture was prepared by adding 1 part anhydride acetate to 2 parts trimethylamine provided in the kit and mixing thoroughly using a Vortex (Scientific Industries, Inc., Bohemia, New York). 15 μL of the acetylating agent mixture was added to each of the eight wells of the polypropylene plate described above, one column at a time, and all control, standard, and plasma samples were acetylated by shaking the plate on a Titer Plate Shaker (Lab-Line Instrument, Inc., Melrose Park, Illinois) at room temperature for 1 minute. After acetylating the last column, the plate was shaken for an additional minute to confirm that the reaction was complete.

[0254] Acetylated control, standard, and plasma sample (100 μL) were transferred to an ELISA plate (n=1). 50 μL of cGMP conjugate was then added to each well, followed by 50 μL of cGMP antibody in each well, except for two nonspecific binding wells to which 50 μL of 1x assay buffer was added. 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 using 200 μL of 1x wash buffer diluted with water from the 5x wash buffer provided in the kit. 200 μL of p-nitrophenyl phosphate (pNpp) was then 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 enzymatic reaction. Next, the plate was read at 405nm using SpectraMax Plus (manufactured by Molecular Devices, Inc., San Jose, California).

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

[0256] Data Analysis The mean absorbance at 405 nm (OD405nm) for the nonspecific binding control was subtracted from the OD at 405 nm for all samples. The subtracted OD at 405 nm for all samples was then normalized by the subtracted mean OD at 405 nm of the maximum binding control to obtain B / B 0%. The B / B 0% of the cGMP standard was then plotted on a logarithmic scale against the cGMP standard concentration, and a standard curve was created using an internally developed 4-parameter nonlinear regression curve fitting template. The total amount of cGMP present in the experimental samples was interpolated using this standard curve in the template. The net change in cGMP (nM) was calculated by subtracting the cGMP value of each animal measured in plasma before administration (before administration, time 0) of each polypeptide, as shown in Table 5, from the cGMP value of the same animal at each time point after administration. Using Microsoft Excel (manufactured by Microsoft Corporation, Redmond, Washington), cGMP (nM, mean ± standard deviation of mean SEM) and net cGMP change (nM, mean ± SEM) at various time points were graphed. Canine cGMP data is shown in Table 5.

[0257] [Table 17]

[0258] [Table 18]

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

[0260] array Sequence ID 1: -rANP (Rat ANP) SLRRSS[CFGGRIDRIGAQSGLGC]NSFRY (Disulfide bond between C7 and C23) Sequence ID 2: -hANP (Human ANP) SLRRSS[CFGGRMDRIGAQSGLGC]NSFRY (Disulfide bond between C7 and C23) Sequence ID 3: -Formula I X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (In the formula, X1 does not exist, or is S or E. X2 is either nonexistent, or L, K, 4-Pal, H, or E. X3 is either nonexistent or R, β-Ala, P, K, E, or G. X9 is G, 4-Pal, or H. X 10 It is G, K, R, or Dap. X 11 These are R, K, G, or Dap. X 13 is either D or G, X 17 is A, H, Dap, K, R, or Orn, X 18 is Q, Y, or 4-Pal, X 26 It is either F or L, X 28 is Y, H, or 4-Pal, and X 29 It either does not exist, is GGP, or is selected from sequence numbers 4-20. Furthermore, the C-terminal amino acid is optionally amidated. Sequence ID 4 SGAPPPE Sequence ID 5 KITAKEDE Sequence ID 6 GPSSGAPPPE Sequence ID 7 GPSSGAPPPS Sequence ID 8 GGSSGAPPPS Sequence ID 9 GGPSSGAPPPS Sequence ID 10 KGPSSGAPPPS Sequence ID 11 GGKSSGAPPPS Sequence ID 12 GGPPS-Aib-KPPPK Sequence ID 13 GSPSSGAPPPS Sequence ID 14 RITAREDKQGYA Sequence ID 15 RITAREDKQGEA Sequence ID 16 GSPSSGAPPPS-PEG24-G Sequence ID 17 SGSPSSGAPPPSG Sequence ID 18 GGESSGEPPPSEE Sequence ID 19 GSGSPSSGAPPPSG Sequence ID 20 SGSPSSGAPPPSEEEG Sequence ID 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 acids are C16-C26 fatty acids. Z1 contains amino acids selected from γGlu, E, and β-Ala. Z2 is either absent or contains a sequence of 4 to 10 amino acids, including amino acids independently selected from E, K, G, P, A, and S. Z3 is either absent or contains polyethylene glycol or the (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moiety. The C-terminal amino acid is arbitrarily amidated. Sequence ID 22 EKEKEKG Sequence ID 23 EPEPEPG Sequence ID 24 APPSG Sequence ID 25 KEKEKG Sequence ID 26 EKEKEKE Sequence ID 27 X1X2X3RSSCFX9X 10 X 11 IX 13 RIGX 17 X 18 SGLGCPSX 26 RX 28 X 29 (In the formula, X1 is either 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 either D or G, X 17 H is, X 18 is Q or Y, X 26 F is, X 28 is H and X 29 This is selected from GGPSSGAPPPS (sequence number 9), GGKSSGAPPPS (sequence number 11), and GSPSSGAPPPS (sequence number 13). The C-terminal amino acid is optionally amidated. Sequence IDs 28-167 Examples 1 to 140 are listed in Table 1. Sequence IDs 168-172 Examples 141 to 145, respectively. Sequence 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 It does not exist, or it is S or E. X ２ It does not exist, or it is L, K, 4-Pal, H, or E. X ３ It does not exist, or it is R, β-Ala, P, K, E, or G. X ９ is G, 4-Pale, or H, X １０ is G, K, R, or Dap, X １１ is R, K, G, or Dap, X １３ is either 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 It does not exist, or GGP, SGAPPPE (Sequence ID 4), KITAKEDE (Sequence ID 5), GPSSGAPPPE (Sequence ID 6), GPSSGAPPPS (Sequence ID 7), GGSSGAPPPS (Sequence ID 8), GGPSSGAPPPS (Sequence ID 9), KGPSGAPPPS (Sequence ID 10), GGKSSGAPPPS (Sequence ID 11), GGPPS-Aib-KPPPK (SEQ ID NO: 12), GSPSGAPPPS (Sequence ID 13), RITAREDKQGYA (Sequence ID 14), RITAREDKQGEA (Sequence ID 15), GSPSGAPPPS-PEG24-G (Sequence ID 16), SGSPSSGAPPPSG (Sequence ID 17), GGESSGEPPPSEE (Sequence No. 18), GSGSPSSGAPPPSG (Sequence ID 19), and Selected from SGSPSSGAPPPSEEEG (Sequence No. 20), (The C-terminal amino acid is optionally amidated.) A polypeptide containing or a pharmaceutically acceptable salt thereof.

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

3.

3. It further contains fatty acids conjugated to amino acids located at the N-terminus of the polypeptide, 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 (In the formula, the fatty acid is C １６ ~C ２６ It is a fatty acid, and is conjugated to an amino acid present at the N-terminus of the polypeptide via the structure Z1-Z2-Z3, and in the formula, Z1 is an amino acid selected from γGlu, E, and β-Ala. Z2 is (EK) ｂ G, (EP) ｂ G, K (EK) ｃ G, (EK) ｃ Selected from E and APPSG (Sequence ID 24) (wherein b is 2, 3, or 4, and c is 1, 2, 3, or 4), and Z3 does not exist, or (polyethylene glycol) ｍ (In the formula, m is an integer selected from 10 to 30) or ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) ｎ A polypeptide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, comprising (wherein n is an integer selected from 1 to 10).

4. X １ is S or E, X ２ is K or 4-Pale, X ３ is R, β-Ala, or K. X ９ G is, X １０ is G or K, X １１ is R or K, X １３ is either D or G, X １７ H is, X １８ is Q or Y, X ２６ F is, X ２８ is H, and X ２９ The polypeptide according to any one of claims 1 to 3, 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 aforementioned fatty acid is C １６ ~C ２２ A polypeptide according to claim 3 or 4, or a pharmaceutically acceptable salt thereof, which is a fatty acid.

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

7. The polypeptide according to 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. The aforementioned Z3 is either absent or (polyethylene glycol) ｍ (wherein m is 12 or 24), and ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)) ｎ A polypeptide according to claim 7 or a pharmaceutically acceptable salt thereof, selected from the formula (wherein n is 4, 6, or 8).

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

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

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

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

13. A pharmaceutical composition comprising a 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. The pharmaceutical composition according to claim 13, which is formulated for subcutaneous (SQ) or intravenous (IV) administration.

15. The pharmaceutical composition according to 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 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.

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

18. The method according to 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 therapeutic purposes.

20. 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 the treatment of CVD.

21. 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 the treatment of heart failure.

22. 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 the treatment of HFpEF.

23. Use of a 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 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, in the manufacture of a drug for treating heart failure.

25. Use of 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, in the manufacture of a drug for treating HFpEF.

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